Hungarian Scientific Society for Food Industry BUDAPEST, HUNGARY
16th International Plant Lipid Symposium Budapest, Hungary 1 - 4 June 2004. (Tuesday-Friday)
Budapest University of Economic Sciences and Public Administration Faculty of Food Science BUDAPEST, HUNGARY
Organised by the Hungarian Scientific Society for Food Industry (MÉTE) and the
Budapest University of Economic Sciences and Public Administration Faculty of Food Science Budapest
ORAL AND POSTER PRESENTATIONS Venue of the Symposium: PESTI CONFERENCE CENTRE H-1054 BUDAPEST, Kossuth Lajos tér 6-8.
1
CONTENT HARTMUT K. LICHTENTHALER
Thirty years of International Symposia of Plant Lipids
I - XXIII
In memoriam Prof. András Farkas PROCEEDINGS OF LECTURES
o-0-1
HARTMUT K. LICHTENTH ALER
Evolution of carotenoid and isopreno id biosynthesis in photosynthetic and non photosynthetic organisms (Terry Gaillard Memorial Lecture)
8 11
1. Session: LIPID BIOSYNTHESIS Chair: Prof. John OHLROGGE
o-1-1
CAHOON, Edgar B.:
o-1-2
. HARTMANN, M.A;. WENTZINGER, L.
Metabolic Redesign of Vitamin E Biosynthesis for Tocotrienol Productiuo and Increased Lipid-Soluble Antioxidant Content Tobacco BY-2 cells, as an useful experimental system for investigating regulation of the sterol pathway
25
30
2.Session: LIPID FUNCTION Chair: Prof. John HARWOOD
o-2-4
R.J. WESELAKE1 , M. MADHAVJI1 , Polyclonal antibodies, aba and abb, were … N. FOROUD 1 , S. SZARKA 2 , within either the third predicted N. PATTERSON2 , W. WIEHLER1 , C. lumenal loop or second predicted NYKIFORUK, T. BURTON 1 , cytosolic loop of BnDGAT1 P. BOORA1 , S. MOSIMANN1 , M. MOLONEY 2 AND A. LAROCHE3
35
3. Session: FATTY ACIDS Chair: Dr. Petra SPERLING 4. Session: REGULATION Chair: Dr. Ivo FEUSSNER 5.Session METABOLISM Chair: Prof. Christoph BENNING
o-5-4
HORNUNG, E.1 , SAALBACH, I.2 and Production of conjugated fatty acids in plants FEUSSNER, I
43
6.Session MEMBRANE LIPIDS Chair: Prof. Peter DÖRMANN
o-6-1
o-6-2
MOORE, Thomas S., MORONEY, James V., YANG, Wenyu
GOMBOS, Zoltán: Phosphatidyglycerol an essential component of photosynthetic membranes for oligomer formation in PSI reaction centers Biosynthesis of Membrane Lipids in Chlamydomonas reinhardtii
2
ppt
49
The roles of lipid bodies and lipid-body protein in the assembly and trafficking of lipids in plant cells Amelioration of sensitivity to UV-b in Arabidopsis by suppression of a putative phospholipase
o-6-4
DENIS J. MURPHY
o-6-5
J.E. THOMPSON, M. LO, C. TAYLOR, L. WANG, L. NOWACK, T.W. WANG
o-6-7
G. TASSEVA*, M.-N. VAULTIER*, Implication of membrane lipids in plant response to a cold shock, from C. CANTREL, F. signalling to adaptation COCHET, J. DAVY de VIRVILLE, C. DEMANDRE, A.-M. JUSTIN, J.-C. KADER, F. MOREAU, E. RUELLAND and A. ZACHOWSKI
56 63
70
7. Session: BIOTECHNOLOGY Chair: Prof Norio MURATA
o-7-3
G.K.A PARVEEZ *; O.ABRIZAH, A.M.Y.MASANI, A. SITI NOR AKMAR A., R. UMI SALAMAH, S. RAVIGADEVI, B. BAHARIAH, A.H. TARMIZI, I., ZAMZURI, A.D. KUSHAIRI, S.C. CHEAH, AND M.W. BASRI.
Genetic engineering for modifying fatty acid composition of palm oil
76
o-7-6
SHAH, FARIDA H.1, 2 ; BHORE, Current status in the genetic alteration of SUBHASH J1 , 2; CHA THYE SAN3 ; fatty acidcomposition in Oil Palm 1 and TAN CHYE LING
84
8. Session: WAXES and STRESS Chair: Prof. Ikuo NISHIDA
o-8-1
o-x-1 o-8-4
J.M. MARTÍNEZ-RIVAS, A. Microsomal oleate desaturase (FAD2) from SÁNCHEZ-GARCÍA, M.D. oilseeds: New advances on the SICARDO, A.B. ESTEBAN AND M. regulation by temperature and MANCHA oxygen TARAN Nataliya , BATSMANOVA Oxidative stress induce leaf lipid changes Ludmila, OKANENKO Alexander A.G. VERESHCHAGIN Occurence of lipophilic micellae formed by fatty alcohols and fatty-acid Na salts in aqueous jojoba-wax hydrolysate
88
95 102
9. Session: LIPOLYTIC ENZYMES Chair: Prof Zoltán GOMBOS
o-9-3
I.M. KOTEL’NIKOVA1 , E.V. NEKRASOV2 , A. V. KRYLOV2
Effect of tobacco mosaic virus infection on phospholipid content, phospholipase d activity and reactive oxygen species production in tobacco leaves
3
105
o-9-4
o-9-7
R. LESSIRE1 , P. COSTAGLIOLI1,2 , C. GARCIA 1 , J. JOUBES1 ., W. DIERYCK 1,2 , J. LAROCHETRAINEAU1 , S. CHEVALIER1 , B. GARBAY1 ERIC TESTET*, JEANNY LAROCHE-TRAINEAU§, ABDELMAJID NOUBHANI*, DENIS COULON*, ODILE BUNOUST‡ RENÉ LESSIRE§ AND JEAN-JACQUES BESSOULE§
Acyl-CoA elongase: genomic studies
113
Characterisation of a lyso PC acyltransferase from S. Cerevisiae
118
PROCEEDINGS OF POSTERS No.
TITLE
AUTHORS 1. POSTER SESSION
BANAS, Antoni; CARLSSON, Anders; HUANG, Bangquan ; NOIRIELl, Alexandre ; BENVISTE, Pierre; SCHALLER, Hubert; BOUVIER-NAVÈ, Pierrette; STYMNE, Sten 1 , S. CHEVALIER1 , A p-1-5... ...C. GARCIA BRETON3 , R. LESSIRE1 AND W. DIERYCK 1,2 .
p-1-3
KOSÁRY, Judit, ; KORBÁSZ, Margit; KISS, Nikoletta; BALOGH, Teréz p-1-18...MAISONNEUVE, Sylvie; CHIRON, Hélène; DELSENY, Michel and ROSCOE, Thomas
p-1-12
p-1-20 p-1-22
Enzymatic properties of an A. thaliana phospholipid: sterol acyltransferase.
126
The acyl-coa elongase in ArabidopsisTthaliana : characterization of a candidate gene presumably encoding the 3-hydroxyacyl-CoC dehydratase Almond lipoxygenases
129
Effect of altered expression of lysophosphatidic acid acyltransferases on triacylglycerol synthesis in ArabidopsisThaliana seeds
NEKRASOV, Edward; KOTEL'NIKOVA, Irina; VASKOVSKY, Victor SHRESTHA, Pushkar, COHEN, Dror; KHALILOV, Ilkhom; KHOZIN –GOLDBERG, Inna and COHEN Zvi 2. POSTER SESSION
The participation of phospholipase D in plant growth processes and response to viral infection Triacylglycerol biosynthesis in microsomes and oil bodies of the oleaginous green alga parietochloris incisa
4
133 138
141 145
p-2-2
BROOKER, Nancy; LONG, James; COLLINS, Josh
p-2-5
FAUCONNIER, Marie -Laure; WELTI, Ruth; DELAPLACE, Pierre; MARLIER, Michel; DU JARDIN, Patrick GONCHAROVA, Svetlana; SANINA, Nina; KOSTETSKY, Eduard
p-2-6
p-218
SANINA, M., GONCHAROVA, S.N.,. KOSTETSKY, E.Y
p-2-9
KOHNO-MURASE, Junko; IWABUCHI, Mari; KOBA, Kazunori; IMAMURA, Jun 3. POSTER SESSION
p-3-1
BHORE, Subhash J; CHA, Thye S; SHAH, Farida H
p-3-3
MIETKIEWSKA, Elzbieta; GIBLIN, E. Michael; WANG, Song; BARTON, Dennis L.; DIRPAUL, Joan; KATAVIC, Vesna and TAYLOR, David C. * GIRISH MISHRA, SATHISHKUMAR RAMALINGAM, M.S.F. LIE KEN JIE AND MEE-LEN CHYE KATAVIC, Vesna; BARTON, L. Dennis; GIBLIN, E. Michael, REED, W. Darwin ; KUMAR, Arvind; TAYLOR, C. David KHALILOV, Ilkhom; KHOZIN – GOLDBERG, Inna and COHEN, Zvi 4. POSTER SESSION
p-3-4
p-3-9
p-3-10
Plant Lipid Derivative Seed Treatments For Managing Fungal Soybean Diseases Lipid and oxilipin profile during storage of potato tubers
149
Seasonal alterations in fatty acid composition of phospho- and glycolipids from Zostera Marina and Laminaria Japonica. Seasonal changes in physico-chemical properties of polar lipids from seagrass Zostera Marina Production of an isomer of conjugated linolenic acid, punicic acid, in rapeseed and rice
159
Particle Bombardment-Mediated Transformation of Elaeis oleifera with Antisense Palmitoyl-ACP Thioesterase Gene Driven by Mesocarp Specific Promoter Seed-specific heterologous expression of a nasturtium FAE gene** in Arabidopsis results in an eight-fold increase in erucic acid content.
168
Characterization of a mutant in Arabidopsis ACBP2
175
Is Ser 282 Crucial for the Function of Brassica napus FAE1 Condensing Enzyme?
178
Initial studies on the fatty acid desaturase genes in the unicellular green algae Parietochloris incisa
181
152
163 166
172
p-4-2
DARNET, Sylvain and RAHIER, Alain
Identification of two novel families of membrane-bound non-heme iron oxygenases involved in plant sterol biosynthesis: a VIGS approach.
184
p-4-3
DÖRTE, Klaus; BAS, Terriet, BASTIAAN BARGMANN AND TEUN MUNNIK
Silencing of a phospholipase d a leads to chlorosis in transgenic tomato leaves
187
p-4-12
ROZENTSVET, Olga; BOSENKO, Olena; GUSCHINA, Irina
Effect of heavy metals on metabolism of lipids in P. perfoliatus
5
202
p-4-16 p-4-17
p-4-18
TONON, Thierry; HARVEY, David; QING, Renwei; LI, Yi; LARSON, Tony; GRAHAM, Ian TROUFFLARD, Stephanie ; BHOGAL, Ramneek; PROST, Isabelle; THOMASSET, Brigitte; RAWSTHORNE, Steve; ROSCHER, Albrecht; PORTAIS, Jean-Charles NAGYNÉ KUTNI, R.; R. SZALAY*, R.; PÁLVÖLGYI, L.
6
Identification of a fatty acid ?11-desaturase from the microalga ThalassiosiraPpseudonana Biochemical changes and carbon supply in linseed developing embryos
190
Breeding High-Oleic Sunflower Lines For Complex Disease Resistance
198
193
Thirty Years of International Symposia on Plant Lipids Hartmut K. Lichtenthaler Botany II (Molecular Biology and Biochemistry of Plants), University of Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany
[email protected] Abstract: After several years of contacts between individual European plant lipid biochemists, the International Symposia on Plant Lipids (ISPL) were started in 1974 by an initiating Plant Acyl Lipid Symposium in Norwich, organized by Terry Galliard. In 1976 the topics were extended at the Karlsruhe Symposium (2nd ISPL) by including all the other plant lipids, such as isoprenoid lipids (sterols, carotenoids and prenyl side chains of chlorophylls and prenylquinones) and lipid polymers. Since then the International Symposia on Plant Lipids (ISPL) have been held every other year. Their goal is to promote scientific cooperation between work groups of different countries, and they have resulted in fast progress in all fields of plant lipid biochemistry. On the occasion of the 16th International Symposium on Plant Lipids in Budapest in June 2004 a brief history of these symposia has been presented here. These symposia also were the start of a series of meetings on plant lipids in Germany, in the USA and in Japan.
List of Contents: 1. 2. 3. 4.
The beginnings……………………………………………………………………..…1 The first international symposium on plant lipids (1st ISPL)…………………….......4 The second international symposium on plant lipids (2nd ISPL)…………………......7 The impact of the Norwich and Karlsruhe symposia for the future plant lipid symposia…………………………………………………………………...…..10 5. The Terry Galliard Memorial Lecture and Medal…………………………………...14 6. The organization of the ISPL by the international board…………………………....15 7. The German-speaking plant lipid workshops (Arbeitstagungen Pflanzliche Lipide).19 8. The meetings of the national plant lipid cooperative NPLC in USA………………..20 9. The Annual Symposia of the Japanese Association of Plant Lipid Researchers (JAPLR)………………………………………………………………...21 10. References…………………………………………………………………………....23 11. Addendum: photos of participants of previous ISPL………………………………...25
1) THE BEGINNINGS In the first two decades after the World War II, modern experimental physiological and bioche mical plant research developed in Europe and the USA due to the availability of new experimental instrumentation and methods. First contacts between scientists of different countries started in the 1960s by individual initiatives. European congresses and meetings, held in most fields of plant biology these days, did not exist at the time. Scientific contacts were greatly enhanced by the First International Photosynthesis Congress in Freudenstadt, Black Forest, Germany, in May 1968. This congress gathered about 230 young as well as established scientists from Eastern and Western Europe and also the leading photosynthesis colleagues from the USA and a few from Japan (as can be seen from the proceedings book 1
I.
“Progress in Photosynthesis” edited by Helmut Metzner in 1969). This congress had a great impact not only on European and international photosynthesis research, but also on many other fields of plant biology, and it started and considerably promoted scientific cooperation, particularly in Europe. The Impact of Photosynthesis Research on Plant Lipid Research Many European photosynthesis scientists either met on their congress in Freudenstadt 1968 for the first time or in the USA as postdocs and since then have stayed in touch. In fact, several plant lipid scientists had started their research in photosynthesis and later extended their research to plant lipid biochemistry. They continued their scientific exchange over the following decades even if they changed or extended their photosynthesis-related research to other topics, such as the photosynthetic biomembrane lipids and their effect or interaction with photosynthetic function. Among them are Andy A. Benson, Hartmut Lichtenthaler, Wilhelm Menke, Norio Murata, Paul-André Siegenthaler and Sjef F.G.M. Wintermans, just to name a few. Their individual contributions to the early plant lipid research is indicated below. The importance of the glyco- and phospholipids as well as the non-acyl lipids, such as the isoprenoid chlorophylls, carotenoids and prenylquinones for the functioning of the photochemically active thylakoids, had become evident through the pioneer paper with the complete lipid composition of thylakoids (Lichtenthaler and Park 1963). On the basis of that thylakoid lipid composition, Lichtenthaler discussed in great detail with Andy Benson, during a visit to La Jolla in the fall of 1963, the possible orientation of chlorophylls, carotenoids and prenylquinones in the acyl lipid moiety of either a mono- or a bimolecular lipid layer of thylakoids. Also at the Freudenstadt Photosynthesis Congress in 1968 several colleagues discussed the significance of acyl lipids and prenyllipids for the photosynthetic function, e.g. Trevor Goodwin and Hartmut Lichtenthaler disputed there with several other colleagues on various aspects of lipid involvement in chloroplast biogenesis with emphasis on the biosynthesis and function of carotenoids and prenylquinones, topics that later became essential parts of the International Symposia on Plant Lipids. The famous photosynthesis researcher Prof. Wilhelm Menke (Cologne) was a pioneer in plant lipid research. In fact, he was the first scientist to isolate spinach chloroplasts by centrifugation (Menke 1938), had checked their relative lipid amounts as compared to cytoplasmic fractions (Menke and Jacob, 1942), and later created the name “thylakoids” for the photosynthetic biomembrane (Menke 1961). In the early 1960s Ernst Heinz started his Ph.D. dissertation on plant acyl lipids with W. Menke and later was coorganizer of the 2nd ISPL in Karlsruhe. The chemist Waldemar Eichenberger from the University of Bern spent his postdoc years 1964-1965 with Menke in Cologne analyzing sterylglycosides and acylated sterols in plants, detecting that chloroplasts did not contain sterols (Eichenberger and Menke 1966). Thus, essential impulses for plant lipid research came from various photosynthesis laboratories that tried to find the function of individual acyl lipids and prenyllipids in the photochemically active thylakoids. The Role of European Postdocs in the USA Another decisive impulse for the establishment of the International Symposia on Plant Lipids came from various young European scientists who had spent one or two postdoc years in well-equipped laboratories in the USA. After their retur n to Europe they kept or searched for contacts with other plant lipid researchers who had been postdoc in the USA. The laboratory of Paul Stumpf in Davis/California was an early center of plant fatty acid and lipid research where in the late 1960s and early 1970s Terry Galliard (Norwich), John Harwood (Cardiff), Lars-Åke Appelqvist (Stockholm), P. Castelfranco (USA), and J. Brian Mudd (USA) spent their postdoc years. Later more scientists, such as J. Joyard (France), R. Lessire (France), D. J. Murphy (England), J. Ohlrogge (USA), J. Sanchez (Spain), M. Yamada (Japan), joined his laboratory. All of them, Paul Stumpf's postdocs of the early and later years, as well as Paul II.
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Stumpf himself, contributed a great deal to the International Symposia on Plant Lipids, some of these former postdocs from the very beginning and others after the ISPL had been established. In fact, those who are underlined later became organizers or co-organizers of one of the ISPL. Another early center for plant lipid research in the USA was the laboratory of Andrew A. Benson (plant sulfolipids), where Joseph F.G.M. Wintermans (Nijmegen) in 1958 (then at State College, Pennsylvania) and later H. Pohl (Kiel) 1974 (then at his laboratories in La Jolla) spent their postdoc time. Andy A. Benson, the co-detector of the photosynthetic carbon fixation cycle (Calvin-Benson-cycle), had extended his research topic in the late 1950s and early 1960s to the detection and investigation of plant sulfolipids (Benson et al. 1959a and b), and his first European postdoc J.F.G.M. Wintermans was later organizer of the 5th ISPL. In the early 1970s Roland Douce (Grenoble), another European postdoc of Andy Benson isolated the chloroplast envelope (Douce et al. 1973) and started to analyze the lipid biosynthesis capacity of the chloroplast envelope (Douce 1974). In 1980 he became coorganizer of the 4th ISPL in Paris. Hartmut Lichtenthaler (Karlsruhe) had been a postdoc in Melvin Calvin’s laboratory in Berkeley/California from 1962 through 1964 and worked out the prenyllipid composition (chlorophylls, carotenoids, plastoquinone-9, phylloquinone K1, tocopherol) of the photosynthetic membrane (Lichtenthaler and Calvin 1964) and also published the first list with the quantitative levels of all thylakoid lipids (Lichtentha ler and Park 1963). Later he was the organizer of the 2nd ISPL in Karlsruhe in 1976. Paul-André Siegenthaler (Neuchâtel) had been a postdoc in Lester Packer’s laboratory at the University of Berkeley from 1963 to 1965 working on the light- induced chloroplast shrinking, where he detected that free fatty acids could uncouple photosynthetic phosphorylation. Since 1980 he has been participating in the ISPL and became the organizer of the 6th ISPL in Neuchâtel in 1984. Having first been in Menke’s laboratory, W. Eichenberger was a postdoc in 1966 with David W. Newman (Oxford, Ohio) performing research on the biosynthesis of steryl glycosides from UDP-hexose (Eichenberger and Newman 1968). W. Eichenberger, then a sterol specialist, participated in the 2nd ISPL in Karlsruhe, and in 1984 he became coorganizer of the 6th ISPL in Neuchâtel /Switzerland. Also D. W. Newman (USA) took part in the Karlsruhe ISPL. After having returned to Germany (to Münster/Westphalia) in 1964 H. K. Lichtenthaler established close contacts with Joseph Wintermans in the neighboring Nijmegen/Netherlands and they co-operated in the lipid composition of the osmiophilic plastoglobuli of chloroplasts (Lichtenthaler 1968). Wintermans was a pioneer in chloroplast acyl lipid research and was the first to determine the phospho-and glycolipid levels of whole leaves and isolated chloroplasts of spinach and related them to the chlorophyll content (Wintermans 1960). He continued to work on the biosynthesis of plant acyl lipids (Wintermans 1966, Wintermans et al. 1969) with particular emphasis in the field of biosynthesis of galactolipids (van Besouw and Wintermans 1978). Later he became the organizer of the 6th ISPL in Groningen/Netherlands. Lichtenthaler, although continuing in photosynthesis and in prenyllipid research (e.g. pigment and prenylquinone composition of the two photosynthetic photosystems (Lichtenthaler 1969), motivated his Ph.D. student Manfred Tevini to perform research on the accumulation of plant acyl lipids during plant and chloroplast development. Both were organizers of the 2nd ISPL in Karlsruhe in 1976. In England Prof. A.T. James at the Unilever research laboratories formed another center for lipid research. Although research there was not directly focused on the particular function of individual plant lipids, he established and arranged contacts with several European plant lipid researchers. He also inspired them to write articles in "Endeavour", e.g. on the osmiophilic plastoglobuli of chloroplasts as a reservoir for excess plastid lipids (Lichtenthaler 1968). Thus, in the early 1970s several European research groups, some of III.
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them headed by former postdocs in USA and also various others, not only became aware of each other but also started to have some contacts and exchange of results and ideas, and later became regular participants of the ISPL. The Years 1972 and 1973 In summer of 1972 the 15th International Conference of Biochemistry of Lipid (ICBL), organized by D.A. van Dorp, took place in Den Haag/Netherlands. Its main topic was “Enzyme reactions in Lipid Biochemistry”. Although it primarily dealt with enzyme reactions concerning general metabolism and also biosynthesis of animal and human acyl lipids, various plant lipid biochemists participated in that congress. Lichtenthaler recalls that he met there again Joseph Wintermans and also Paul Mazliak with his coworker Jean-Claude Kader, as well as A.T. James, Terry Galliard and several others. Also Friedrich Spener, Münster, who later participated in many of the ISPL, remembers that it was his first participation in such an international congress where he became acquainted with leading lipid biochemists. On this ICBL the discussion started among the few plant lipid scientists on the need of a small European and international symposium dedicated solely to plant lipids, their accumulation, metabolism, intracellular localization, biosynthesis and function. Yet it took some more time before Terry Galliard would organize the first international symposium on plant acyl lipids in Norwich in 1974. In 1973, when Paul Stumpf visited the United Kingdom, Terry Galliard and Mike Gurr organized a meeting of all U.K. plant lipid biochemists at Unilever Colworth House in Sharnbrook/Bedford, which dealt with the biosynthesis and biochemistry of plant fatty acids and acyl lipids. John Harwood recalls that the Unilever laboratory “in those days was the only laboratory to rival Stumpf’s”. At the meeting, the idea of an international symposium on plant lipids was again proposed, and Terry Galliard and Mike Gurr put together a meeting in Norwich in 1974. 2) THE FIRST INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS (1st ISPL) This meeting, organized by Terry Galliard as a joint international symposium of the “Phytochemical Society” and “The Lipid Group of the Biochemical Society”, was held with the title “RECENT ADVANCES IN THE CHEMISTRY AND BIOCHEMISTRY OF PLANT LIPIDS”. We know little on Terry Galliard’s preparations of this first ISPL in Norwich and how he selected and activated the speakers for this plant acyl lipid symposium. He achieved financial support from the Royal Society, the Potato Marketing Board, Tato & Lyle Ltd. as well as Unilever (UK) Ltd. so that, effectively, there was no registration fee. A small “registration fee” of 2.50 pounds paid for coffee and tea in the breaks. John Harwood recalls: “The inexpensive nature of the meeting meant that many students attended; about one half of the participants were Ph.D. students or very young post-doctorals” mainly from UK. There were receptions for all participants which were provided by the Trustees of the John Innes Charity, the University of East Anglia and the Food Research Institute. An additional symposium dinner costed only 3.30 pounds! It was Terry Galliard’s personal initiative and engagement to organize such a symposium. As a former postdoc of Paul Stumpf in the USA he knew well the need for an international plant lipid discussion forum. He had various contacts to other plant lipid scientists within Europe and the USA as well. These were at that time only a rather small number of persons, and he activated these colleagues to participate in the 1974 Norwich symposium. This was a great achievement. All members of the today “international family of plant lipid scientists” are IV.
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grateful to Terry Galliard for finally having started this international symposium on plant acyl lipids which was the first ISPL of many others to follow. Officially, this ISPL had been organized by Terry Galliard (Hull) with the help of M. J. Gurr (Unilever, Sharnbrook), E. J. Mercer (Aberystwyth) and M.J.C. Rhodes (Norwich), yet Terry Galliard was the driving force for this symposium that was arranged at the University Residences, Fifers Lane, Norwich. That Terry Galliard was the effective motor and organizer of this ISPL has now been confirmed by Mike Gurr who recently commented “I was only a very minor adviser”. The symposium lasted two and a half days (April 8 to April 10, 1974) and 12 lectures were given. Although the symposium was rather short and brought together only about 25 established plant lipid scientists out of the total of 90 participants, it was the essential breakthrough and starting point for the many ISPL to come (see Table 1 on the next page) and for the later formation of the international plant lipid family as we know it today. The following were lecturers at Norwich: L.-Å. Appelquist (Stockholm), Harry Beevers (Santa Cruz), Terry Galliard (Norwich), F.D. Gunstone (St. Andrews), John L. Harwood (Cardiff), C. Hitchcock (Unilever), M. Kates (Ottawa), P.E. Kolattukudy (Pullman), P.C.J. Kuiper (Wageningen), Paul Mazliak (Paris), J.B. Mudd (Riverside), Paul K. Stumpf (Davis). The titles of their lectures are given in Table 2. About 90 scientists and Ph.D. students, most of them from the U.K., participated in this symposium, but only a smaller number of them were directly involved in plant lipid research. Among them were, besides the above lecturers, the plant lipid scientists J. Friend (Hull), T.W. Goodwin (Liverpool), Ernst Heinz (Köln), A.T. James (Unilever), R. G.O. Kekwick (Birmingham), Rachel M. Leech (York), D.M. Lösel (Sheffield), H.K. Mangold (Münster), Alfons Radunz (Köln), Eva Selstam (Göteborg), Manfred Tevini (Karlsruhe), D.R. Threlfall (Hull), A. Trémolières (Paris). Those underlined among the lecturers and participants named above were organizers or coorganizers of future international symposia on plant lipids. Table 2. Lecturers and titles of lectures given at the first international plant lipid symposium in Norwich in April 1974. C. Hitchcock
Structure and distribution of plant lipids
J.L. Harwood
Fatty acid biosynthesis
P.K. Stumpf
Biosynthesis of chloroplast lipids
F.D. Gunstone
Determination of the structure of fatty acids
L.-Å. Appelqvist
Biochemical and structural aspects of storage and membrane lipids in developing seeds
H. Beevers M. Kates
Organelles from fatty seedlings: biochemical roles in gluconeogenesis and phospholipid biosynthesis Biosynthetic pathways for phospholipid synthesis in spinach leaves
J.B. Mudd
Biosynthesis of glycolipids
P.E. Kolattukudy
Biochemistry of surface lipids
P. Mazliak P.J.C. Kuiper
Exchange processes between organelles involved in membrane lipid biosynthesis Role of lipids in water and ion transport
T. Galliard
Degradation of plant lipids by hydrolytic and oxidative enzymes. V.
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Table 1. List of the International Symposia on Plant Lipids, the ISPL. Nr.
Year
Place
Main Organizer
1)
1974: April 8-10
Norwich (UK)
Terry Galliard
2)
1976: July 18-21
Karlsruhe (Germany)
Hartmut Lichtenthaler
3)
1978: August 28-30
Göteborg (Sweden)
Conny Liljenberg
4)
1980: July 4-7
Paris (France)
Paul Mazliak
5)
1982: June 7-10
Groningen (Netherlands)
Josef F.G. Wintermans
6)
1984: July 16-20
Neuchatel (Switzerland)
Paul-André Siegenthaler
7)
1986: July 27-August 1
Davis (USA)
Paul K. Stumpf
8)
1988: July 25-28
Budapest (Hungary)
Peter Biacs
9)
1990: July 9-13
Wye (UK)
Peter J. Quinn
10)
1992: April 27-May 2
Djerba (Tunisia)
Abdelkader Cherif
11)
1994: June 26-July 1
Paris (France)
Jean-Claude Kader
12)
1996: July 7-12
Toronto (Canada)
John P. Williams
13)
1998: July 5-10
Sevilla (Spain)
Juan Sánchez
14)
2000: July 23-28
Cardiff (UK)
John Harwood
15)
2002: May 12-17
Okazaki (Japan)
Norio Murata
16)
2004: June 1-4
Budapest (Hungary)
Peter Bia cs
17)
2006: July 15-21
East Lansing (USA)
Christoph Benning
The contributions of the lectures of this first ISPL in 1974 were published as a proceedings book with T. Galliard and E.I. Mercer as editors in 1975 (see Table 2). It was natural that this book was reviewed by Andy A. Benson (La Jolla) who, as detector of the plant sulfolipid and of phosphatidyl- glycerol (Benson et al. 1959 a and b), was one of the pioneers of plant acyl lipid research. His extremely positive review started with the sentenc e: “In its 12 chapters the book covers fatty acid structure methodology, biosynthesis and their roles in plant lipid function” and ending with “The book will be a standard reference for a long while” (Benson 1976) and, in fact, it was. The plant lipid scientists among the participants in Norwich discussed a continuation of such international plant lipid symposia and agreed with the proposal of the German participants Manfred Tevini, Ernst Heinz and H.K. Mangold that the next symposium might possibly be held in Karlsruhe/Germany in 1976. Hartmut Lichtenthaler (who could not participate in Norwich but had sent Manfred Tevini) acted then together with Manfred Tevini as the main organizers for this 2nd ISPL. VI.
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Table 3. Proceedings Books of the International Symposia on Plant Lipids. (The years given indicate the year of the symposium, some of the books appeared one year later. The last proceedings of the 16th ISPL are published only in the web.) Year
Publisher
Editors
Title
Dedicated to
1974
Academic Press
T. Galliard E.J. Mercer
Recent Advances in the Chemistry and Biochemistry of Plant Lipids
1976
Springer Verlag
M. Tevini H.K. Lichtenthaler
Lipids and Lipid Polymers in Higher Plants
1978
Elsevier
L.-A. Appelquist C. Liljenberg
Advances in the Biochemistry and Physiology of Plant Lipids
1980
Elsevier
P. Mazliak et al.
Biogenesis and Function of Plant Lipids
J. Wintermans
1982
Elsevier
J. Wintermans P. Kuiper
Biochemistry and Metabolism of Plant Lipids
Paul.K. Stumpf
1984
Elsevier
P. Siegenthaler W. Eichenberger
Structure, Function and Metabolism of Plant Lipids
Morris Kates
1986
Plenum Press
P. Stumpf, J. Mudd, W. Nees
Metabolism, Structure and Function of Plant Lipids
Andrew Benson
1988
Plenum Press
P.A. Biacs et al.
Biological Role of Plant Lipids
Hartmut. K. Lichtenthaler
1990
Portland Press
P.J. Quinn J.L. Harwood
Plant Lipid Biochemistry, Structure and Utilization
J. Brian Mudd
1992
Centre National
A. Cherif et al.
Metabolism, Structure and Utilization of Plant Lipids
Paul Mazliak
1994
Kluwer Academic
J.-C. Kader P. Mazliak
Plant Lipid Metabolism
Terry Galliard
1996
Kluwer Academic
J.P. Williams et al.
Physiology, Biochemistry and Molecular Biology of Plant Lipids
G. Roughan
1998
University Sevilla
J. Sánchez et al.
Advances in Plant Lipid Research Ernst Heinz
2000
Portland Press
J.L. Harwood P.J. Quinn
Recent Advances in the Biochemistry of Plant Lipids
Norio Murata
2002
Kluwer Academic
N. Murata M. Yamada et al.
Advanced Researches of Plant Lipids
Jean-Claude Kader
2004
Méte Budapest
P. Biacs
Proceedings of the 16th International Plant Lipid Symposium
Tibor Farkas
3) THE SECOND INTERNATIONAL SYMPOSIUM ON PLANT LIPIDS (2nd ISPL) The 2nd ISPL was held at the University of Karlsruhe from July 18 to 21, 1976, with the topic “Lipids and Lipid Polymers in Higher Plants”. H. Lichtenthaler and M. Tevini (both Karlsruhe) organized this 1976 symposium with the support of H.K. Mangold (Münster) and VII.
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E. Heinz (Köln) at the University Campus Karlsruhe. The intentions were to bring together in one place scientists from very different fields of plant lipids, such as fatty acids, glycolipids, phospholipids and lipid polymers, but in addition also those working on prenyllipids and sterols who had not participated in Norwich. The emphasis was placed on biosynthesis, distribution and function of plant lipids and their role in biomembranes and epidermal cell walls. This 2nd ISPL was sponsored by two larger grants from (i) the Deutsche Forschungsgemeinschaft (German Research Council), Bonn, and (ii) the Erwin- RieschFoundation, Tübingen, as well as (iii) by donations of several private companies. Broadening of Treated Topics Lichtenthaler recalls: "It was our intention to broaden the topics of the symposium and to invite not only the colleagues working on fatty acids and acyl lipids of plants, but also those that were performing research with prenyllipids, such as sterols, carotenoids, prenylquinones and the C20 isoprenoid chain of chlorophylls. For these non-acyl plant lipids I had proposed the name prenyllipids which was first used in the ISPL 1976 in Karlsruhe (see Lichtenthaler 1977, Goodwin 1977). Moreover, we also wanted to include lipid polymers, such as epicuticular waxes and cutins. Many of these “non-acyl lipid plant scientists“ worked more or less isolated in their individual countries not having discussion partners in their own country. We were hoping to provide them with a joint forum for discussing their research results with their colleagues working on acyl lipids and to increase the number of participants. And, in fact, this worked our very well.” Main Lecturers and Participants 17 main lectures were given at the Karlsruhe symposium and 47 short oral contributions with original data. Of the 12 lecturers of the first ISPL in Norwich seven also presented their results at the Karlsruhe symposium. The sessions, lecturers and titles of the main lectures in Karlsruhe are summarized in Table 4, their contributions were published as a book by Springer Verlag (Springer Publishers) in 1977 with M. Tevini and H.K. Lichtenthaler as editors (see Table 3). Additional contributors and participants of the Karlsruhe symposium were many colleagues who participated and contributed also to the subsequent ISPL. Those participants of the Karlsruhe ISPL underlined below were organizers or co-organizers of ISPL following the one in Karlsruhe: G. Akoyunoglou (Athens), L.-Å. Appelqvist (Stockholm), T.J. Bach (Karlsruhe), P. Benveniste (Strasbourg), M. Bertrams/Frentzen (Köln), F.G. Czygan (Würzburg), R. Douce (Grenoble), P. Gregory (Ithaca), E. Heftman (Berkeley), T. Galliard (Norwich), M. Gleizes (Bordeaux), E. Hartmann (Mainz), M.A. Hartmann (Strasbourg), J.L. Harwood (Cardiff), K.P. Heise (Göttingen), P.J. Holloway (Bristol), J. Joyard (Grenoble), P. Karunen (Turku), R. Kekwick (Birmingham), H.W. Kircher (Tucson), H. Kleinig (Freiburg), P.J.C. Kuiper (Groningen), R. Lessire (Talence), B. Liedvogel (Freiburg), C. Liljenberg (Göteborg), P. Mazliak (Paris), J.D. Mikkelsen (Copenhagen), D.J. Morré (West Lafayette), D.J. Murphy (York), D.W. Newman (Oxford, USA), C. Péaud-Lenoel (Marseille), P. Pohl (Kiel), R. Pont Lezica (Argentina), A. Radunz (Köln), J. Rétey (Karlsruhe), M. Sancholle (Toulouse), E. Selstam (Göteborg), M. Signol (La Garenne), C. Sironval (Liège), F. Spener (Münster), D.R. Threlfall (Hull), A. Trémolière (Paris), A.F. van Besouw (Nijmegen), A. Weber (Hamburg), P. von Wettstein-Knowles (Copenhagen), R. Wilkinson (Griffin, USA), J. Williams (Toronto) and J.F.G.M. Wintermans (Nijmegen). Peter Biacs (Budapest), who had been scheduled as a speaker in Karlsruhe, could not make it the very last moment, but he participated in future symposia and was the organizer of the 8th ISPL and now of the 16th ISPL in Budapest 2004. VIII.
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Table 4. Lecturers and titles of overview lectures of the Karlsruhe international symposium on plant lipids in July 1976 (2nd ISPL). These lectures were published in the book “Lipids and Lipid Polymers in Higher Plants”, M. Tevini and H.K. Lichtenthaler (eds.), Springer Verlag, Heidelberg 1977 (see Table 3). Opening Session P. Sitte , Freiburg T.W. Goodwin, Liverpool
Functional organization of biomembranes The prenyl lipids of the membranes of higher plants
P. Mazliak, Paris
Glyco- and phospholipids of biomembranes in higher plants
I. Physiology and Biochemistry of Fatty Acids and Glycerides P.K. Stumpf, Davis
Biosynthesis of fatty acids by chloroplasts
H.K. Mangold, Münster
The cyclopentenyl fatty acids
II. Physiology and Biochemistry of Phospho- and Glycolipids E. Heinz, Köln M. Tevini, Karlsruhe
Characterization of enzymatic reactions in glycolipid biosynthesis Light, lipids and plastid development
III. Physiology and Biochemistry of Plant Steroids L.J. Goad, Liverpool W. Eichenberger, Bern
Biosynthesis of plant sterols Steryl glycosides and acylated steryl glycosides
IV. Physiology and Biochemistry of Prenyllipids F.W. Hemming, Liverpool The biosynthesis and physiological significance of prenols and their phosphorylated derivatives B.H. Davies, Aberystwyth Higher pla nt carotenoids D. Siefermann-Harms , Tübingen The xanthophyll cycle H.K. Lichtenthaler, Karlsruhe Regulation of prenylquinone synthesis in higher plants C. Liljenberg, Göteborg
Chlorophyll formation, the phytylation step
V. Lipid Polymers in Higher Plants P.E. Kollatukudy, Pullman P.J. Holloway, Bristol
Biochemistry of lipid polymers The intermolecular structure of some plant cutins
Activation of Colleagues In order to attract all colleagues working on lipid-soluble plant compounds to the 2nd ISPL in Karlsruhe it was clear that we had (i) to offer a good scientific framework program, inviting well-known speakers, (ii) to reduce the registration fee to an absolute minimum (30 Deutsch Marks) and (iii) to provide travel assistance to as many participants as possible. The registration fee (30 DM = ca. 7 pounds = 12 US $ at that time) included book of Abstracts, coffee and tea at the breaks as well as the bus excursion to the Black Forest with dinner. Following the principles of the Erwin-Riesch-Stiftung, we supported with its donation the travel and/or accommodation expenses of young researchers and Ph.D. students and waived their registration fee, which is essential since they are the guarantors for the future meetings. We also took advantage of the fact that the 10th International Congress on Biochemistry (ICB) was held in London during the last week of July in 1976, and decided to organize the Karlsruhe Symposium just one week earlier. We proposed to several overseas colleagues, whom we expected to come to the ICB in London, that we would pay their airfare from London to Karlsruhe. This then guaranteed a broader participation of our US-colleagues. IX.
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Paul Stumpf was already in Germany on a sabbatical leave working with H.K. Mangold at the Federal Fat Research Institute in Münster /Westphalia. Trevor W. Goodwin, Liverpool agreed to participate and motivated some of his former co-workers in the field of isoprenoid lipids, such as L.J. Goad, B.H. Davies, F.W. Hemming and D.R. Threlfall. During his sabbatical in 1975 in Göteborg/Sweden, Lichtenthaler became acquainted with the Swedish colleagues Conny Liljenberg, Eva Selstam (both Göteborg) and Lars-Åke Appelqvist (Stockholm). All three Swedish colleagues participated in the Karlsruhe Symposium in 1976 and became the organizers of the 3rd ISPL in Göteborg in 1978. “Thus, by activating the plant lipid colleagues in the different European countries and in the USA in a very personal way and many of them by providing travel aid, we could assemble a lot of them at our Karlsruhe symposium. The participation consisted of 140 plant lipid scientists and Ph.D. students from 14 countries, and all of them were involved in plant lipid research which enormously stimulated the scientific discussion on all levels. This was a clear improvement to the 1 st ISPL in Norwich, where many of the participants were not involved in plant lipid research but came to listen to what was known about plant lipids.”
4) THE IMPACT OF THE NORWICH AND KARLSRUHE SYMPOSIA FOR THE FUTURE PLANT LIPID SYMPOSIA The Birth of the International Plant Lipid Family Lichtenthaler further recalls: "Some of the Karlsruhe participants already knew each other, e.g. from the Norwich symposium or earlier meetings or their stay as postdocs in USA, however others were fully new. We wanted to stimulate scientific discussion and cooperation by making sure that all participants became well known to each other. For this purpose we tried to mix people as often as possible by additional evening activities at different locations. Thus, the registration on Sunday afternoon and evening took place at the casino restaurant of the Federal Constitutional Court (Bundesverfassungsgericht) near the Karlsruhe Schloss (Karlsruhe Palace), there was a city hall reception on Monday evening, a joint dinner on Tuesday evening at the Heinrich Hertz-Gastdozentenhaus and, on late Wednesday afternoon, a bus excursion to the Black Forest and the wine country south of Baden-Baden followed by dinner for all participants in the wine village of Neuweier. The scientific sessions and discussions, the common daily lunches, the joint social activities as well as the special programs for the accompanying persons gave all the participants the feeling to belong to a larger scientific community that had much in common and mutual interests. In this way and in continuation of the Norwich symposium the international plant lipid family was born that, from now on, would meet every two years in a different country and grow with each further ISPL.” Development of Cooperation between Plant Lipid Working Groups The Norwich and Karlsruhe symposia on plant lipids were a strong stimulus for fast further progress in plant lipid research and for cooperation. Many participants realized that there were colleagues who worked either with other plant lipids or on different detailed aspects but often on the same scientific question. By exchanging their methods and combining their expertise they could make much faster progress than each individual laboratory, and moreover, they even could approach, in a joint cooperative effort, more complicated scientific problems. A few examples are given. Thus, Roland Douce and Hartmut Lichtenthaler with their colleagues started to investigate if and which prenylquinones were present in the chloroplast envelope and determined their levels with respect to the level of the envelope X.
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carotenoids (Lichtenthaler et al. 1981). Later Wintermans' group cooperated with R. Douce on the localization of galactolipid and acyl transferases in the outer envelope of spinach chloroplasts (Hemskerk et al. 1986). Norio Murata, Japan, a photosynthesis researcher, who first participated in the 6th ISPL in Neuchâtel in 1984, was inspired to further his plant lipid research; and over the years he accepted many European researchers in his laboratory in Okazaki and later became the organizer of the 15th ISPL in 2002. The large impact of the biannual ISPL between 1976 and 1998 on plant lipid research and cooperation is well documented for the lipids involved in photosynthesis in the review book by P.A. Siegenthaler and N. Murata (eds.) 1998. Another example are Mitsuhiro Yamada, Japan, who worked in the 1980s during three exchange stays in the laboratory of Jean-Claude Kader in Paris, whereas the latter worked during several research programs in the laboratory of M.Yamada in Japan in a “friendly and fruitful, but hard competition on the LTPs, the lipid transfer proteins” as Jean-Claude Kader recalls. Also the contributions in the proceedings books of the ISPL (Table 3) demonstrate the increasing scientific cooperation of plant lipid research groups with each further ISPL. After a few ISPLs were held, those who had participated, advertised these plant lipid meetings that were run in an open, familiar and cooperative discussion style. Many young scientists came for the first time to the following ISPL and kept participating in all the future ones. In this context I would like to mention John Ohlrogge as an example who will be a co-organizer of the 17th ISPL in East Lansing in 2006. He recalls when being asked how he learned about the ISPL and what attracted him: “The first ISPL I attended was Neuchâtel in 1984. I learned about this plant lipid meeting through other colleagues, such as Paul Stumpf. This ISPL was one of the most important meetings for my career because for the first time I was surrounded by other plant lipid biochemists. At all other meetings that I attended, such as the American Society of Plant Biology, only a few people attended sessions on lipids. In fact, I had the best discussions during my career at the Neuchâtel meeting, I met many of the people whose papers I had read, and I have attended every ISPL meeting since that time. I particularly remember meeting the European colleagues, such as Sten Stymne, Penny von WettsteinKnowles, Hartmut Lichtenthaler, Ernst Heinz, John Browse, Toni Slabas, and many others whose work I admired”. This ISPL- induced development also promoted the exchange of young colleagues who could learn or introduce new technologies to another European laboratory. In addition, at the following ISPL two years later all participants of the previous ISPL wanted to come up with further progress as well as completely new scientific results and approaches in order to show their colleagues and friends their scientific knowledge and competence. Thus, cooperation and competition between the European plant lipid research groups was greatly stimulated, and fast progress was made in our knowledge on biosynthesis, function and interaction of plant lipids. Part of this success was due to the fact, that the proceedings books were soon published after the symposia and that, starting with the 3rd ISPL in Göteborg in 1978, the oral contributions of all participants were included in the proceedings books. Four of the proceedings books of the ISPL from 1978 to 1984 appeared with Elsevier Science Publishers, which also published books on general and animal lipids. The Elsevier contact partner, who had always participated in the ISPL collecting the manuscripts, noticed the increasing progress the plant lipid scientists had made. Then at the 6th ISPL in Neuchâtel in 1984 he informed us that, despite originally being clearly behind the animal lipid research, the plant lipid scientists were now leading and trendsetters in many topics of general lipid biochemistry.
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Continuation of the International Symposia on Plant Lipids In Norwich and Karlsruhe the participants from the different countries pleaded for a regular continuation of these plant lipid symposia, and an international group of senior scientists with members of various countries discussed this in detail. The decision was to hold these international symposia on plant lipids, the ISPL, every two years. The Swedish colleagues Conny Liljenberg, Lars-Åke Appelqvist, and Eva Selstam proposed to organize the 3rd ISPL in Göteborg in 1978. Paul Mazliak, with the support of Roland Douce and Pierre Benveniste, was ready to organize the 4th International Symposium on Plant Lipids in Paris in 1980. Afterwards the Dutch colleagues around J.F.G.M. Wintermans and P.J.C. Kuiper as well as others thought of possibly holding one of the future ISPL. And the international steering board of senior lipid scientists (see below Chapter 6) agreed to proceed in this sequence. It was also agreed that future organizers should very early signalize their readiness to organize one of the ISPL so it could be decided not only two but even four years ahead on the subsequent organizers. This was found particularly useful to give an organizer enough time to raise funds and to prepare the symposium. Also several basic principles of the Norwich and Karlsruhe ISPL, such as a reduced fee for students, a joint symposium dinner, an excursion on one afternoon and special evening events including a reception were found very stimulating and were recommended to integrate in future ISPL. The European Character of the ISPL At the beginning these international symposia on plant lipids were thought to be European symposia with international participation in order to stimulate plant lipid research in Europe and to provide the European colleagues with a forum where they could discuss and exchange their results. Certain European countries and their rather modest national societies, were just too small to provide such a discussion forum for their few plant lipid scientists, this could only be done on a European level. Various of the European senior scientists had been participants of meetings of the ASPP in the USA and knew the benefit of a larger discussion forum. Thus, the international advisory board of senior plant lipid scientists and group leaders that was dominated by the European plant lipid researchers, propagated that the ISPLs were “International European Symposia” that should primarily be held in Europe. The colleagues from overseas (USA, Canada) agreed. This was similar to the international Photosynthesis Congresses initiated by Helmut Metzner in Europe with the 1968 Freudenstadt Congress, which were considered to be primarily a forum for the photosynthesis researchers of Europe and open to strong international participation from overseas. An equivalent of the ASPP as a European organization, e. g. the Federation of European Societies of Plant Physiology, FESPP, did not exist at that time, the latter was founded in 1978, i.e. clearly after the start of the ISPL (see Lichtenthaler 2004). Later it turned out that even within the FESPP Congresses there was little space or echo for special plant lipid sessions, one major reason being that the plant biochemists (and in fact many plant lipid researchers were biochemists) did not participate in the FESPP Congresses of plant physiologists. Also, the US colleagues in plant lipid research, biochemists and plant physiologists, did not find a proper forum within their ASPP and therefore started a separate series of biannual glycerolipid meetings in 1993 (see below National Plant Lipid Cooperative, Chapter 8). The great success of the first six ISPLs showed that it had been an excellent idea to establish these European plant lipid symposia with international participation. The Decisions for the Location of Future ISPL This European character of the ISPL always came up at the meetings of the international advisory board of senior plant lipid scientists (see below: Chapter 6 on the function of this board), when, on the basis of a new proposal, another European laboratory and country was selected for the organization of the next ISPL. Thus, after the first two ISPL in England and XII.
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Germany the next ones were held in Sweden, France, the Netherlands, and Switzerland. In view of the regular contributions of our US colleagues it became clear that the ISPL could not always be held exclusively in Europe, where they were badly needed to stimulate communication between the lipid scientists of the many small European countries. Thus, the international board decided unanimously to hold the 7th ISPL in the USA. This was done with pleasure to honor Paul Stumpf's great achievements in plant lipid biochemistry (see e.g. Stumpf 1987) and in the activation of international cooperation. However, due to the high airfare costs from Europe to Davis/California, only the group leaders and senior scientists of the many European plant lipid laboratories could afford to participate in the ISPL in the USA. Yet, at the same time this was a chance for a large number of American colleagues to take part in this ISPL. For this reason the total number of participants in the Davis ISPL in 1986 rose to more than 300, whereas in Europe usually 150 to 200 people had partic ipated. The following ISPL were held again in Europe (Hungary and England as shown in Table 1), where once again it was possible for the young European researchers and Ph.D. students to participate. The decision to go to Tunisia with the 10th ISPL was made because the main organizer, Abdel Cherif, and his colleagues in Tunis were well-known members of the international plant lipid family. Adel Cherif had got his Ph.D. with Paul Mazliak (Paris) and he and his Tunisian co-organizers had spent one or more research stays in Paul Mazliak's laboratory. The tourist island of Djerba was chosen as the location for that ISPL because most of the European colleagues could make arrangements for direct and fairly inexpensive flights and accommodation. Thus, the ISPL in Djerba in 1992 was as easy and inexpensive to participate in as any ISPL in Europe. The international board considered several times to hold one of the ISPL in Israel, following the proposals of Ya’acov Leshem (Ramat Gan), Zvi Cohen (Sde Boker) and other Israeli members of the international plant lipid family. However, due to the political instability and the assumed high security risks it was postponed for a later point in time. However, the offer of John P. Williams to hold the 12th ISPL in Toronto was approved although it was known that once again fewer European plant lipid scientists would be able to participate in comparison to an ISPL held in Europe. After two further ISPLs in Europe, Sevilla (1998) and Cardiff (2000), the 15th ISPL took place in Japan for the first time. The international advisory board made that decision in order to acknowledge the great contributions of our Japanese plant lipid scientists, such as Mitsu Yamada (Tokyo), Norio Murata (Okazaki), and various others to the progress in pla nt lipid research. The 16th ISPL had to be held again in Europe and was held in 2004 once more in Budapest by Peter Biacs. On the proposal of Christoph Benning, supported by John Ohlrogge and other US colleagues, the 17th ISPL will be held again in the USA (see Table 1). Thus, these initially exclusively “European ISPL” have become truly international symposia on plant lipids. There are further offers to hold one of the coming ISPL in France, Sweden, and Australia. In view of the fact that these ISPL started as “European international symposia”, most European plant lipid scientists favour the idea to possibly hold every second ISPL in Europe. Dedication of the ISPL-Proceedings Books The first two proceedings books of the ISPL 1974 and 1976 contained only the contributions of the plenary lectures. Starting with the third ISPL in Göteborg in 1978 the proceedings books became more comprehensive by including also the content of short contributions and of various posters. At the 1980 ISPL in Paris, Hartmut Lichtenthaler proposed to dedicate the proceedings book of the 4th ISPL to Sjef Wintermans, on the occasion of his approaching 60th birthday, for his great merits in the early plant lipid research. Paul Mazliak (Paris), then organizer and editor of the proceedings book, took up this idea and the international board of XIII.
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senior plant scientists agreed to dedicate the future proceedings books to a meritorious plant lipid scientist. So far the choice to whom the books would be dedicated has always been made by the ISPL organizers and editors of the corresponding proceedings book. The colleagues that were chosen were well-known for their contributions to plant lipid biochemistry and physiology, usually colleagues who had participated in several ISPLs. After Wintermans the next dedications were made to Paul Stumpf, Morris Kates, and Andrew Benson. These and all the further are listed in Table 3. “It is a great honor when your colleagues and friends dedicate a proceedings book to you”, responded one of the recipients. In 1994 the eighth dedication was made post- mortem to Terry Galliard, the founder of the ISPL series, who had passed away much too early. 5) THE TERRY GALLIARD MEMORIAL LECTURE AND MEDAL The lecture: On 31st March 1993, Professor Terry Galliard, the organizer of the first ISPL in Norwich in 1974, passed away at the age of 53 years after a brave fight against an illness that had been diagnosed two years earlier. The chemist T. Galliard had received his Ph.D. in 1963 at the Medical Biochemistry Department, University of Birmingham. From 1964 through 1966 he spent two of his postdoc years with Paul Stumpf in Davis/California. His very early death was a great loss for his friends of the international plant lipid family. Jean-Claude Kader who, in the early 1980s had spent two years as a postdoc in Terry Galliard’s laboratory, was particularly shocked. At that time J.-C. Kader was the organizer of the 11th ISPL in Paris in 1994. He decided to hold a Terry Galliard Memorial Lecture, a scientific lecture of a renowned colleague to be given at the opening of the 11th ISPL. He nominated Norio Murata to give this memorial lecture. The international board of plant lipid scientists agreed to continue with these T. Galliard lectures on all future ISPL. Organizers make a proposal and select, usually in cooperation with the international board of plant lipids scientists, the T. Galliard lecturer. So far six Terry Galliard lectures have been held, the names and lecture titles are shown in Table 5. Practically all T. Galliard le cturers were members of the plant lipid familiy and had participated in several of the ISPL. Table 5. Names of the Terry Galliard Medal Lecturers on the international symposia on plant lipids and the titles of their lecture. Year
Lecturer & Title
1994
Norio Murata (Okazaki) The cyanobacterial desaturases: aspects of their structure and regulation. John Shanklin (Upton, NY) Structure-function studies on desaturases and related hydrocarbon hydroxylases. John L. Harwood (Cardiff) Life and stress: a plant’s standpoint. John Ohlrogge (East Lansing) Fatty acid synthesis: from CO2 to functional genomics. Ernst Heinz (Hamburg) Sterol glucosides and ceramide glucosides: cloning of enzymes contributing to their biosynthesis. Hartmut Lichtenthaler (Karlsruhe) Evolution of carotenoid and isoprenoid lipid biosynthesis in photosynthetic and non-photosynthetic organisms.
1996 1998 2000 2002 2004
XIV.
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The Terry Galliard Medal: Later, another addition was made to this tradition. John Harwood, a close personal friend of Terry Galliard and a regular participant in all ISPLs, had the idea to provide a Terry Galliard Medal to each of the T. Galliard lecturers. He started this tradition in 2000 when he organized the 14th ISPL in Cardiff. The medal shows the face of Terry with the text “TERRY GALLIARD MEDAL” on one side and “PLANT LIPID BIOCHEMISTRY” on the reverse where, additionally, the name of the lecturer is engraved, see Fig. 1 for the last medal given in 2004. The first T. Galliard medals were conferred on the ISPL 2000 in Cardiff in presence of his widow Annona and his son Ian Galliard (Fig.2). The next T. Galliard lecturers are shown in Fig. 3. The honor to give the T. Galliard medal lecture is reserved for meritorious colleagues in plant lipid biochemistry.
Fig. 1. Terry-Galliard-Medal for Plant Lipid Biochemistry, which is conferred to those plant lipid scientists who give the Terry Galliard Lecture.
6) THE ORGANIZATION OF THE ISPL BY THE INTERNATIONAL BOARD The International Board of Plant Lipid Scientists At the first ISPL in Norwich 1974 some of the participating plant lipid scientists started to talk about a future international symposium on plant lipids and decided for another meeting in Karlsruhe in 1976. At the 2nd ISPL in Karlsruhe this group of senior scientists and leaders of research groups of different European countries as well as the USA came together now forming an unofficial international board of plant lipid scientists. They discussed the proposals for future plant lipid symposia and finally decided on the place and organizer of the next two ISPL in Göteborg in 1978 and in Paris in 1980. The concern of this international advisory and steering board was to make symposia available with a low registration fee and inexpensive accommodation as well as to hold them at the right place and time in order to make sure that many young co-workers and Ph.D. students would have the chance to participate. They agreed that there should be a lower registration fee for Ph.D. students, and if possible student dormitories should be available for students and lipid scientists alike. All proposals for future topics and developments have been made in this board. Members of this advisory board are usually one or two colleagues representing a larger country and only one for a smaller country. Yet, also the former organizers belong to this steering board as well as several colleagues from the country running the future ISPL. The organizers invite these XV.
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Fig. 2. The first four Terry Galliard lecturers on the 14th International Plant Lipid Symposium in Cardiff, July 2000, where they received the Terry Galliard Medal, here with Terry’s widow and his son. From left to right: John Shanklin (Toronto 1996), Norio Murata (Paris 1994), Ian Galliard, Annona Galliard, John Harwood (Sevilla 1998) and John Ohlrogge (Cardiff 2000).
Fig. 3. The fifth and sixth Terry Galliard lecturers Ernst Heinz (left, Okazaki 2002, here on a photo from 1993) and Hartmut Lichtenthaler (right, Budapest 2004 during his lecture). XVI.
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board members during the ISPL to an unofficial round table one evening or during lunch. On the 6th ISPL in Neuchâtel Paul- André Siegenthaler had invited the international advisory board of lipid colleagues to his beautiful house and garden above Lake Neuchâtel where we spent a wonderful summer evening and unanimously accepted the offer of Paul Stumpf to hold the 7th ISPL in Davis/California. We never had very stringent rules on the number of persons in the board discussion but we tried to keep it small. The board members would then communicate the essentials of the discussion to the colleagues in their country. In addition, the decision was made public in the final discussion round of the running ISPL and/or at the evening banquet. At the Karlsruhe ISPL in 1976 the following colleagues participated in the international board discussion: Terry Galliard and John Harwood (both UK), Paul Mazliak and Roland Douce (both France), Conny Liljenberg and Eva Selstam (future organizers for Sweden), J. Wintermans and P. Kuiper (Netherlands), John Williams (Toronto), Paul Stumpf (USA), W. Eichenberger (Switzerland), Penny von Wettstein-Knowles (Denmark) as well as the German organizers H.K. Lichtenthaler, M. Te vini, E. Heinz and H.K. Mangold. The Independence of the International Advisory Board of Plant Lipid Scientists Since its founding in 1974 (Norwich) and 1976 (Karlsruhe) the international steering board of plant lipid scientists representing the whole international plant lipid family remained independent and free of any scientific organizational structure or society. There were several attempts to incorporate our very successful plant lipid family into the frame of a society organization. This was particularly strong during the 1984 ISPL in the USA where a large chemical parent organization offered us to become a member under their guidance. This would, however, have meant that we had to pay a regular annual membership fee, had to follow the statutes of that society-type organization and were no longer free to decide where, when and by whom the next symposium was held, etc. The only advantage would have been to obtain organizational help from them for future ISPL and the availability of addresses. An international scientific group like the plant lipid family, however, performs interdisciplinary research and comprises biochemists, plant physiologists, organic and structural chemists, molecular biologists and electron microscopists as members from many countries. Such an open international science group cannot be pressed into the frame of a national scientific society or of one professional organization, such as biochemists or plant physiologists. In fact, the ongoing great success of the international family of plant lipid scientists is due to its freedom of decision-making in all topics, its openness for everybody, i.e. for new members, and those who might perform plant lipid research for a limited number of years and others who are involved in continuous pla nt lipid research. Independent of any administrative obligations everybody can be a member as long as he is involved in plant lipid research. This also means that each new organizer of an ISPL can bring in his own ideas and impetus. For these reasons, the international advisory board of the ISPL remained independent and should continue to remain independent in the future. The list of participants is always passed from one organizer to the other in order to facilitate the flow of information and to reach all members of the international plant lipid family. Parallel Sessions or only one Oral Session for Everybody? For the first seven ISPL we had only one oral session for all the participants. Thus, all lipid scientists and Ph.D. students were in the same lecture hall and everybody listened to the main lectures and short oral contributions regardless if the session topic dealt with biosynthesis or function of sterols, prenyllipids, waxes, fatty acids or particular plant acyl lipids. It turned out to be very useful to learn from colleagues of the different plant lipid research areas, to integrate them in the international plant lipid family and to sensitize them to new research XVII.
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approaches and techniques of a certain plant lipid field that could also be applied to other fields of plant lipid research. Holding only one oral session enormously stimulated the scientific discussion, gave rise to cooperation and provided fast progress. With the increasing number of participants not everybody could give an oral presentation, therefore, at the 3rd ISPL in Göteborg in 1978 and the following ISPL, poster presentations including poster discussions at special hours were added to the oral sessions. At the 8th ISPL in Budapest in 1986 an attempt was made to have two parallel sessions held on two different floors of the symposium building. This had been done in order to increase the number of oral presentations and to reduce the number of posters. This was, however, a very negative experience since one session was overcrowded, whereas the speaker in the parallel session had only a few listeners. Moreover, a specialist in one field, who was expected to be in the particular session of his field, might be found in the other session because he worked in the other field as well or wanted to extend his knowledge and learn a new technique or approach for his further research. Thus, many speakers did not get the proper feedback on their research. This attempt also showed that a fast move from one session to the other for one presentation and back to the first one never works well, since the time schedule of oral presentations including discussion can never be completely adhered to. Depending on the relevance and topical interest of a presentation a shorter or longer discussion may take place. Based on this rather negative experience the international advisory board decided to have only one oral session per future ISPL. It is better to limit the number of speakers and increase the number of posters than having parallel sessions. Since the 9th ISPL the organizers strictly followed these guidelines, and this is strongly recommended also for the future ISPL. Number of Speakers and Participants in the ISPLs Whereas the first ISPL in Norwich assembled ca. 25 plant lipid scientists from seven countries (and many others that followed the 12 lectures), 140 plant lipid scientists and Ph.D. students from 14 countries participated in the 2nd ISPL in Karlsruhe in 1976 with 57 oral contributions (no posters). The 3rd ISPL had a participation of 130 scientists from 17 countries with 61 contributions. In Groningen in 1982 (5th ISPL) the number of participants had increased to 160 with 109 papers (oral presentations and posters), as shown in the proceedings book. On the 11th ISPL in Paris in 1994 there were 285 lipid scientists from 26 countries with 54 lectures and 165 posters, and 161 contributions were selected for the proceedings book. 225 plant lipid colleagues from 29 countries participated in the 15th ISPL in Japan in 2002 with 68 lectures and 93 posters. Thus, the number of plant lipid scientists including Ph.D. students has steadily been increasing from symposium to symposium and so has the duration of the ISPL. At the first five ISPL there were three actual lecturing days, whereas the number of days has increased to five days for the most recent seven or eight ISPL. The high number of Ph.D. students and young plant lipid scientists at the 16th ISPL in Budapest in 2004 is a very positive sign for the continuity of the ISPL and the international plant lipid family, also in the years to come. In fact, there exist many open scientific questions in the field of biochemistry and physiology of plant lipids and new questions are arising with each progress. Thus, a great deal of research has to be performed, and plant lipid biochemistry will remain an attractive field of science for young colleagues.
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7) THE GERMAN-SPEAKING PLANT LIPID WORKSHOPS (Arbeitstagungen Pflanzliche Lipide) At the 2nd ISPL in Karlsruhe in 1976 the German plant lipid scientists decided to hold, in those years in between the international lipid symposia, biannual meetings on plant lipid research that became known as the “Arbeitstagungen Pflanzliche Lipide ”, the Plant Lipid Workshops. These meetings were organized at different universities in Germany and once in Switzerland (see Table 6). Speakers at these workshops were always established plant lipid researchers, but primarily young scientists, Ph.D. and diploma students who learned how to present their first scientific results. The topics were biosynthesis, physiology and function of all plant lipids including prenyllipids, sterols, waxes etc., the same as in the ISPL. The German-speaking colleagues from the Netherlands also participated in these meetings, e.g. J.F.G.M. Wintermans, and Waldemar Eichenberger and other colleagues from Switzerland. These workshops originally lasted for one day and were later extended to two and sometimes three days (Table 6). The number of participants was initially around 40 but then increased to 50 to 70 persons. Each organizer took the right to invite a few foreign colleagues to give review lectures on special topics. In 1993, at the 9th German plant lipid workshop in Karlsruhe, 85 persons participated with Trevor Goodwin (Liverpool), Paul Mazliak (Paris) and Waldemar Eichenberger (Bern) as guest speakers. Guy Ourisson (Strasbourg) had been invited, too, had to cancel the last minute, but sent his review manuscript. Of all the presentations 31 (including 10 reviews) were submitted as manuscripts, reviewed and published as a special double issue “Plant Lipids” of the Journal of Plant Physiology with Andrea Golz and Hartmut Lichtenthaler (1994) as guest editors (J. Plant Physiology, Vol. 143, issues 4 and 5, 397-580, 1994). Among them the review papers Mazliak 1994, Ourrisson 1994, Goodwin 1994, Schorr et al. 1994 and Golz et al. 1994 have been of high and lasting interest. Later some of the German colleagues participating in these workshops obtained grants by the German Ministry of Research, BMBF, for particular research on plant lipids and new oil plants. Their research within this program was presented at the meetings of the German Society for Fat Research DGF. Initially, the meetings of the DGF were held separately from and usually before the biannual workshops of the Plant Lipid Group. Because the topics were overlapping and those colleagues did not want to present their data twice, it was decided in 1997 to hold the DGF meetings together with the workshops of the Plant Lipid Group. In 2002 the Germa n Society for Fat Research (DGF) in Frankfurt/Main, formed a larger European unit by cooperating or merging with similar societies in France, Great Britain and the Netherlands. In the meantime it has expanded its European membership to an even larger number of countries. It changed its name to Euro Fed Lipid which stands for “European Federation for the Science and Technology of Lipids”. The reason for the foundation was the realization that the national societies of each individual European country were too small to survive in the future. Based on this new structure, the former workshops of the German Plant Lipid Group are now held by Euro Fed Lipid as the “European Symposia on Plant Lipids”, ESPL. The first ESPL took place in Aachen, Germany, in 2003 (Table 6), with Margit Frentzen as the organizer who had participated in the German plant lipid workshops and in most of the international ISPL. This 1st ESPL had 132 participants from 25 countries. The 2nd ESPL will be organized by Penny von Wettstein-Knowles, Copenhagen, who has been a regular participant in the international symposia on plant lipids since the 2nd ISPL in Karlsruhe in 1976. XIX.
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Table 6. List of German language plant lipid meetings (Arbeitstagungen Pflanzliche Lipide) that started in 1977, one ye ar after the 2nd International Symposium on Plant Lipids (ISPL) in Karlsruhe in 1976. These plant lipid workshops of the Plant Lipid Group for the young, German speaking scientists and Ph.D. students were always held in the years between the international ISPL and took place at different German universities and once also in Switzerland. Various Swiss and Dutch colleagues participated regularly in these meetings of the Plant Lipid Group, and on special occasions also colleagues from France, England and the USA. __________________________________________________________________________ Year University Organizer _______________________________________________________________________________________________________________
1977 1979 1981 1983 1985 1987 1989 1991 (Sept. 10-13) 1993 (Sept. 5 – 8) 1995 (Sept. 3 – 6)
Mainz Köln Ulm Münster Freiburg Hannover Göttingen Wuppertal Karlsruhe Bern
Elmar Hartmann Ernst Heinz Armin R. Gemrich Friedrich Spener Hans Kleinig Gernot Schulz Klaus-Peter Heise Roland R. Theimer Hartmut K. Lichtenthaler & Wilhelm Boland Waldemar Eichenberger
From 1997 to 2001 the meetings of the Plant Lipid Group were held together with the German Society for Fat Research DGF: 1997 (Oct. 6 –8) Bonn Wilhelm Boland 1999 (May 31-June 2) Göttingen R. Töpfer, W. Friedt, F. Spener 2001 (July 15-18) Meisdorf Ivo Feussner __________________________________________________________________________ Since 2003 the meetings of the German language Plant Lipid Group are held in English as European Symposia on Plant Lipids, organized by Euro Fed Lipid, and will take place in different European countries: 2003 (Sept. 10-13)
Aachen
Margit Frentzen
2005 (Aug. 17-20)
Copenhagen Penny von Wettstein-Knowles
(1st European Symposium on Plant Lipids) (2nd European Symposium on Plant Lipids)
_______________________________________________________________________________________________________________
8) THE MEETINGS OF THE NATIONAL PLANT LIPID COOPERATIVE NPLC Two American colleagues, Jan Jaworski and John Ohlrogge, who regularly participated in the International Symposia on Plant Lipids, ISPL, held in Europe most of the time, started their own series of meetings on plant glycerolipids in the USA. These biannual meetings of the National Plant Lipid Cooperative, held in between the international ISPL, started in 1993. The first one was held in Minneapolis as a “satellite” meeting associated with the American Society of Plant Biology's (ASPB) annual meeting. All other meetings have been held in early June at Fallen Leaf Lake near South Lake Tahoe at the “Sierra Camp” which is a beautiful XX.
20
mountain resort facility of the Stanford University Alumni, termed Stanford Sierra Conference Center (see Table 7). John Ohlrogge recalls: “We began these meetings in part because not so many North Americans could afford to attend the international meetings in Europe. We originally had 5 years of funding from a grant for Cooperative Research in Plant Biology. This funding allowed us to pay part of the expenses of students and postdocs to attend the meeting. I have always regretted that we used the term “national” in our organization. This term was used only because the grant which we first wrote emphasized funding for US collaborations, and because the granting agencies did not like the idea of sending US tax dollars outside the US. We have tried to emphasize that our meetings are open to anyone and we always invite speakers from outside the U.S.” Table 7. US American biannual meetings on plant glycerolipids organized by the National Plant Lipid Cooperative NPLC and now held at the Sierra Camp in California (CA). ___________________________________________________________________________ Year Location Organizers 1993 (July 29-31) Minneapolis Jan Jaworski, John Ohlrogge 1995 (June 1- 4) Sierra Camp, Lake Tahoe, CA Jan Jaworski, John Ohlrogge 1997 (June 4-8) Sierra Camp, Lake Tahoe, CA Jan Jaworski, John Ohlrogge 1999 (June 9-13) Sierra Camp, Lake Tahoe, CA Jan Jaworski, John Ohlrogge 2001 (June 6-10) Sierra Camp, Lake Tahoe, CA Jan Jaworski, John Ohlrogge 2003 (June 4-8) Sierra Camp, Lake Tahoe, CA Jan Jaworski, John Ohlrogge ___________________________________________________________________________ The focus of these NPLC meetings has always been on glycerolipids, rather than the broader topics covered in the International ISPL meetings. Attendance is limited to 125 participants by the facility and has been between 100-120 over the past several meetings. Since 1998 there has no longer been any official grant support. Therefore, J. Jaworski and J. Ohlrogge have raised a small amount of money from private industry sponsors to support the meetings. Jan Jaworski has taken the major role in the organization of the meetings. Both organizers also appointed a program committee to choose speakers, etc. Participants at these NPLC meetings are the US colleagues and their students including foreign guests. Usually nine lectures are given at each of the three morning sessions, followed by poster-viewing, a free afternoon and after dinner a plenary lecture at 8:00 P.M. In 2003 the opening presentation was given by Andy A. Benson (San Diego), and John Harwood (Cardiff, U.K.) participated as a foreign guest speaker.
9) THE ANNUAL SYMPOSIA OF THE JAPANESE ASSOCIATION OF PLANT LIPID RESEARCHERS (JAPLR) The international symposia on plant lipids, the ISPL, with their stimulating role for discussion, cooperation and future research on plant lipids gave also rise to an own series of plant lipid meetings in Japan. The Japanese colleagues Mitsu Yamada and Norio Murata, who had participated in several of the international ISPL created in 1988 this Japanese plant lipid symposia series which is held at Japanese universities or research institutions as shown in Table 8. Usually 50 to 60 persons participate in these symposia and sometimes also a few more. Norio Murata writes about the origin of the Japanese Symposia on Plant Lipids: XXI.
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“In 1986, some leading Japanese plant lipid researchers including Norio Murata, Jiro Sekiya, Mikio Tada, Mitsu Yamada discussed the necessity of having a series of annual scientific meetings on plant lipids. This was because, although they had annual meetings held by the Japanese Conference on the Biochemistry of Lipids (JCBL), these meetings were strongly orientated toward lipid researches on human disease and animal science, and the number of presentations by plant lipid researchers was decreasing year to year. Accordingly, a decision was made to initiate a series of plant lipid symposia, which has continued to have the 17th meeting in November 2004. In the past symposia, we sometimes invited plant lipid researchers from abroad and also domestic animal and yeast lipid researchers. In the former case, English was used as the official language. In 1998, Mitsu Yamada and other leading plant lipid researchers also discussed the necessity of an official organization unifying plant lipid researches and the eligibility of members and secretaries for the proposed organization. Eventually, the Japanese Association of Plant Lipid Researchers (JAPLR) was established in 1999. The first president was Professor Mitsu Yamada (1999-2003) and the second is Professor Norio Murata (2004-present).”
Table 8. The annual symposia of the Japanese Association of Plant Lipid Researchers (JAPLR). No. Year Date
University / Institute (City)
Organizer
1
1988 15. Jan
Okayama University (Okayama)
J. Sekiya and M. Tada
2
1989 9-10. Jan
NIBB (Okazaki)
A. Kawaguchi and N. Murata
3
1990 17-18. Jan
NIBB (Okazaki)
M. Yamada
4
1991 27. Mar
Kyoto Prefectural University (Kyoto)
K. Ichihara
5
1991 9-10. Dec
Tsukuba Bioscience Center (Tsukuba) K. Kasamo
6
1993 16-17. Jul
University of Tokyo (Tokyo)
7
1994 18-19. Sep
Hokkaido Tokai University (Sapporo) M. Yamada, H. Okuyama, G. Sakaki
8
1995 8-9. Sep
Kinki University (Nara)
O. Hirayama
9
1996 29-30. Nov
Kyoto University (Kyoto)
S. Shimizu
10
1997 29-30. Nov
Kyushu University (Fukuoka)
H. Wada
11
1998 27-28. Nov
University Tokyo (Tokyo)
I. Nishida
12
1999 26-27. Nov
NIBB (Okazaki)
N. Murata and K. Mikami
13
2000 24-25. Nov
Nagoya University (Nagoya)
Y. Sasaki
14
2001 30. Nov-12. Dec NIBB (Okazaki)
N. Murata
15
2002 29-30. Nov
Tokyo Inst. of Technol. (Yokohama)
K. Takamiya and H. Ohta
16
2003 28-29. Nov
Yamaguchi University (Yamaguchi)
K. Matsui
17
2004 26-27. Nov
NAIST (Tsukuba)
Y. Kamisaka
A. Kawaguchi
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Acknowledgements: I would like to thank various colleagues and friends of the international plant lipid family, such as Andy A. Benson (San Diego), Peter Biacs (Budapest), Waldemar Eichenberger (Bern), Margit Frentzen (Aachen), Ernst Heinz (Hamburg), Jean-Claude Kader (Paris), Conny Liljenberg (Göteborg), Norio Murata (Okazaki), John Ohlrogge (East Lansing), Paul- André Siegenthaler (Neuchâtel), Fritz Spener (Münster), and Sjef Wintermans (Nijmegen) for valuable information, and in particular John Harwood (Cardiff) for his details on the first international symposium on plant lipids in Norwich as well as Frank Amoneit (Frankfurt) for information on the European Federation for the Science and Technology of Lipids.
10) REFERENCES Benson, A.A. (1976). Lipids in Plants (Book Review). Phytochemistry 15, 45. Benson, A.A., Daniel H. and Wiser R. (1959a). A sulfolipid in plants. Proc. Natl. Acad. Sci. USA 45, 1582-1587. Benson, A.A., Wintermans, J.F.G.M. and Wiser, R. (1959b). Chloroplast lipids as carbohydrate reservoirs. Plant Physiol. 34, 315-317. Douce, R. (1974). Site of synthesis of galactolipids in spinach chloroplasts. Science 183: 852853. Douce, R., Holtz R.B. and Benson A.A. (1973). Isolation and properties of the envelope of spinach chloroplasts. J. Biol. Chem. 248: 7215-7222. Eichenberger, W. and Menke, W. (1966). Sterole in Blättern und Chloroplasten, Z. Naturforschg 21b: 859-867. Eichenberger, W. and Newman, D.W. (1968). Hexose transfer from UDP- hexose in the formation of steryl glycosides and esterified steryl glycosides. Biochem. Biophys. Res. Commun. 32: 366-374. Golz, A. and Lichtenthaler, H.K., eds. (1994). Recent advances in plant lipid research. Special issue of J. Plant Physiol. 143, 397- 580. Golz, A., Focke, M. and Lichtenthaler, H.K. (1994). Inhibitors of de novo fatty acid biosynthesis in higher plants. J. Plant Physiol. 143, 426- 433. Goodwin, T.W. (1994). Plant carotenoid research 1945-1985. J. Plant Physiol. 143, 440- 443. Hemskerk, J.W.M., Wintermans, J.F.G.M., Joyard, J., Bloch, M.A., Dorne, A.-J. and Douce, R. (1986). Localization of galactolipid: galactolipid galaytosyl transferases in outer envelope membrane of spinach chloroplasts. Biochim. Biopyhs. Acta 877: 281-289. Lichtenthaler, H. K. (1968). Plastoglobuli and the fine structure of plastids. Endeavour, Vol. XXVII, 144-149. Lichtenthaler, H. K. Localization and functional concentrations of lipoquinones in chloroplasts. Photosynth. Research, Vol. I, H. Metzner (ed.), pp. 304-314, Tübingen 1969. Lichtenthaler, H.K. (2004). A history of the Federation of European Societies of Plant Physiology FESPP since its foundation in 1978 – including notes on the renaming as the Federation of European Societies of Plant Biology (FESPB) in 2002. J. Plant Physiol. 161, 635-639. Lichtenthaler, H. K., and Park, R. B. (1963). Chemical composition of chloroplast lamellae from spinach. Nature 198, 1070-1072. Lichtenthaler, H. K. and Calvin, M. (1964). Quinone and pigment composition of chloroplasts and quantasome aggregates from Spinacia oleracea. Biochim. Biophys. Acta 79, 30-40. Lichtenthaler, H. K., Prenzel, U., Douce, R. and Joyard, J. (1981). Localization of prenylquinones in the envelope of spinach chloroplasts. Biochim. Biophys. Acta 641, 99105. XXIII.
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Lichtenthaler, H. K., Meier, D., Retzlaff, G. and Hamm, R. (1982). Distribution and effects of bentazon in crop plants and weeds. Z. Naturforsch. 37c, 889-897. Mazliak, P. (1994). Desaturation processes in fatty acid and acyl lipid biosynthesis. J. Plant Physiol. 143, 399- 406. Menke, W. (1938). Untersuchungen über das Protoplasma grüner Pflanzenzellen. I. Isolierung von Chloroplasten aus Spinatblättern. Z. Physiol. Chem. 257, 43-48. Menke, W. (1961). Über die Chloroplasten von Anthoceros punctatus. Z. Naturforsch. 16b, 344- 336. Menke, W. and Jacob, E. (1942). Untersuchungen über das Protoplasma grüner Pflanzenzellen IV. Die Lipoide der Spinatchloroplasten. Z. Physiol. Chem. 272, 227-231. Metzner, H. (ed.). Photosynthesis Research, Volumes I, II and III, Tübingen 1969. Ourisson, G. (1994). Pecularities of sterol biosynthesis in plants. J. Plant Physiol. 143, 434439. Schorr, R., Mittag M., Müller, G., and Schweizer E. (1994). Differential activities and intramolecular location of fatty acid synthase and 6- methylsalicylic acid synthase components. J. Plant Physiol. 143, 407- 415. Siegenthaler, P.-A. and Murata, N. (eds.). Lipids in Photosynthesis, Structure, Function and Genetics (Advances in Photosynthesis Vol. 6), Kluwer Academic Publishers, Dordrecht 1998. Stumpf, P.K. (ed.). The Biochemistry of Plants, Vol. 9, Lipids: Structure and Function, Academic Press, New York 1987. Van Besouw, A. and Wintermans J.F.G.M. (1978). Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation. Biochem. Biophys. Acta 529, 44- 53. Wintermans J.F.G.M. (1960). Concentration of phosphatides and glycolipids in leaves. Biochem. Biophys. Acta 44, 49-54. Wintermans J.F.G.M. Changes in Lipid components in isolated chloroplasts. In: Goodwin T.W. (ed.), Biochemistry of Chloroplasts, Vol. I, p. 115, Academic Press, New York. 1966. Wintermans J.F.G.M., Helmsing P.J., Polman B.J.J., van Gisbergen J., and Collard J. Galactolipid transformations and photochemical activities of spinach chloroplasts. In: Metzner H. (ed.), Progress in Photosynthesis, Vol- I, pp. 332-337, Tübingen 1969.
Origin of photos:
Figs. 1 and 3: from Hartmut Lichtenthaler, Karlsruhe Fig. 2: from John Harwood, Cardiff
XXIV.
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7
In memoriam Tibor Farkas (1929-2003)
Tibor Farkas, a Member of the Hungarian Academy of Sciences, was a highly respected scientist who was regarded as one of the leading figures in the field of lipid research. He considered himself a fortunate man in having the opportunity to enjoy his work throughout his entire life: he was dealing with what he loved. Tibor Farkas was born in Budapest, Hungary, on 8 June 1929. On receiving his degree in 1955 from zoophysiology at Lóránd Eötvös University in Budapest, he joined the staff of the Biologial Research Institute of the Hungarian Academy of Sciences at Tihany, where he remained until 1971. During his time in Tihany, he spent various periods abroad: in 1956, he participated in a several- month study-trip in the German Democratic Republic, and in 1963, he was invited to work for a full year at the Institute of Pharmacology at the University of Milan. He continued such journeys later, with visits to institutes dealing with marine biology and lipid research in the Soviet Union, and between 1972 and 1973, he carried out research at UCLA, the University of California in Los Angeles. He was awarded the titles of Candidate and of Doctor of the Hungarian Academy of Sciences in 1970 and 1990, respectively. He was a research professor at the Institute of Biochemistry at the Biological Research Center of the Hungarian Academy of Sciences in Szeged from 1971 until his death. He received wide-ranging professional recognition from the end of 1980s. He was elected to membership of the American National Academy of Sciences in 1989. He was elected a Corresponding Member and then a Full Member of the Hungarian Academy of Sciences in 1990 and 1998, respectively. He was Vice-President of the Szeged Academic Committee of the Hungarian Academy of Sciences from 1993, and later a member of the National Scientific Qualifications Committee and the Doctoral Awards Council. He had been elected to membership of the German Lipid-Science Society in 1959, and of The American Oil Chemistry Society in 1991. He was awarded the Széchenyi State Prize in 1998. His main research field was the biochemistry and physiolo gy of lipids, with particular regard to the adaptation of membranes to changes in temperature. The story of thermal adaptation at the level of cell membranes stretches back to the second half of the 1950s and to his work in Tihany. His interest in lipids began with his passion to familiarize himself with the lower forms of animal life to be found in the waters of Lake Balaton. After collecting a bucketful of lakewater containing such creatures, he often observed that a layer of oil covered the surface of this water. As no-one was able to provide a satisfactory answer to his question of why there was so much oil in these organisms, his enthusiasm led him to begin his lipid researches. This was a determining decision in his life, for he was laying the foundation-stone of the current lipid school. In the course of a visit to Tihany, Hans Paul Kaufmann, one of the world’s greatest lipid chemists, became acquainted with the activities of Tibor Farkas and recognized the significance of his researches. On returning home, through the columns of a high- level German professional journal he reported to the world the techniques developed by Farkas for the separation of fatty acids. 8
This line of research subsequently continued for several decades. The first serious result was achieved in conjunction with Sándor Herodek, Géza Tóth and László Csáky, with the demonstration that radioactive acetic acid administered into fish appeared in their fatty acids. In the early 1960s, Farkas and Herodek published their interesting observation that lower-order organisms to be found in Lake Balaton have the ability to adapt the physical state (melting points) of their lipids very sensitively to the temperature of their environment, thereby ensuring that their bioprocesses can occur even at temperatures that are so low that they would otherwise die. This Hungarian discovery contributed to the famous Sinensky principle of homeoviscous membrane adaptation, confirmed in a broad sector of the biosphere some 15 years later, and this basic research and discovery are still frequently referred to in the literature today. The analysis of samples from methodically selected organisms from the arctic, temperature and subtropical zones not only proved the validity of homeoviscous adaptation, but also revealed the molecular bases of the adaptational response at the level of the phospholipids and the biomembranes. These discoveries led to the truly wide recognition of the activities of Tibor Farkas, and contributed to the election of this Szeged scientist to membership of the American National Academy of Sciences. Farkas was already a researcher of international repute when in 1971 Brunó F. Straub, a Member of the Hungarian Academy of Sciences, invited him to join the staff of the Biological Research Center, inaugurated that year in Szeged. Here, Farkas was able to continue his research work under conditions that were outstandingly good on an Eastern European scale. For nearly 10 years, he was active in the world of plants, striving to identify the nature of the connection between plant cell membranes and frost resistance. Further, he utilized the earlier observations to investigate the membrane-adaptive solutions made use of by frost-resistant plants in order to survive extremely low temperatures. Together with his former students Ibolya Horváth and László Vígh, he succeeded in incorporating certain phospholipid precursor molecules into frost-sensitive wheat cultivars. Following their incorporation, the frost-sensitive plants became able to adapt to changes in the external temperature and hence to acquire protection against the early frosts. Patents based on this work were taken out in 15 countries. After this decade of digression, Tibor Farkas returned to work on marine animals. He demonstrated that, among others, the order of the membranes at a given temperature is regulated by certain lipid molecules, this holding true for cold-blooded animals living in the Arctic Ocean, in the subtropical zone, or in freshwater. His name is also associated with the sensational recognition that the freshwater Hypophthalmichthys contains the same type of oil as various marine fish, its consumption therefore being suitable for the prevention of diseases of the vascular system. The emphasis in his work was placed on the molecular composition and architecture of the membranes, with special regard to the roles of the special phospholipid molecular species in the thermal adaptation and in the development of different diseases. The last decade of his life too was characterized by great enthusiasm and devotion. In this period, a large part of his energy was spent in studying the roles played by long-chain polyunsaturated fatty acids in the central nervous system. His team carried out intensive researches on docosahexaenoic acid (DHA), one of the most important and most commonly occurring structural elements of nerve membranes, and its functions in relation to learning and memory in response to different diets. Together with his enthusiastic young colleagues, he established that DHA influences the expression of a number of genes connected with the cognitive functions. Diets containing different n-3 fatty acids exerted nearly the same effect on the pattern of cerebral gene expression. Among others, changes occurred in the expression of genes which play roles in the development of synaptic plasticity and the cytoskeleton, in
9
signal transduction processes, in the energy metabolism and in the membrane association of certain proteins. This widely acknowledged discovery indicated that the effects of the n-3 fatty acids on the cerebral gene expression are exerted in part directly, and in part via changes in membrane composition. The action is independent of the length of the fatty acid carbon chain; the n-3 structure is the determining factor. In a later publication, they described how the well-know DHA decrease in the elderly can be reversed to restore the level typical in the brain in the young: by administration of a simple fish oil-containing diet. Accordingly, the n3 fatty acids may play a great part in the future in the supplementary therapy of geriatric and neurodegenerative diseases. Over and above a good number of collaborative programmes in Hungary, Tibor Farkas and his colleagues built up an extensive network of international connections. Mention may be made of the Institute of Marine Research in Helsinki, Finland; the Alfred Wegener Institute für Polar und Meeresforschung in Bremerhaven, Germany; the WilhelmsUniversität in Münster, Germany; the Stazione Zoologica in Naples, Italy; the National Institute of Oceanography in Dona Paula, Goa, India; and the Universidad de las Islas Baleares in Palma de Mallorca, Spain. Until the end of his life, Tibor Farkas was constantly compiling and systematizing his data. He was open with everything and with everyone: he gladly shared his ideas with his colleagues. His chain of thought was always simple and clear. The consequences of the many decades of activities by this internationally acclaimed scientist will live on: not only did he create the lipid- membrane school in the country, but his name is associated with the training of new generations of Hungarian lipidologists. He devoted special care to the polishing of the thought processes of his students. Those who had the opportunity to work under his guidance consider themselves specially favoured. Fate not only brought them together with a genius researcher, but also furnished them with a fine example of humanity and affection. Up to the final minute of his life, he was able to work and to create. Throughout his many-sided, colourful and rich scientific career, he was alwasy an optimist. As concerns his final illness, his only complaint was that the operation and the days of convalescence were keeping him from his work. And then he would speak of many other things that were of more importance to him than his condition. It is almost impossible for those of us he has left behind to fully grasp that he has gone. All of us who worked with him tended to regard him in the same way as a child looks on a parent: someone with whom a serious problem can never arise. Tibor’s words: ”I never wanted to be something or somebody. I was simply interested in why things are the way they are”. In the name of the colleagues and former students Laszlo Vigh
10
EVOLUTION OF CAROTENOID AND ISOPRENOID BIOSYNTHESIS PHOTOSYNTHETIC AND NON-PHOTOSYNTHETIC ORGANISMS
IN
HARTMUT K. LICHTENTHALER
Botanisches Institut II, University of Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany Email:
[email protected]
Abstract Sterols and carotenoids are typical representatives of the group of isoprenoid lipids in plants. All isoprenoids are synthesized by condensation of the two active C5 -units: dimethylallyl diphosphate, DMAPP, and isopentenyl diphosphate, IPP. Like animals, higher plants form their sterols via the classical cytosolic acetate/mevalonate (MVA) pathway of IPP biosynthesis. Plants as photosynthetic organisms, however possess a second, nonmevalonate pathway for IPP biosynthesis, the DOXP/MEP pathway. The latter operates in the chloroplasts and is responsible for the formation of carotenoids and all other plastidic isoprenoid lipids (phytol, prenylquinones). Although there exists some cooperation between both IPP producing pathways, one can never fully compensate for the other. Thus, in higher plants sterols are primarily made via the MVA pathway and carotenoids via the DOXP pathway. This also applies to several algae groups, such as red algae and Heterokontophyta. In the large and diverging group of 'Green Algae' the situation is more complex. The more advanced evolutionary groups (Charales, Zygnematales) possess, like higher plants, both IPP forming pathways and represent an evolutionary link to these. In contrast, the proper Chlorophyta, often single cell organisms (Chlorella, Scenedesmus, Trebouxia), represent a separate phylum and synthesize sterols and carotenoids via the DOXP pathway whereas the MVA pathway is lost. The common ancestor of both groups, Mesostigma viride, again exhibits both IPP pathways. In the photosynthetic Euglenophyta the situation is inverse, both the sterols and the carotenoids are formed exclusively via the MVA pathway, the DOXP pathway is lost during the secondary endosymbiosis. Also Fungi synthesize sterols and carotenoids via the MVA pathway. Animals possess only the MVA pathway for sterol biosynthesis. In contrast, the malaria parasite Plasmodium and other Apicomplexa have lost the MVA pathway and synthesize their isoprenoids only via the DOXP pathway of their plastid-type apicoplast. In evolutionary terms the DOXP/MEP pathway shows up first in photosynthetic and heterotrophic bacteria, whereas Archaea possess the MVA pathway. The early anoxigenic photosynthetic bacteria (one photosynthetic light reaction) and the later Cyanobacteria (two light reactions and oxigenic photosynthesis) that form a link to the endosymbiontic chloroplasts contain the DOXP/MEP pathway. The latter is also present in many heterotrophic pathogenic bacteria. Some bacteria possess, in addition to the DOXP/MEP pathway, some genes of the MVA pathway that they obtained apparently by lateral gene transfer. A few others have evidently lost the DOXP/MEP pathway and acquired the MVA pathway. Some members of the Streptomycetes, in turn, have both IPP producing routes, one for 'housekeeping' (DOXP/MEP pathway) and the other (MVA pathway) for synthesis of secondary isoprenoid products. When viewing the evolutionary trends it is clear that 1) the two pathways of IPP biosynthesis evolved independently, 2) lateral gene transfer has occurred especially on the bacteria level, 3) primary endosymbiosis has taken place and secondary endosymbiosis partially with differing results, and 4) a loss of the genes of the DOXP pathway took place in some organisms and in others a loss of the genes for the MVA pathway. On the basis of the available evidence an evolutionary view of IPP formation is presented. 1. Introduction Plants, animals and microorganisms contain various primary and partially also secondary isoprenoid compounds that are made of the C5 -units of ‘active isoprene’, known today as isopentenyl diphosphate (IPP). The ‘biogenetic isoprene rule’ was first detected in 1885 by Wallach [65] and the head-to-tail addition of the ‘active C5 units’ was pointed out by Ruzicka [52, 53]. In the early 1950s acetate [42] and acetyl-CoA [44] were detected as precursors, as well as mevalonic acid (MVA) as an intermediate [69] and finally isopentenyldiphosphate (IPP) as the active cellular biosynthetic C5 -unit [14]. For more than three decades it had been believed that all isoprenoids of living cells were made via this acetate/MVA pathway. Despite the fact that more and more inconsistencies in the labeling of plastidic isoprenoids (carotenoids, phytol, plastoquinone-9) showed
11
up, as reviewed by Lichtenthaler [35, 40], the acetate/MVA pathway was regarded as the only biosynthetic pathway for IPP biosynthesis in living organisms. In the early 1990s when labeling with 13 C-glucose and applying high resolution NMR spectroscopy a second, biochemically fully independent, non-mevalonate IPP-biosynthesis pathway, known today as DOXP/MEP pathway, was detected: first in bacteria [51], then in all photosynthetic oxygen evolving organisms, such as green algae [38, 56, 57] and higher plants [6, 39, 40, 76]. This demonstrated that green plants (with the exception of some green algae) possess two IPP synthesizing pathways, the acetate/MVA pathway in the cytosol (e.g. for sterols) and the DOXP/MEP pathway in the plastids (e.g. for carotenoids and phytol). This was confirmed by various other authors as reviewed by Lichtenthaler [36] and Rohmer [50]. Heterotrophic organisms such as Archaea, fungi and animals do not possess the DOXP/MEP pathway of IPP biosynthesis, they synthesize their IPP and isoprenoids via the acetate/MVA pathway. The larger part of bacteria contains the DOXP/MEP pathway, whereas a few others possess the MVA pathway and a few Streptomycetes contain even both pathways as reviewed in [10]. Higher plants with their chloroplasts that are derived from cyanobacteria-like endosymbionts possess the DOXP/MEP pathway for carotenoid biosynthesis and the MVA pathway for sterol biosynthesis. This applies for several algae groups as well [36, 17]. However, the photosynthesising organism Euglena does not possess the DOXP/MEP pathway, whereas in Chlorophyta the cytosolic MVA pathway is missing [58, 60]. Moreover, the malaria parasite Plasmodium, as a heterotrophic organism, unexpectedly exhibits the DOXP/MEP pathway [29]. So what are the principles for the distribution of both IPP producing pathways in living organisms, where did the DOXP/MEP and the MVA pathway originate from? This topic is shortly reviewed here. 2. The MVA and the DOXP/MEP pathways and their inhibition The present knowledge of the biochemical enzymatic steps of the two independent IPP producing pathways is shown in Fig. 1. The classical acetate/MVA starts from 3 acetyl-CoA, requires 6 enzymes, 2 NADPH and 3 ATP to finally yield isopentenyl-diphosphate. All enzymes have been cloned from plants. The regulatory step is the HMG-CoA reductase (enzyme 3) that can specifically be blocked by the statin mevinolin as first shown for plants in [8, 9], whereby the active part of the inhibitor mevinolin is a structural analogue of the endogenous substrate intermediate as shown in Fig. 2. The final product of this pathway IPP is transferred by IPP isomerase to its isomer dimethylallyl diphosphate (DMAPP). The latter is the starter molecule for terpenoid biosynthesis to which one or several IPP molecules are added in a head-to-tail addition response depending on the final terpenoid product. In contrast, the non-mevalonate, plastidic DOXP/MEP pathway of IPP biosynthesis starts from pyruvate and glycerinaldehyde-3-phosphate, comprises 7 enzymes, requires 3 ATP equivalents (ATP or CTP), 3 NADPH and yields in the last enzymatic step, catalysed by HMBPP reductase (Lyt B), both substrates, IPP and its isomer DMAPP (usually in a ratio of 5:1 or 3:1), as indicated in Fig. 1. The first enzyme is the DOXP synthase yielding DOXP that is reduced by DOXP reductoisomerase to MEP [59]. An essential regulatory step of the DOXP/MEP pathway is this DOXP reductoisomerase that can efficiently be blocked by fosmidomycin and its derivative FR900098 as is independently shown for plants [59, 71] and for bacteria [31]. Fosmidomycin is a structural analogue to 2-C-methylerythrose 4-phosphate, the intermediate in the enzymic reaction of DXR, as shown in Fig. 2. The seven enzymes involved in the DOXP/MEP pathway have been isolated and their genes (dxs, dxr, ygbP, ychB, ygbB, gcpE and lytB) have been cloned in plants and bacteria. This has been summarized for the first 5 enzymes in Table 2 (see below). Enzyme 3 (ygbP) catalyses an activation of MEP by CTP to form CDPmethyl-D-erythritol [27, 48]. The function of HMBPP synthase (gcpE) in the DOXP/MEP pathway was demonstrated by several authors [2, 13, 61, 47]. Evidence for the 7th enzyme HMBPP reductase (lytB) came from several groups [1, 3, 25, 49].
12
MVA Pathway
DOXP/MEP Pathway
Pyruvate + Glycerinaldehyde-3-P 1) DOXP-Synthase (dxs)
2 x Acetyl- CoA - CoA
CO2
1) Acetoacetyl-CoA Thiolase O
OH
O
OP
S-CoA
OH
O
1-Deoxy- D-xylulose-5-P (DOXP)
+ A cetyl-CoA
2) DOXP-Reduktoisomerase (dxr)
2) HMG-CoA Synthase
- CoA
OH
1
O HO
O
HO
3
2
OH
4
OP
OH
S-CoA
2- C-Methyl- D-erythritol-4-P (MEP)
3-(S)-Hydroxy-3-methylglutaryl -CoA
3) CDP-ME-Synthase (ygbP)
+ CTP
OH
(HMG-CoA)
O CDP + 2 NADPH
OH
3) HMG-CoA Reduktase
OH CDP-Methyl- D-erythritol (CDP-ME)
- CoA
4) CDP-ME-Kinase (ychB)
+ ATP
O HO
OP
HO
OH
O CDP OH
3-(R)-Mevalonate
OH
CDP-Methyl- D-erythritol-2-phosphate (CDP-ME2P)
(MVA)
CMP
5) MEcPP-Synthase (ygbB)
4) Mevalonate Kinase
+ 2 ATP
OPPO
5) Mevalonate-5-phosphate Kinase OH
O HO HO
5 4
Mevalonate-5-diphosphate
3
OH
(MVAPP)
- CO2
6) HMBPP-Synthase (gcpE)
+ NADPH
OPP
+ ATP
OH
2-C-Methyl- D-erythritol -2,4 -cyclo-diphosphate (MEcPP)
2
1
O PP
4-Hydroxy -3-methyl-2-( E )-butenyl -diphosphate (HMBPP)
6) Mevalonate-5-diphosphate Decarboxylase
+ NADPH
7) HMBPP-Reduktase (lytB) 5
5
OPP Isopentenyl-diphosphate (IPP)
4
Isomerase
3
2
1
OPP
4
3
2
1
O PP
Dimethylallyl-diphosphate
Isopentenyl-diphosphate
(DMAPP)
(IPP)
Figure 1. Scheme of the two pathways for isopentenyl diphosphate (IPP) and isoprenoid biosynthesis: the acetate/mevalonate pathway (MVA) and the 1-deoxy-D -xylulose-5-phosphate/methyl-D -erythrithol (DOXP/MEP pathway). The enzymes of both pathways are numbered. In the plastidic DOXP/MEP pathway the genes of the corresponding enzymes are indicated in parenthesis.
3. Cross-talks between the plastidic DOXP/MEP and the cytosolic MVA pathway of IPP biosynthesis in plants In the cells of higher plants and several algae groups the two IPP producing biochemical pathways operate in parallel. The MVA pathway in the cytoplasm is responsible for the biosynthesis of sterols, sesquiterpenes, polyterpenes and can efficiently be blocked by mevinolin, whereas the accumulation of plastidic isoprenoids is not affected [9]. The DOXP/MEP pathway of IPP formation operates in the chloroplast and the other plastid forms and provides the C5 -units for the biosynthesis of carotenoids, phytol (side-chain of chlorophylls), the nona-prenyl side-chain of plastoquinone-9, as well as for isoprene emission [70] and other plastidic isoprenoids as indicated in Fig. 3. This DOXP/MEP pathway and consequently the biosynthesis of carotenoids, phytol etc., can efficiently be blocked by the herbicide fosmidomycin [71] whereas the biosynthesis of the cytosolic sterols is not affected.
13
In view of the two cellular IPP producing pathways, the question arises whether IPP or any other isoprenoid chain produced by the IPP pathway of one cellular compartment can be used by the isoprenoid biosynthesis machinery of the other compartment. Can the two IPP pathways complement each other if necessary? In other words, is there a cross-talk between the IPP pathways in the cytosol and that in the plastids? These questions can be answered by studying the incorporation of intermediates of each IPP pathway, such as labeled MVA and labeled DOXP or its non-phosphorylated form DOX, into typical plastidic isoprenoids, such as ß-carotene or phytol (chlorophyll). The results of such a study for two algae and a higher plant, all of which possess both IPP forming pathways, is shown in Table 1. TABLE 1. Contribution of the DOXP/MEP pathway and the MVA pathway to isoprenoid biosy nthesis in the two green algae Klebsormidium (‚Charophyceae’) and Mesostigma (‚Prasinophyceae’) and in the higher plant Lemna gibba L. The incorporation of precursors of the DOXP/MEP pathway [2-14 C]-1deoxy-D-xylulose (14 C-DOX) and the MVA pathway [5-3 H]-mevalonolactone (3 H -MVL) into the plastidic phytol and the cytosolic sterols was studied [60]. Organism / Isoprenoid Klebsormidium flaccidum Phytol Sterols Mesostigma viride (SAG 50-1) Phytol Sterols Lemna gibba Phytol Sterols
14
Applied precursor 3 C-DOX H-MVL
Ratio C / 3H
14
257.7 114.2
2.7 174.9
95.4 0.7
1600.0 618.9
14.9 535.7
107.4 1.2
359.6 28.3
15.4 46.5
23.4 0.6
As expected the applied 14 C-DOX is incorporated into the phytol chain of chlorophyll in the two ‘green alga’ Klebsormidium and Mesostigma to a very high extent, whereas the incorporation of the 3 H-mevalonolactone (3 H-MVL) into the phytol fraction occurred only in trace amounts (Table 1). In fact, the incorporation rate of 14 C-DOX into phytol was 95 and 107 times higher in both algae than that of the 3 H-MVL label. In the higher plant Lemna the preferred incorporation of 14 C-DOX into phytol was, however, only 23.4 times higher than that of 3 H-MVL. In contrast to phytol, the labeling of the cytosolic sterols by 3 H-MVL proceeded at higher rates as expected. However, the sterol fraction was also labeled from 14 C-DOX to a relatively high degree in the two green algae (Table 1), whereas the sterols of the higher plant showed only some labeling by 14 C-DOX. The results clearly demonstrate that in the two green algae the plastidic pathway DOXP/MEP contributes considerably to the biosynthesis of sterols, however only to some extent in the higher plant Lemna. In contrast, the MVA -pathway only contributes little to the biosynthesis of the plastidic isoprenoid phytol. Also several early observations indicate at least some exchange or cooperation between both isoprenoid pathways. One example is the very low labeling rate of plastidic isoprenoids from applied 14 C-MVA, whereas sterols are labeled at high rates. This had already been detected in 1958 by Goodwin [23] and was noticed by a number of other authors, see review [40]. Moreover, in the 13 C-labeling of the diterpene ginkgolide from 13 Cglucose, three isoprene units were found to be labeled via the MVA pathway and the fourth C5 -unit in a different way [55] which is now known as labeling pattern of the DOXP/MEP pathway [39, 40]. In the liverwort Heteroscyphus the first three isoprene units of phytol were shown to be labeled from 13 C-MVA whereas the fourth C5 -unit was not labeled [45]. Both observations point to the import of a cytosolic farnesyl diphosphate (FPP) or a non-phosphorylated isoprenoid C15 -unit into the plastid to which a fourth plastidic IPP (derived fro m the DOXP/MEP pathway) was added. In our labeling studies of phytol and carotenoids from 13 C-glucose in algae and higher plants, performed in the light under photosynthesis conditions, we did not detect such an import of FPP into the plastid. However, one has to consider that a low labeling of a plastidic isoprenoid via the cytosolic pathway of clearly less than 10 % would not have been seen in the NMR spectra.
14
Inhibitor
Intermediate
A. HMG-CoA Reductase (HMGR) HO
HO
O COOH OH
O H
CoA
Mevaldyl-CoA thiohemiacetal
Mevinolin
H
COOH OH
S
B. DOXP-Reductoisomerase (DXR) O HO
O HO
N
N
O
HO
PO 3H2
PO 3H2
Fosmidomycin
O HO PO 3H 2
2-C-Methylerythrose4-phosphate
FR-900098
Figure 2. Inhibitors of the acetate/MVA pathway (Mevinolin) and of the DOXP/MEP pathway (fosmidomycin and its methyl derivative FR900098) for biosynthesis of isopentenyl diphosphate (IPP). The structural analogy of the active inhibitor (site) with the natural enzyme intermediates is indicated.
Cytoplasm
Plastid GA -3P + Pyruvate DXS
3 Acetyl -CoA
1-Deoxy - D -xylulose -5-P HMG -CoA
Fosmidomycin
HMGR
Mevinolin
DMAPP +IPP
IPP
IPP
? FPP
+IPP
2x
Sesquiterpenes Sterols Triterpenes Mitochondria Ubiquinone-9 Ubiquinone-10
Monoterpenes
GPP (C 10 ) +IPP
Diterpenes, Phytol
+IPP
GGPP (C
+IPP
FPP (C 15 )
IPP Isoprene
Mevalonate
DMAPP
DXR
20 )
2x Carotenoids
+5 IPP
Plastoquinone-9
(C 40 ) (C 45 )
Polyterpenes
Figure 3. Compartmentation of IPP and isoprenoid biosynthesis in higher plants between cytosol (MVA pathway) and the plastid (DOXP/MEP pathway). The specific block of the enzyme HMG-CoA reductase (HMGR) by the antibiotic mevinolin (a statin) and of the DOXP reductoisomerase (DXR) in the plastid by fosmidomycin is indicated (based on [36]). IPP = isopentenyl diphosphate, DMAPP = dimethylallyl diphosphate, GPP = geranyl diphosphate, FPP = farnesyl diphosphate, GGPP = geranylgeraniol diphosphate.
Another possibility to detect cross-talk is to compare the biosynthesis of selected plastidic and cytosolic isoprenoids when one of the two IPP pathways is blocked by inhibitors, such as shown in Fig. 2. This has been done with cell cultures of green tobacco cells where the uptake of inhibitors and substrates works better than in intact whole plants. Also, this investigation demonstrated that the sterols, normally labeled via the MVA
15
pathway, can be formed via the DOXP/MEP pathway when the cytosolic MVA pathway is blocked [26]. During an inhibition of the DOXP/MEP route of tobacco TBY-2 cells by fosmidomycin an incorporation of [213 C]-MVA into plastoquinone-9 was observed. A similar attempt using fosmidomycin and a mevinolin-type statin as an inhibitor of the MVA pathway, was made in Arabidopsis seedlings [34]. Inhibition of the cytosolic MVA pathway caused only a transient reduction of sterol levels indicating that the plastidic DOXP/MEP pathway might partially compensate for the lack of cytosolic IPP needed for the biosynthesis of sterols. In any case, the currently available data of labeling and inhibitory studies of several laboratories demonstrate that there exists a substantial cross-talk between both cellular IPP biosynthesis pathways. Especially the plastidic DOXP/MEP pathway delivers isoprenoid building units for the biosynthesis of sterols in the cytosol. It may contribute to the formation of other cytosolic isoprenoids, such as polyterpenes, as well; however, this has yet to be investigated. The inverse cooperation, the import of isoprenoid building blocks into the chloroplast, apparently proceeds to a much lower degree than the export. The whole exchange apparently depends on the physiological state and the developmental stage of the plant cells. At high photosynthetic rates it sounds reasonable that the chloroplast exports isoprenoid building blocks for the cytosolic isoprenoid formation. Only in the case of non-green plant tissues the partial import of cytosolic isoprenoid chains into the plastid might play a certain role. Despite all cross-talk indications it appears to be clear that in intact plants neither the cytosolic MVA nor the plastidic DOXP/MEP pathway of IPP formation can fully compensate the other isoprenoid route when one of the two is partially blocked or operates at a low rate. What isoprenoid compounds can be imported from the cytosol or exported from the plastid is not known at all. The question if they are prenyl diphosphates, such as IPP, GPP or FPP, or rather non-phosphorylated isoprenoid chains, which would be more likely, cannot be answered. Transporters for isoprenoid intermediates or prenyl diphosphates in the plastid envelope have not yet been detected, but this is certainly a promising research topic.
4. Distribution of the DOXP/MEP and MVA pathways in photosynthetic algae and bacteria The compartmentalization of the isoprenoid biosynthesis with the cytosolic MVA pathway and the plastidic DOXP/MEP pathway of IPP formation (Fig. 3) exists in all higher plants tested so far [35, 36, 40]. This is further emphasized by the presence of all the genes required for the performance of the DOXP/MEP pathway in higher plants as indicated for the first 5 enzymes [37] in Table 2. The enzymes of the MVA pathway are present as well [7]. The genes of the DOXP/MEP pathway are bound to the nucleus, yet the proteins operate in the plastids. In contrast to the bacterial DOXP/MEP enzymes, the plant enzymes possess an additional transit peptide sequence that directs them to their proper organelle, the plastid. 4.1. Photosynthetic algae In the various photosynthetic algae groups possessing a differential photosynthetic pigment apparatus with either chlorophyll a and b (usually addressed as ‘green algae’ but representing a polyphyletic group), chlorophyll a and phycobilisomes (red algae, rhodophytes) or with chlorophyll a and c (heterokontophytes), we found the same dichotomy of the cellular isoprenoid biosynthesis as in higher plants: the MVA pathway for biosynthesis of the cytosolic sterols and the DOXP/MEP pathway for the biosynthesis of carotenoids, phytol and isoprene [17, 36, 39, 58, 70]. This also applies to the marine diatoms Nitschia and Phaeodactylum belonging to the heterokontophytes [15]. One of two exceptions of this rule was found in the case of the chlorophytes Chlorella, Scenedesmus and Chlamydomonas [56, 57], where not only the plastidic isoprenoids are made via the DOXP/MEP pathway but also the cytosolic sterols. A detailed further investigation of this unexpected phenomenon resulted in the detection that the commonly termed polyphyletic group ‘green algae’ consists of the Streptophyta (including higher plants) with both IPP pathways, and the proper Chlorophyta that have lost their MVA pathway for IPP and isoprenoid biosynthesis [60]. This is indicated in Fig. 4. Chlorophyta have also lost certain cytosolic enzymes of their sugar metabolism [54]. Moreover, chlorophyta also form a separate group from the streptophyta based on differences in the 18S-rRNA composition and ultra-structural characteristics of their cells and flagellae [21]. Further evidence for the absence of the MVA pathway is the fact that statins (mevinolin, cerivastatin) do not inhibit the growth of chlorophyta and also that genes of the MVA pathway could not be detected [60]. In addition, in the pigment-free chlorophyte Prototheca wickerhamii ergosterol is synthesized via the DOXP/MEP pathway [72]. The prasinophyte Mesostigma viride that is a common precursor of Chlorophyta and Streptophyta contains both the MVA and the DOXP/MEP pathway for IPP biosynthesis (Fig. 4) indicating that chlorophytes and streptophytes, including higher plants, are derived from the same
16
evolutionary ancestor. Another exception from the existence of two IPP routes in the cell are the Euglenophytes. In the flagellate Euglena gracilis sterols and the plastidic isprenoids (carotenoids, phytol) are formed via the classical acetate/MVA pathway [17]. Euglena originated from a colorless flagellate that incorporated a green alga (possibly a chlorophyte) in a secondary endosymbiosis , during which the plastidic DOXP/MEP pathway of the endosymbiont was lost (see Fig. 5). A secondary endosymbiosis may not necessarily lead to a loss of the IPP producing pathway of the endosymbiont as is seen in the example of the heterokontophytes in which both IPP producing pathways were maintained (see Fig. 5). TABLE 2. Distribution of the genes for the first five enzymes of the DOXP/ MEP pathway in plants, protozoa and eubacteria (based on [37, 41]). Organism Plants Arabidopsis thaliana Capsicum annuum Chlamydomonas reinhardtii Mentha piperita Oryza sativa Protozoan parasite Plasmodium falciparum Toxoplasma grandii Eubacteria Photosynthetic Bacteria: Synechocystis sp. Synechoccocus leopoliensis Chlorobium tepidum Rhodobacter capsulatus Pathogenic Bacteria: Escherichia coli Haemophilus influenzae Helicobacter pylori Chlamydia pneumoniae Mycobacterium tuberculosis Vibrio cholerae
dxs
dxr
ygbP
ychB
ygbB
Q38854 O78327 O81954 O64904 O22567
AJ242588
AF230737
AAC32234
AAF07360
O96694 +
O96693 +
P73067 Y18874 + P26242
Q55663 AJ250721 +
P77488 P45205 Q9ZM94 Q9Z6J9 O07184 +
P45568 P44055 AAD05777 Q9Z8J8 Q10798 +
AF116825
AF179283
+ +
+ +
P74323
P72663
+ Q08113
+
Q46893 O05029 AAD5981 AAD18718 P96864 +
AAC71873 +
P73426
Q08113 P24209 P45271 AE001363 + +
P36663 P44815 O25664 + P96863 +
Recently research has concentrated on the Chlorarachniophytes (Bigelowiella natans), green amoeboflagellate algae that acquired their plastid by a secondary endosymbiosis of a green alga [5]. Like the plastids of heterokonts, haptophytes, apicomplexa and cryptomonads, their plastids are surrounded by four envelope membranes, whereas the plastids of euglenids and dinoflagellates are surrounded by only three envelope membranes [5]. In contrast to heterokonts, the photosynthetic apparatus of Bigelowiella contains chlorophyll a and b as do euglenids, green algae and higher plants, and must be derived from a ‘green alga’. It is of great interest to know if Bigelowiella contains both IPP producing pathways, the DOXP/MEP and MVA pathway, or if it has lost one of them during or after the secondary endosymbiosis of a green alga. A very special taxonomic group are the Apicomplexa comprising the sporozoa parasites Plasmodium falciparum and Toxoplasma gondii. These contain a special cell organelle, the apicoplast, a non-green plastid with a genome similar to that of chloroplasts of green algae. The apicomplexa arose from a secondary endosymbiosis (the apicoplast envelope consists of 4 membranes) by either the incorporation of a green alga [22] or a red alga [4, 68] (see also Fig. 5) whereby the plastid obtained an envelope of four membranes. Whether Apicomplexa, Heterocontophyta, Haptophyta, Cryptophyta and Dinoflagellates have a common ancestor [4, 68] is still a matter of debate. In any case, Plasmodium possesses the DOXP/MEP pathway of IPP and isoprenoid biosynthesis [29], and its development can be blocked by fosmidomycin whereas the MVA pathway is missing. This also applies to Toxoplasma gondii as has been recently established [19]. Cyanobacteria possess the DOXP/MEP pathway of IPP formation as has been shown for Synechocystis [17, 40, 46]. This is in agreement with the endosymbiosis theory of chloroplasts according to which cyanobacteria-like photosynthetic organisms were taken up by a flagellate in a primary endosymbiosis as is shown in Fig. 5.
17
Phylogeny
1
Investigated species
Isoprenoid pathway
“Prasinophyceae“
Tetraselmis striata
Trebouxiophyceae
Trebouxia asymmetrica Chlorella saccharophila
Chlorophyceae
Scenedesmus obliquus, Chlamydomonas reinhardtii, a Chlorella fusca
Ulvophyceae
Gloeotilopsis planctonica
Embryophytes
Lemna gibba , Daucus carota Hordeum vulgare b
Zygnematales *
Spirogyra sp.
Klebsormidiales *
Klebsormidium flaccidum
b
2
Cytosol
Plastid
DOXP/ MEP
DOXP/ MEP
MVA
DOXP/ MEP
b
Charales * “Prasinophyceae“
1
Chlorophyta
Mesostigma viride
2
Streptophyta
Figure 4. Distribution of the DOXP/MEP pathway and the MVA pathway of IPP biosynthesis in Chlorophyta (1) and Streptophyta, including higher plants (2). The distribution correlates with the current phylogeny of green algae and higher plants [21] based on 18S-rRNA sequences, ultrastructural characteristics of flagellate cells and of cell mitosis. The chlorophyta have lost the MVA pathway, whereas the common ancestor Mesostigma viride has both IPP pathways. * These clades of ‘green algae’ are summed up by most of the recent authors as ‘Charophyceae’, but are not monophyletic. a,b IPP and isoprenoid biosynthesis of these organisms has been studied before [17, 40].
4.2. Photosynthetic bacteria In contrast to plastids, mitochondria as symbiontic bacterial organisms have lost their capacity for their own isoprenoid biosynthesis [43] and are dependent on the cytosolic MVA pathway for the synthesis of the isoprenoid chains of ubiquinones -9 and -10 [18]. The photosynthetic Cyanobacteria possess two photosynthetic light reactions (photosystems 1 and 2), split water and evolve oxygen as do the different algae groups and higher plants. They perform an oxigenic photosynthesis. A more primitive photosynthesis with only one light reaction is found on one hand in the green sulfur photosynthetic bacteria, such as Chlorobium tepidum (Chlorobacteriaceae) (see Table 2), and on the other hand in the purple bacteria such as Rhodobacter. Both photosynthetic phototroph bacteria have bacteriochlorophylls, perform an anoxigenic photosynthesis (no oxygen evolution) and possess the DOXP/MEP pathway for the biosynthesis of their carotenoids and isoprenoid chains (ubiquinones, menaquinones, phytol) as is indicated in Table 2, has been demonstrated for Rhodobacter capsulatus [24], and is also outlined in [10]. The MVA pathway does not occur in these photosynthetic phototroph bacteria. Thus, from all photosynthetic organisms the DOXP/MEP pathway of IPP biosynthesis shows up first in the prokaryotic photosynthetic bacteria with only one light reaction. Since the cyanobacteria represent photosynthetic organisms, with a higher complexity of their photosynthetic apparatus, their two photosystems apparently derive from Chlorobacteriaceae (photosystem 1) and purple bacteria (photosystem 2), in evolutionary terms they must have emerged later than the photosynthetic bacteria with only one light reaction and anoxigenic photosynthesis. Thus, the origin of the DOXP/MEP pathway in chloroplasts of algae and higher plants goes back to the early photosynthetic bacteria as is summarized in Fig. 5.
18
Plastids with Chlorophyll a and b
Plastids with Chlorophyll a and Phycobilisomes
DOXP
DOXP + MVA
MVA Euglenophyta Euglena
DOXP + MVA
Higher plants Chlorophyta Lemna, Chlorella Daucus, Scenedesmus, Hordeum Chlamydomonas
Rhodophyta Cyanidium
SECONDARY ENDOSYMBIOSIS
Flagellate (MVA)
Plastids with Chlorophyll a and c DOXP + MVA Heterokontophyta Ochromonas, Phaeodactylum, Nitzschia
SECONDARY ENDOSYMBIOSIS
DOXP Cyanobacteria Synechocystis
[?]
Plasmodium falciparum Malaria parasite DOXP (SECONDARY ENDOSYMBIOSIS)
PRIMARY ENDOSYMBIOSIS
Flagellate (MVA)
Flagellate (MVA)
Cyanobacteria-like ancestors
Photosynthetic bacteria
DOXP
DOXP
Figure 5. Putative evolution of photosynthetic bacteria, algae and higher plants with particular emphasis on the occurrence of the DOXP and/or the MVA pathway for IPP-biosynthesis. During evolution the MVA pathway for IPP-biosynthesis was lost in Chlorophyta, whereas the DOXP pathway was lost in Euglenophyta. The scheme lists also the malaria parasite (Plasmodium falciparum) that contains the DOXP pathway in a plastid-like apicoplast, and originated by incorporation of either a red or green alga in a secondary endosymbiosis process. The scheme is based on [36].
5. Distribution of the DOXP/MEP pathways in Bacteria. According to our present knowledge the DOXP/MEP and MVA pathways of IPP formation is a very early biosynthesis pathway that originated during the evolution of bacteria. From there the DOXP/MEP pathway was transferred via photosynthetic cyanobacteria-like ancestors of cyanobacteria by primary and later also secondary endosymbiosis to algae and higher plants. It is of interest in this respect that the sequences of the plant genes of the DOXP/MEP pathways differ to a higher degree from that of the cyanobacteria than from other bacteria [32] indicating that the presently existing cyanobacteria have gone through a long evolutionary development. Today most of the members of the bacteria, such as E. coli, use exclusively the DOXP/MEP pathway of IPP formation. The photosynthetic cyanobacteria belong to this group, as well as green sulphur bacteria (Chlorobium), the purple bacteria within the proteobacteria (e.g. Rhodobacter), and many non-photosynthetic bacteria, such as members of the Aquificales, Thermotogales, Chlamydiae, Bacteroides and Gram-positive ones with either low or high G+C as reviewed in [10]. An exception from this general rule is a minority of bacteria that 1) either lost the DOXP/MEP pathway and replaced it by the MVA pathway or 2) acquired, in addition to the DOXP/MEP route, the full or part of the genes of the MVA pathway. All this apparently occurred by lateral gene transfer, a process that happened rather frequently during the evolution of the large diverging phylum of bacteria. Thus Borrelia burgdorferi, Myxococcus fulvus [10], Paracoccus zeaxanthinifaciens [20], some Grampositive bacteria with low G+C content [67], including Lactobacillus plantarum and the green phototroph nonsulfur bacterium Chloroflexus aurantiacus [10], belong to the first group and only possess the MVA pathway. The second group is represented by members of the Gram-positive bacteria with a high G+C content, i.e. several Streptomyces species possessing both the MVA and the DOXP/MEP route. In Streptomyces aeriouvifer primary isoprenoids, such as the electron carrier menaquinone, are formed during the exponential growth phase via the DOXP/MEP pathway, whereas in the stationary phase the antibiotic naphpertin is synthetized via the acetate/MVA pathway [62]. The accumulation of naphpertin could be blocked by the mevinolin-like statin pravastatin without affecting the growth of the bacterium. A similar partition of isoprenoid biosynthesis in primary isoprenoids (DOXP/MEP pathway) and secondary isoprenoids via the MVA route has also been
19
detected in the actinomycete Actinoplanes [63]. Bacteria evidently were the play ground of the evolution in those cases where a full or partial loss of gene sequences and the acquirement of others by lateral gene transfer has occurred. Thus, the DOXP/MEP route is present and some genes of the MVA route and vice versa. This has been reviewed in detail in [10]. The DOXP/MEP pathway of isoprenoid biosynthesis and its genes are also found in many pathogenic bacteria, e.g. those causing lung disease, tuberculosis, leprosy, cholera, and ulcer, i.e. Chlamydia pneumonia, Mycobacterium tuberculosis, M. leprae, Vibrio cholerae and Helicobacter pylori, respectively, as is shown in Table 2 and [41]. Within the Bacteria the phylogenesis of the different groups is presently unresolved. Thus, except for Aquifacales and Thermotogales, that had branched off relatively early from the common unknown ancestors, it is not known which group is more advanced or more primitive than others. The photosynthetic bacteria with only one photosystem (one light reaction) and the mandatory anaerobics must have developed earlier than the cyanobacteria with two photosystems that evolve oxygen. However this is not reflected in the tree of bacteria usually shown [e.g. 12]. One can assume that during the evolution various photosynthetic bacteria have lost their pigment apparatus and with it their competence for photosynthetic quantum conversion. Therefore, the question arises which of the present heterotrophic bacteria are derived from former photoautotrophic bacteria. Are pathogenic bacteria, such a set of former phototropic bacteria that survived the loss of the photosynthetic (photoautotrophic) competence by turning into pathogens that live and multiply in a host organism? Or did they originate from other earlier heterotrophic bacteria? Such questions are summarized in Fig. 6 and must be a matter of further research. Also, the possible relationships between photosynthetic bacteria and early nonpathogenic bacteria have to be investigated.
6. Isoprenoid biosynthesis in Archaea and Eucarya Heterotrophic eukaryotes, such as animals and fungi [16, 66], only use the MVA pathway of IPP and isoprenoid biosynthesis. The chloroplast containing photo-autotrophic plants and most algae groups possess in their cytosol the MVA pathway being used for biosynthesis of sterols, sesquiterpenes [see reviews 35, 36] and the side-chain of the mitochondrial ubiquinones [18], whereas the plastids contain the DOXP/MEP pathway of IPP formation. Thus, all Eucarya possess the MVA pathway, except for the proper chlorophyta within the ‘green algae’ that have lost this pathway [60]. The prokaryotic Archaea (formerly archaebacteria) form an independent evolutionary group that developed in their own way, independently of Bacteria and Eucarya. Like non-photosynthetic eukaryotes, archaea possess only the MVA pathway to synthesize their isoprenoids [11]. All archaea have the genes for HMG-CoA synthase, HMG-CoA reductase and the mevalonate kinase, whereas any further genes for the formation of IPP are missing [11, 32, 64]. Evidently, the genes of the enzymes for the transformation of MVA phosphate to IPP show no similarities to those of other organisms with the MVA pathway. Also, the usual IPP isomerase was not found in the genome of Archaea, but instead a ‘class 2’ IPP isomerase resembling that found in the bacterium Streptomyces [30]. Genes of the DOXP/MEP pathway were not found in Archaea, except for the sequence of a CDP-ME synthase in Pyrococcus honkoshii which seems to have been acquired by lateral gene transfer, possibly from a bacterium [10]. On the basis of various other biochemical and genetic informations the Archaea are somewhat closer to the Eucarya than the Bacteria [11, 33]. A putative scheme of a phylogenetic tree of the evolution of Archaea, Bacteria and Eucarya, originally based on the analysis of rRNA, is shown in a modified form in Fig. 7 and summarizes the presently known distribution of the DOXP/MEP and the MVA pathways of IPP formation. Acknowledgements: I wish to thank Martin Knapp and Dr. Christian Müller for assistance during the preparation of the manuscript.
20
?
Bacteria
Photosynthetic Bacteria
(DOXP /MEP)
(DOXP /MEP pathway)
Loss of Photosynthesis ?
Cyanobacteria
Secondary Endosymbiosis
Endosymbiosis
Pathogenic Bacteria e.g. Helicobacter Yersinia pestis Vibrio cholerae Mycobacterium tuberculosis
Most Algae, Higher Plants
(red or green alga?)
(with Chloroplasts)
Apicomplexa Parasites (with Apicoplast) Plasmodium Toxoplasma
Figure 6. Putative evolution and transfer of the DOXP / MEP pathway of IPP biosynthesis from early heterotrophic or photosynthetic bacteria to pathogenic bacteria, cyanobacteria and by endosymbiosis to algae, higher plants and the parasitic apicomplexa.
Bacteria
Archaea
Eucarya
DOXP/MEP
MVA
MVA
Green Gramsulfur positives bacteria Chlorobium Purple bacteria Rhodobacter
Green non-sulfur bacteria Chloroflexus (only MVA)
Mitochondria
Animals Fungi Euryarchaeota Crenarchaeota
Plants + DOXP/MEP
Algae
Euglena (only MVA)
(no IPP pathway)
Cyanobacteria + Chloroplasts
Flagellates Trichomonads
Flavobacteria Microsporadia
Thermotogales Aquifex
‘Cenancestor’ Figure 7. Simplified phylogenetic tree of the domains Bacteria , Archaea and Eucarya with indication of the presently known predominant distribution of the DOXP/MEP and the MVA pathways of IPP and isoprenoid biosynthesis. There are a few exceptions from this scheme, e.g. Chlorophyta (only DOXP/MEP pathway) and Apicomplexa (only DOXP/MEP pathway), whereas some Bacteria can have also the MVA pathway. The photosynthetic Bacteria and Eucarya are underlined by a green bar. (Branch lengths have no particular meaning in this tree).
7. References
21
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[52] Ruzicka, L. (1938) Die Architektur der Polyterpene. Angew. Chemie 51, 5-11. [53] Ruzicka, L., Eschenmoser, A. and Heusser, H. (1953) The isoprene rule and the biogenesis of isoprenoid compounds. Experientia 9, 357-396. [54] Schnarrenberger, C., Jacobshagen, S., Müller, B. and Krüger, I. (1990) Evolution of isozymes of sugar phosphate metabolism in green algae. in Z. I. Ogita and C. L. Market (eds.), Isozymes. Structure, function and use in biology and medicine. Wiley Liss, New York, pp. 743-764. [55] Schwarz, M. K. (1994) Terpen-Biosynthese in Ginkgo biloba: Eine überraschende Geschichte. Dissertation, Eidgenössische Hochschule Zürich. [56] Schwender, J., Lichtenthaler, H.K., Seemann, M. and Rohmer, M. (1995) Biosynthesis of isoprenoid chains of chlorophylls and plastoquinone in Scenedesmus by a novel pathway. in P. Mathis (ed.), Photosynthesis: from Light to Biosphere. Kluwer Academic Publishers, Amsterdam, pp. 1001-1004. [57] Schwender, J., Seemann, M., Lichtenthaler, H.K. and Rohmer M. (1996) Biosynthesis of isoprenoids (carotenoids, sterols, prenyl sidechains of chlorophyll and plastoquinone) via a novel pyruvate/glycero-aldehyde-3-phosphate non-mevalonate pathway in the green alga Scenedesmus. Biochem. Journal 316, 73-80. [58] Schwender; J., Zeidler, J., Gröner, R., Müller, C., Focke, M., Braun, S., Lichtenthaler, F.W. and Lichtenthaler, H.K. (1997) Incorporation of 1-deoxy-D -xylulose into isoprene and phytol by higher plants and algae. FEBS Letters 414, 129-134. [59] Schwender, J., Müller, C., Zeidler, J. and Lichtenthaler, H.K. (1999) Cloning and heterologous expression of a cDNA encoding 1deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana. FEBS Letters 455, 140-144. [60] Schwender, J., Gemünden, C., and Lichtenthaler, H.K. (2001) Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2 -Cmethylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta 212, 416-423. [61] Seemann, M., Campos, N., Rodríguez-Concepción, M., Hoeffler, J.-F., Grosdemange-Billiard, C., Boronat, A. and Rohmer, M. (2002) Isoprenoid biosynthesis via the methylerythritol phosphate pathway: accumulation of 2-C-methyl-D -erythritol 2,4-cyclodiphosphate in a gcpE deficient mutant of Escherichia coli. Tetrahedron. Lett. 43, 775-778. [62] Seto, H., Watanabe, H. and Furihata, K. (1996) Simultaneous operation of the mevalonate and non-mevalonate pathways in the biosynthesis of isopentenyl diphosphate in Streptomyces aerio uvifer. Tetrahedron Lett. 37, 7979-7982. [63] Seto, H., Orihara, N. and Furihata, K. (1998) Studies on the biosynthesis of terpenoids produced by Actinomycetes. Part 4. Formation of BE -40644 by the mevalonate and nonmevalonate pathways. Tetrahedron Lett. 39, 9497-9500. [64] Smit, A. and Mushegian A. (2000) Biosynthesis of isoprenoids via mevalonate in Archaea: the lost pathway. GenomeRes. 10, 14681484. [65] Wallach, O. (1885) Zur Kenntnis der Terpene und der ätherischen Öle. Justus Liebigs Ann. Chem. 227, 277-302. [66] Wang, Y., Dreyfuss, M.; Ponelle, M., Oberer, L. and Riezman, H. (1998) A glycosylphosphatidylinositol-anchoring inhibitor with an unusual tetracarbocyclic sesterterpene skeleton from the fungus Codinaea simplex. Tetrahedron 54, 6415-6426. [67] Wilding, E. I., Brown, J. R., Bryant, A. P., Chalker, A. F., Holmes, D. J., Ingraham, K. A., Iordanescu, S., So, C. Y., Rosenberg, M. and Gwynn, M. N. (2000) Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate in Gram-positive cocci. J. Bacteriol. 182, 4319-4327. [68] Wilson, R. J. M. (2002) Progress with parasite plastids. J. Mol. Biol. 319, 257-274. [69] Wolf, D. E., Hoffmann, C.H., Aldrich, P.E., Skeggs, H.R., Wright, L.D. and Folkers, K. (1956) ß-hydroxy-ß-methyl-d-dihydroxy-ßmethylvaleric acid (divalonic acid), a new biological factor. J. Am. Chem. Soc. 78, 4499. [70] Zeidler, J.G., Lichtenthaler, H. K., May, H.U. and Lichtenthaler, F. W. (1997) Is isoprene emitted by plants synthesized via the novel isopentenylpyrophosphate pathway? Z. Naturforsch. 52c, 15-23.
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[71] Zeidler, J.G., Schwender, J., Müller, C., Wiesner, J., Weidemeyer, C., Beck, E., Jomaa, H. and Lichtenthaler, H.K. (1998) Inhibition of the non-mevalonate 1-deoxy-D -xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Z. Naturforsch. 53 c, 980-986. [72] Zhou, W. X. und Nes, W. D. (2000) Stereochemistry of hydrogen introduction at C-25 in ergosterol synthesized by the mevalonateindependent pathway. Tetrahedron Lett. 41, 2791-2795.
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METABOLIC ENGINEERING OF VITAMIN E BIOSYNTHESIS FOR TOCOTRIENOL PRODUCTION AND INCREASED ANTIOXIDANT CONTENT EDGAR B. CAHOON United States Department of Agriculture -Agricultural Research Service Plant Genetics Research Unit Donald Danforth Plant Science Center 975 North Warson Road St. Louis, Missouri 63132 USA
Introduction Tocotrienols and tocopherols comprise the Vitamin E family of lipid soluble antioxidants in plants. Tocotrienols are the primary form of Vitamin E in the seed endosperm of most monocots, including agronomically important cereal grains such as wheat, rice, and barley. Tocotrienols are also found in the seed endosperm of a limited number of dicots, including Apiaceae species and certain Solanaeceae species, such as tobacco [1]. These molecules are found only rarely in vegetative tissues of plants. Tocopherols, by contrast, occur ubiquitously in plant tissues and are the exclusive form of Vitamin E in leaves of plants and seeds of most dicots [1]. Tocopherols also occur in photosynthetic microbes such as Synechocystis. Tocotrienols and tocopherols are plastid localized molecules that consist of a polar chromanol head group linked to a long-chain hydrocarbon tail. Tocotrienols differ structurally from tocopherols by the presence of three trans double bonds in the hydrocarbon tail (Fig. 1). In addition, four different forms of tocotrienols and tocopherols can occur in plants. These are designated a, β, γ, and d, and differ with regard to the numbers and positions of methyl groups on the aromatic head group. X1
X1 HO
HO O
X2
O
X2
CH 3
CH 3
Tocotrienol α-Tocotrienol/Tocopherol β-Tocotrienol/Tocopherol γ-Tocotrienol/Tocopherol δ-Tocotrienol/Tocopherol
Tocopherol X1, X 2= -CH 3 X1= -CH3; X 2= -H X 1= -H; X 2= -CH 3 X 1= -H; X 2= -H
Figure 1. Chemical structures of tocotrienols and tocopherols.
What is the Biochemical and Genetic Origin of Tocotrienols in Monocot Seeds? The committed step in the biosynthesis of tocopherols is the condensation of homogentisate from the shikimate pathway with phytyl diphosphate from the plastidic non-mevalonate isoprenoid pathway. This reaction is catalyzed by homogentisate phytyltransferase (HPT), and cDNAs for this enzyme have been isolated from several plant species [2, 3]. The product of this reaction 2-methyl-6-phtylbenzoquinol is then modified by cyclization and methylation reactions to form a tocopherol molecule. HPTs from spinach and Arabidopsis have been shown to have strong, if not exclusive, substrate preference for phytyl diphosphate [2, 4]. It has generally been assumed that the committed step in tocotrienol biosynthesis is the condensation of homogentisate with geranylgeranyl diphosphate, based on the structural similarity of this isoprenoid and the tocotrienol side chain.
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It has been proposed that this reaction is catalyzed by a prenyltransferase that has substrate preference for geranylgeranyl diphosphate rather than phytyl diphosphate [5]. Based on these substrate properties, such an enzyme can be designated homogentisate geranylgeranyl transferase (HGGT). The activity of HGGT has yet to be demonstrated in plants, and prior to our recent report [6], cDNAs for HGGT had not been isolated. We hypothesized that HGGT is a structurally divergent form of HPT. To test this hypothesis, we attempted to isolate HGGT cDNAs from developing seeds of barley and rice by PCR using degenerate oligonucleotides based on partially conserved amino acid sequences in HPTs. As is the case with most monocots, the seed endosperm of these plants is enriched in tocotrienols. The degenerate oligonucleotides corresponded to the amino acid sequences Y(I/V)VG(I/L/F/M)NQ and FIW(K/N)(I/L/M)FYA. Through the use of this approach, full-length cDNAs for structurally divergent forms of HPT were isolated from barley and rice seeds. A cDNA for a closely related polypeptide was subsequently identified from wheat among an EST population generated from developing seeds. To determine whether the cDNAs for these polypeptides correspond to HGGTs, the cDNAs were expressed under control of a cauliflower mosaic virus 35S promoter in tobacco callus. The primary form of Vitamin E in these cells is a-tocopherol, and these cells contain no detectable tocotrienols. Consistent with the expected activity of an HGGT, expression of the cDNAs from barley, wheat, and rice resulted in the production of tocotrienols in tobacco callus. Notably, expression of the barley HGGT yielded approximately a 10-fold increase in the content of Vitamin E antioxidants in tobacco callus (Fig. 2).
Tocopherol+Tocotrienol Content (ng/mg DW)
180
Tocotrienol
160
Tocopherol
140 120 100 80 60 40 20 0
1
2
3
4 5
+Vector
1
2
3
4
5
+Barley HGGT
Figure 2. Production of tocotrienols in tobacco callus by expression of a barley HGGT cDNA under control of a cauliflower mosaic virus 35S promoter. Shown are measurements of tocopherols and tocotrienols in extracts from five independent calli transformed with either the binary vector (+Vector) or the barley HGGT cDNA (+Barley HGGT).
Similarly, expression of the barley cDNA conferred tocotrienol production to Arabidopsis leaves and resulted in a 10- to 15-fold increase in the content of Vitamin E antioxidants [6]. Overall, these results demonstrate that the cDNAs isolated from barley, wheat, and rice encode HGGTs. These results also show that expression of HGGT alone is sufficient to confer tocotrienol production to plants such as Arabidopsis that do not normally produce this form of Vitamin E. As such, enzymes that cyclize and methylate the HPT product 2-methyl-6-
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phtylbenzoquinol in the tocopherol biosynthetic pathway are also able to modify the HGGT product 2-methyl-6geranylgeranylbenzoquinol to form tocotrienols. We have also initiated studies to determine whether HGGTs are also associated with tocotrienol synthesis in seeds of dicots that accumulate tocotrienols. The degenerate oligonucleotides described above have been used to PCR amplify cDNAs for divergent HPTs from seeds of tobacco and the Apiaceae coriander. These studies have resulted in the isolation of partial cDNAs for polypeptides that are most closely related to monocot HGGTs but more distantly related to monocot and dicot HPTs (Fig. 3). We are currently isolating full-length cDNAs for the tobacco and coriander polypeptides for functional characterization in transgenic plants. Structural Properties of HGGTs The HGGTs from barley, wheat, and rice share 80 to 90% amino acid sequence identity, but =60% identity with HPTs from rice, soybean, and Arabidopsis. Like HPTs, HGGTs are members of the UbiA family of prenyltransferases, which includes enzymes such as chlorophyll synthase and 4-hydroxybenzoate octaprenyl transferase, which is associated with ubiquinone biosynthesis (Fig. 3).
Chlorophyll Synthase
Uncharacterized UbiA Prenyltransferases
Oat At1g4446
At3g11950
At1g60600
(Putative homogentisate
At4g23660
solanesyltransferase)
Synechocystis HPT
At2g44520
Tobacco
Rice
Coriander
Putative Dicot HGGTs
At2g18950 Soybean Wheat Barley
HPTs
Maize Rice 0.1
Monocot HGGTs Figure 3. Phylogenetic analysis of the UbiA prenyltransferase family in plants.
HGGTs, like HPTs, are presumed to be localized in plastids. In agreement with this, the ChloroP program predicts that the N-terminal 62 amino acids of the barley HGGT are structurally consistent with a plastid transit peptide. In addition, using the SOSUI analysis algorithm (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), the barley HGGT is predicted to contain seven transmembrane helices (Fig. 4). A similar membrane topology is predicted for the Arabidopsis HPT as well as many other members of the UbiA prenyltransferase family.
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H2N
COOH
Figure 4. Predicted membrane topology of the barley HGGT.
Substrate Specificity of HGGTs Results from the expression of the barley HGGT in transgenic plants suggest that this enzyme has strong substrate specificity for geranylgeranyl diphosphate, rather than phytyl diphosphate. Expression of this enzyme in tobacco calli and Arabidopsis leaves, for example, resulted in the accumulation of Vitamin E antioxidants in the form of tocotrienols (prinicipally as γ-tocotrienol), but generated little or no change in the content of tocopherols [6]. To examine the substrate specificity of the barley HGGT further, we have expressed this enzyme in a mutant line of Arabidopsis that contains an inactive form of HPT (kindly provided by Dr. Peter Dörmann). As a result of the mutation, these plants produce only trace amounts of tocopherols. Upon expression of the barley HGGT in the Arabidopsis HPT mutant, approximately 90% of the Vitamin E detected was in the form of tocotrienols and the remaining 10% was in the form of tocopherols (primarily a-tocopherol). This result is consistent with a strong, but not exclusive, substrate specificity of the barley HGGT for geranylgeranyl diphosphate. We are currently in the process of generating recombinant barley HGGT and Arabidopsis HPT in insect cells using a baculovirus expression system. In vitro assay of the recombinant enzyme should clarify the substrate specificity of the monocot HGGT. References: [1] Kamal-Eldin, A. and Appelqvist, L.A. (1996) The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31, 671701. [2] Collakova, E. and DellaPenna, D. (2001) Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 127, 1113-1124. [3] Savidge, B., Weiss, J.D., Wong, Y.-H. H., Lassner, M.W., Mitsky, T.A., Shewmaker, C.K., Post-Beittenmiller, D., and Valentin, H.E. (2002) Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 129, 321-332. [4] Soll, J., Kemmerling, M. and Schultz, G. (1980) Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions. Arch. Biochem. Biophys. 204, 544-550.
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[5] Soll, J. and Schultz, G. (1979) Comparison of geranylgeranyl and phytyl substituted methylquinols in the tocopherol synthesis of spinach chloroplasts. Biochem. Biophys. Res. Commun. 91, 715-720. [6] Cahoon, E.B., Hall, S.E., Ripp, K.G., Ganzke, T.S., Hitz, W.D., and Coughlan, S.J. (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nature Biotechnol. 21, 1082-1087.
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TOBACCO BY-2 CELLS AS AN USEFUL EXPERIMENTAL SYSTEM FOR INVESTIGATING REGULATION OF THE STEROL PATHWAY M.A. HARTMANN and L. WENTZINGER Institut de Biologie Moléculaire des Plantes (UPR CNRS 2357), Département des Isoprénoïdes, Strasbourg, France.
1. Introduction Sterols, which are members of the vast family of isoprenoids, are essential molecules for all eukaryotes. In addition to their widely recognized roles as architectural components of cell membranes and their importance for regulating membrane fluidity and permeability, sterols are also able to modulate a variety of metabolic and ontogenetic events. This is particularly true for cholesterol, the most notorious sterol, whose signaling functions in cell division, cell growth, cell death and various developmental processes have been extensively studied in mammals (Edwards and Ericsson, 1999). In comparison, such a wealth of information about plant sterols is still far from being available (Hartmann, 2004). Whereas animals and fungi usually synthesize a major sterol end product – cholesterol and ergosterol, respectively – plants produce a bewildering array of sterols, with sitosterol, stigmasterol and 24-methylcholesterol as the most represented compounds. In higher plants, two distinct pathways concomitantly operate to form isopentenyl diphosphate (IPP), the common precursor of all isoprenoids and its isomer, dimethylallyl diphosphate (DMAPP) (Rohmer, 1999; Lichtenthaler, 1999) (Fig. 1).
Acetyl -CoA
CYTOSOL Pyruvate + Glyceraldehyde
PLASTID
3-P
HMG-CoA
HMGR 2-C -Methyl -D- erythritol -4-P (MEP) MVA
IPA Cytokinins
IPP
DMAPP
Isoprene IPP
Sesquiterpenes
FDS
Dolichols
GGPP
Monoterpenes
2x
Diterpenes Carotenoids
FPP
Farnesylated proteins
GPP
3x
IPP
DMAPP
SQS FPP SQUALENE
Cyt a et a 3 Ubiquinones
Sterols
MITOCHONDRIA
Brassinosteroids
Figure 1- Isoprenoid biosynthesis in higher plants
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Plastoquinone
ABA
In plastids, IPP is synthesized via the intermediate formation of 2-C-methyl-D-erythritol-4-phosphate (MEP pathway). After conversion to geranyl diphosphate (GPP) or geranygeranyl diphosphate (GGPP), isoprene units are used for the synthesis of mono- and diterpenes, carotenoids, abcissic acid (ABA) or the prenyl chains of chlorophylls and plastoquinone. In the cytosol, IPP is formed via the classical acetate/mevalonate (MVA) pathway, giving rise to a completely different set of compounds, as represented by cytokinins, isopentenyl adenine (IPA), sterols, sesquiterpenes, ubiquinone, heme a, dolichols, and farnesylated proteins. The key enzyme in this pathway has been identified as 3-hydroxy -3-methylglutaryl coenzyme A reductase (HMGR). Farnesyl diphosphate (FPP), the product of the reaction catalyzed by the farnesyl diphosphate synthase (FDS), occupies a central position. The first dedicated step of the sterol branch is the formation of squalene, a reaction catalyzed by squalene synthase (SQS). The conversion of squalene into squalene oxide, a reaction mediated by squalene epoxidase (SE), then into sterols represents a sequence of near 20 reactions (Fig. 2), which are all catalyzed by membrane proteins, probably organized as multi-enzymatic complexes. Sterols as the mixture of sitosterol, stigmasterol and 24-methylcholesterol are the major end products of this multi-branched pathway, suggesting that their biosynthesis is likely regulated separately from the synthesis of other isoprenoids.
SE
SQS OPP
FPP
oxidosqualene O
squalene
cycloartenol HO
SMT1 24-methylene cycloartanol HO
cycloeucalenol HO
COI obtusifoliol HO
C-14 DEMETHYLASE HO
4α -methyl ergosta8,14,24-trien-3 β-ol
HO
24-ethylidene lophenol HO
SMT2
HO
HO
24-methylene lophenol
HO
HO
HO
SITOSTEROL
24-METHYLCHOLESTEROL HO
STIGMASTEROL
Figure 2- Plant sterol biosynthetic pathway Solid arrows mean one reaction, and doted arrows, multiple steps. SQS, squalene synthase; SE, squalene epoxidase; SMT1, SMT2, sterol methyltransferases 1 and 2; COI, cycloeucalenol-obtusifoliol isomerase. End products are sitosterol, stigmasterol and 24-methylcholesterol.
To gain some insights into the regulatory mechanisms controlling the sterol branch, we have chosen to use tobacco (Nicotiana tabacum L.) Bright Yellow-2 (BY-2) cells. This pale-yellow cell line is rapidly proliferating (cell cycle of about 14 h), with considerable homogeneity (Nagata et al., 1992). Consequently, these cells exhibit high metabolic flux rates, which facilitate incorporation experiments and measurements of individual enzyme reactions. HMGR as well as other sterol biosynthesis enzymes are actively expressed, resulting in a high level of sterol production and membrane biogenesis. Our approach has been to feed BY-2 cells with various inhibitors or metabolic intermediates, in order to deregulate the sterol pathway and thus to induce
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significant fluctuations in enzyme activities and expression of the corresponding genes. The present study was focused on the role played by HMGR and SQS, in response to a depletion of endogenous sterols induced by several blockages in the sterol pathway, at the level of SQS, squalene epoxidase (SE) and obtusifoliol C-14 demethylase (see Fig. 2). The corresponding enzymes were inhibited by a 24-h-treatment of 3-d-old BY-2 cells with squalestatin-1 (also called zaragozic acid A), terbinafine and Lab 170250F, respectively. The principal findings are the following. 2. HMGR is able to respond to a selective depletion of endogenous sterols In mammalian cells, HMGR is the major rate-limiting step for cholesterol synthesis and is subject to feedback regulation at multiple molecular levels (Goldstein and Brown, 1990). Tobacco plants overexpressing HMGR has been shown to produce higher amounts of sterol intermediates and end products (Chappell et al., 1995; Schaller et al., 1995), clearly indicating that this enzyme also controls the carbon flux toward sterols in higher plant cells. Overexpression of FDS, an enzyme located downstream of HMGR, in Arabidopsis plants does not trigger any increase in the carbon flux toward sterols (Masferrer et al., 2002). Thus, it appears as if plants accumulate higher levels of sterols only when the limitation imposed by the HMGR bottleneck is overcome. Conversely, we have shown that, in BY-2 cells, HMGR is also able to respond to a selective depletion of endogenous sterols. Thus, a treatment of BY-2 cells with squalestatin-1, a very potent inhibitor of SQS (IC50 of 5.5 nM), triggers a 3-fold increase in both HMGR activity and mRNA levels (Wentzinger et al., 2002), suggesting that the level of squalene and/or other intermediates downstream acts as a signal to modulate HMGR activity and expression. In an attempt to identify such a signal, we created additional blockages in the post-squalene pathway. For instance, inhibition of squalene epoxidase (SE), the enzyme operating next to SQS, by terbinafine was found to induce a 3-fold increase in HMGR activity, but not in the corresponding transcripts (Wentzinger et al., 2002). However, a different situation is encountered if the blockage is located more downstream in the pathway, as illustrated by the inhibition of obtusifoliol 14-demethylase (CYP 51). Following treatment of BY-2 cells with Lab 170250F HMGR activity was indeed strongly and rapidly decreased. In this case, the depletion in endogenous ∆5 -sterols was accompanied by an accumulation of obtusifoliol and other 14α-methyl ∆8 -sterols, suggesting that either a biosynthetic intermediate or an unusual sterol might serve as a negative feedback regulator for HMGR activity. One can conclude that the level of end products may be not the unique regulatory signal of the pathway. The lack of an early intermediate (cycloartenol, the first cyclic intermediate?) in the post-squalene pathway might induce a positive compensatory response of HMGR whereas an accumulation of intermediates more downstream of the pathway might act as negative effectors. The consequences of an inhibition of terminal steps in the pathway, which are actually under investigation, should give additional insights into regulation of the overall pathway and maybe allow us to identify the compounds, which could serve as positive or negative effectors. 3. SQS plays a major role in cell growth but is not a rate -limiting step for sterol biosynthesis SQS is the first enzyme of the cytoplasmic isoprenoid pathway dedicated to sterol synthesis (Figs 1 and 2). It mediates the reductive head-to-head condensation of two molecules of FPP to form squalene via presqualene diphosphate. This reaction takes place in the endoplasmic reticulum (ER), as do all subsequent steps involved in sterol biosynthesis. Because of its particular position at the interface between hydrophilic and hydrophobic intermediates, it was assumed that SQS might constitute a major control point for regulating the sterol branch in directing FPP molecules into either sterols or non-sterol isoprenoids in response to changing cellular requirements. SQS is actively expressed in proliferating BY-2 cells, coordinately with other enzymes of the sterol biosynthetic pathway (Wentzinger et al., 2002). In the presence of squalestatin, cell growth is rapidly impaired. Inhibition of SQS directly affects cell division as attested by the arrest of the cell cycle, specifically in the G1 /G0 phase, but with no cytotoxic effect or cell death (Hemmerlin et al., 2000). These data clearly emphasize the major role played by SQS in plant growth. However, our results also indicate that SQS is not a rate-limiting step for sterol synthesis. Treatment of BY-2 cells with terbinafine induces not only a decrease in the amount of final ∆5 -sterols, but also an impressive accumulation of squalene, that does not inhibit either SQS activity or steady-state mRNA levels (Wentzinger et al., 2002). In this context, it is important to note that squalene was found not to accumulate in the ER membranes, but in lipid droplets throughout the cytosol. Thus, SQS could not “sense” the excess of squalene. Not even SQS inhibition itself by squalestatin triggers a change in mRNA levels. Finally, SQS activity is not affected by the inhibition of HMGR, an upstream enzyme, or obtusifoliol 14-demethylase, a downstream enzyme. Taken together, these data indicate that SQS does not participate in controlling the carbon flow toward sterols, at least in the case of BY-2 cells not challenged by a pathogen (Vögeli and Chappell, 1988).
32
4. Deregulation of the sterol pathway may trigger the unwanted liberation of toxic intermediates An intriguing question related to SQS inhibition (or FDS overexpression) concerns the fate of FPP molecules, which do not contribute to the build-up of sterols. As shown in Fig. 1, FPP serves as a substrate for a variety of non-sterol isoprenoids. As a consequence, a redirection of FPP toward other branches might occur. We have previously shown that exogenous farnesol, after conversion in FPP, can be incorporated into sterols, but also into the prenyl side-chain of ubiquinone Q10 (Hartmann and Bach, 2001). When farnesol was given to BY-2 cells in the presence of squalestatin, to block the sterol route, no additional incorporation of isoprene units into ubiquinone was observed, indicating that no redirection of FPP molecules toward this compound took place. Instead, a significant part of FPP molecules were hydrolyzed into farnesol, a compound known to have deleterious effects, especially when it is present at a concentration higher than 20 µM (Hemmerlin and Bach, 2000). Cell death/senescence-like responses resulting from overexpression of FDS in Arabidopsis have been also reported (Masferrer et al., 2002). Thus, it is clear that the FPP level in the cytosol has to be closely controlled. In the same context, an accumulation of oxygenated sterol intermediates resulting from the inhibition of the C-4 or C-14-demethylation reactions, located more downstream of the sterol pathway, might also be toxic for membrane functions. In sharp contrast to SQS inhibition, SE inhibition by terbinafine did not affect cell growth, despite the important accumulation of squalene, which is stored inside lipid droplets (Wentzinger et al., 2002). This situation is similar to that reported for a sterol-overproducing tobacco mutant, in which sterols in excess are converted in steryl esters, then stored in steroleosomes (Gondet et al., 1994). Interestingly, we have shown that the accumulated squalene can be reused as a precursor for sterol biosynthesis in response to a depletion of endogenous end products, clearly emphasizing that lipid droplets do constitute a metabolically active cell compartment. Thus, deregulation of the sterol pathway can trigger deleterious effects only when there is no available sink for toxic intermediates. 5. Crosstalk between sterol and triacylglycerol biosynthetic pathways Because sterols actively participate in membrane biogenesis, there must be coordination between lipid metabolic enzymes and transport processes that distribute lipids within the different membranes of the cell. Inhibition of the sterol pathway in BY-2 cells was found to induce changes in other lipid biosynthetic pathways. An example is given by the inhibition of SE by terbinafine, which triggers, concomitantly to squalene accumulation, a dosedependent increase in the triacylglycerols (TAG) content and rate of synthesis (Wentzinger et al., 2002). TAG are also accumulated in lipid droplets. The mechanisms whereby the inhibition of SE induces a synthesis of TAG still remain to be elucidated. However, TAG formation might represent a way to divert fatty acids from membrane phospholipids in the absence of sterol biosynthesis. An interesting point to note is that TAG are remobilized toward membrane biogenesis as soon as sterol biosynthesis is no longer inhibited. It would be interesting to investigate whether or not inhibition of the sterol pathway in BY-2 cells also induces changes in phosphatidylserine and glucosylceramide biosynthetic pathways like those occurring in leek seedlings treated by fenpropimorph (Hartmann et al., 2002).
6. References Chappell, J., Wolf, F., Proulx, J., Cuellar, R. and Saunders, C. (1995) Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol 109, 1337-1343. Edwards, P.A. and Ericsson, J. (1999) Sterols and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway. Annu Rev Biochem 68, 157-185. Goldstein, J.L. and Brown, M.S. (1990) Regulatio n of the mevalonate pathway. Nature 343, 425-430. Gondet, L., Bronner, R. and Benveniste, P. (1994) Regulation of sterol content in membranes by subcellular compartmentation of steryl esters accumulating in a sterol-overproducing tobacco mutant. Plant Physiol 105, 509-518. Hartmann, M.A. and Bach, T.J. (2001) Incorporation of all-trans-farnesol into sterols and ubiquinone in Nicotiana tabacum L. cv Bright Yellow-2 cell cultures. Tetrahedron Lett 42, 655-657. Hartmann, M.A., Perret, A.M., Carde, J.P., Cassagne, C? and Moreau, P. (2002) Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation oftriacylglycerols. Biochim Biophys Acta 1583, 285-296. Hartmann, M.A. (2004) Lipid Metabolism and Membrane Biogenesis, Top Curr Genet, Vol 6, G. Daum (ed), Springer, pp. 183-211. Hemmerlin, A. and Bach, T.J. (2000) Farnesol- induced cell death and stimulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in tobacco cv Bright Yellow-2 cells. Plant Physiol 123, 1257-1268. Hemmerlin, A., Fischt, I. And Bach, T.J. (2000) Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells. Physiol Plant 110, 342-349. Lichtenthaler, H.K. (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Ann Rev Plant Physiol Plant Mol Biol 50, 47-65.
33
Masferrer, A., Arró, M., Manzano, D., Schaller, H., Fernández-Busquets, X., Moncaleán, P., Fernández, B., Cunillera, N., Boronat, A. and Ferrer, A. (2002) Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsisinduces a cell death/senescence-like response and reduced cytokinin levels. Plant J 30, 123-132. Nagata, T., Nemoto, Y. and Hasezawa, S. (1992) Tobacco BY-2 cell line as the “Hela” cell in the cell biology of higher plants. Int Rev Cytol 132, 1-30. Rohmer, M. (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat Prod Rep 16, 565-574. Schaller, H., Grausem, B., Benveniste, P., Chye, M.L., Tan, C.T., Song, Y.H. and Chua, N.H. (1995) Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 in tobacco results in sterol overproduction. Plant Physiol 109, 761-770. Vögeli, U. and Chappell, J. (1988) Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor. Plant Physiol 88, 1291-1296. Wentzinger, L., Bach, T.J. and Hartmann, M.A. (2002) Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Physiol 130, 334-346.
34
POLYCLONAL ANTIBODIES, ABA AND ABB, WERE … WITHIN EITHER THE THIRD PREDICTED LUMENAL LOOP OR SECOND PREDICTED CYTOSOLIC LOOP OF BnDGAT1 R.J. WESELAKE1 , M. MADHAVJI1 , N. FOROUD1 , S. SZARKA 2 , N. PATTERSON2 , W. WIEHLER1 , C. NYKIFORUK, T. BURTON1 , P. BOORA 1 , S. MOSIMANN1 , M. MOLONEY2 AND A. LAROCHE3 1 Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4 2 Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 3 Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada T1J 4B1
1.
Introduction
The process of triacylglycerol bioassembly has been shown to involve both acyl-CoA-dependent [1] and acylCoA-independent processes [2, 3]. Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is an integral membrane enzyme of the endoplasmic reticulum (ER) that catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA [1]. cDNA encoding DGAT1 was first cloned from Mus musculus liver [4] and shortly thereafter from developing seeds of Arabidopsis thaliana [5-8] and microsporederived (MD) cell suspension cultures of Brassica napus L. cv Jet Neuf [9, 10]. A second cDNA encoding a B. napus DGAT, which represents a truncated form of DGAT1, has been designated BnDGAT2 [10, 11]. More recently, a second family of DGAT genes, unrelated to the aforementioned DGAT family, was cloned from Mortierella ramanniana with members in fungi, plants and animals [12]. The hydropathy plots of DGAT1 from various species are very similar in that the polypeptides all display a hydrophilic N-terminal segment followed by a number of potential transmembrane membrane intrapolypeptide segments [10]. The general characteristic of the mammalian DGAT1 hydropathy plot is also similar to that of acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.2.26), which uses cholesterol as an acyl acceptor instead of sn-1, 2-diacylglycerol [10, 13]. The current report presents structure/function studies conducted with BnDGAT1 from MD cell suspension cultures of B. napus and a recombinant polyhistidine-tagged N-terminal truncation of the enzyme [BnDGAT1(1-116)His 6 ] using immunochemical and biophysical approaches. Proteinase K treatment of microsomal vesicles combined with Western blotting that used polyclonal antibodies recognizing different segments of the BnDGAT1 polypeptide provided some insights into the topology of this enzyme. The recombinant N-terminal fragment of BnDGAT1 interacted with acyl-CoAs displaying positive cooperativity and appeared to self-associate to form dimers and possibly tetramers. This finding was consistent with recent reports on the self-association of human DGAT1 [14] and mammalian ACAT1 [15]. 2.
Materials and Methods
Microsomes were prepared from MD cell suspension cultures of B. napus L. cv Jet Neuf according to Byers et al. [16]. Protein determination was conducted using the Bradford method [17]. Immunochemical and molecular genetic strategies were adapted from [18] and [19], respectively. Rabbits were immunized with the following peptides conjugated to keyhole limpet haemocyanin: 21-LDRLHRRKSSSDSSN-35 and 278CYQPSYPRSPCIRKG-292. A cysteine residue was added to the first peptide to facilitate conjugation. Antisera recognizing the first and second segments, respectively, were referred to as antibody A (AbA) and antibody B (AbB). cDNA encoding the first 116 amino acid residues of BnDGAT1 was cloned into the expression vector pET26b(+) (Novagen, Madison, WI, U.S.A.) for production of the poly-histidine-tagged derivative BnDGAT1(1-116)His 6 in Escherichia coli BL21 (DE3) (Stratagene, La Jolla, CA, U.S.A.) [20]. Pelleted bacterial cells were resuspended in 30 mL of ice-cold 10 mM HEPES-NaOH buffer (pH 7.4) containing 1mM 2-mercaptoethanol and 100 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i. with 30 s on ice between each cycle. The suspension of ruptured bacteria was centrifuged at 13,000 x g for 15 min at 4ºC to obtain a clarified lysate, which served as an initial extract in the purification of BnDGAT1(1-116)His 6 using immobilized nickel ion affinity chromatography [20]. Purified BnDGAT1(1-116)His 6 was concentrated by pressure ultrafiltration and dialyzed into various buffer environments for crystallization,
35
acyl-CoA binding assays, gel filtration chromatography on Superdex 200 HR 10/30 (Amersham Pharmacia Biotech, Baie d’Urfé, PQ, Canada) and cross-linking experiments. [1-14 C]Acyl-CoAs (~50 Ci/mol) were synthesized from radiolabeled free fatty acids, oleic acid (cis ∆9 18:1) and erucic acid (cis ∆13 22:1), using acylCoA synthetase [21]. The interaction between radiolabeled acyl-CoA and BnDGAT1(1-116)His6 was assessed by Lipidex-1000 (Packard Instrument Company, Meriden, CT, U.S.A.) binding assays at 30ºC using 0.2 µM protein according to the method of Rasmussen et al. [22]. Cross-linking of BnDGAT1(1-116)His 6 was conducted using the reagent dimethyl suberimidate [23]. 3.
Results and Discussion
3.1. Probing the Topology of BnDGAT1 using an Immunochemical Approach An immunochemical approach was used to probe the topology of BnDGAT1 in microsomal vesicles. Polyclonal antibodies, AbA and AbB, were raised against chemically synthesized amino acid sequences representing the peptide segment 21-LDRLHRRKSSSDSSN-35 and peptide segment 278CYQPSYPRSPCIRKG-292 within BnDGAT1. AbA and AbB were used in Western blotting experiments with microsomal preparations of cell suspension cultures. Both antibodies recognized a polypeptide of about 60 kDa in SDS-solubilized microsomes (Figure 1, lane 1). AbB, however, reacted with a polypeptide of considerably lower molecular mass, which might correspond to BnDGAT2. Following treatment of microsomal vesicles with proteinase K, immunochemical reactions for both antibody preparations were no longer detected (Figure 1, lane 2). The loss of the AbA signal supports a previous suggestion that the N-terminus of A. thaliana DGAT1 is found on the cytosolic side of the ER [5]. The loss of the AbB signal suggests that the AbB-binding region is also located on the cytosolic side of the ER.
AbA
AbB
BnDGAT1 →
→
→
1
2
1
2
Figure 1. Western blot of BnDGAT1 from microsomes with AbA or AbB without Proteinase K treatment (lane 1) and after Proteinase K treatment (lane 2). Microsomes containing 1 mg protein were treated for 30 min on ice with 10 µg Proteinase K. The hydrolytic reaction was terminated with 0.1 mg phenylmethylsulfonyl fluoride per mg Proteinase K and incubated on ice for a further 5 min based on protocols described by Morimoto et al. [24]. Samples were diluted to 1 mL with 10 mM HEPES-NaOH (pH 7.4) and centrifuged for 20 min at 220,000 x g at 4°C. The pellet was treated with 150 µL SDS-PAGE sample buffer and boiled for 2 min and 25 µL were subjected to SDS-PAGE (12% total monomer concentration with 1.1% cross-linker). Western blotting conditions were similar to those described by Nykiforuk et al. [10].
Two possible topology scenarios for BnDGAT1 are shown in Figure 2. Analysis of the BnDGAT1 protein sequence using a computer program (TMHMM2.0) for predicting transmembrane helices [25] suggested that the polypeptide could contain up to10 transmembrane segments (Figure 2A). The 10 transmembrane model, however, places the AbB recognition site on the lumenal side of the ER. Since this site was accessible to Proteinase K, the alternative 9 transmembrane model in Figure 2B seems more appropriate.
36
AbA epitope
A AbB epitope
AbA epitope
B Figure 2. Two membrane topology scenarios for BnDGAT1. The top cartoon (A) represents a scenario in which all 10 hydrophobic regions are transmembrane helices whereas the bottom cartoon (B) represents a scenario in which 9 hydrophobic regions are transmembrane helices. From left to right the cylinders represent the respective hydrophobic regions 1 to 10. The corresponding amino acid residues for these predicted hydrophobic regions are 116-135, 159-178, 190-212, 217-239, 259-279, 298-320, 346-368, 408-430, 440-462, and 474-503. The solid gray rectangle represents the lipid bilayer with cytosolic and lumenal face above and below the lipid bilayer, respectively. The black lines represent the loops in the cytosol or the lumen. The white rectangles represent peptide segments that would be recognized by AbA and AbB, respectively.
3.2. Crystallization of BnDGAT1 ( 1-116)His6 Analysis of purified BnDGAT1(1-116)His6 using dynamic light scattering indicated that the protein preparation was largely monodisperse and potentially suitable for crystallization. Small plate-like monoclinic crystals were grown from preparations of BnDGAT1(1-116)His 6 purified by immobilized nickel ion affinity chromatography (Figure 3). The largest microcrystals were too small (0.07 x 0.07 x 0.01 mm) for x-ray diffraction studies using a rotating anode x-ray source. These crystallization trials have set the foundation for a more in depth structural analysis of BnDGAT1(1-116)His 6
Figure 3. Microcrystals of BnDGAT1(1-116)His6 . Protein solution was dialyzed against 10 mM glycine-NaOH (pH 9.0) containing 20 mM NaCl and 1 mM EDTA. Screening for crystallization using vapor diffusion and hanging drops resulted in production of microcrystals within 1-2 weeks. .
3.3. Acyl-CoA Binding Properties of BnDGAT1 ( 1-116) His6 A previous study over a limited range of [1-14 C]18:1-CoA concentrations indicated that this thioester interacts with BnDGAT1(1-116)His 6 [20]. In the current study, the range of thioester concentrations used in Lipidex-1000 binding assays was extended up to near 30 µM for 18:1-CoA (Figure 4). As well, binding studies were conducted with [1-14 C]22:1-CoA for comparative purposes. The binding of both ligands to BnDGAT1(1-116)His 6 occurred in a sigmoidal fashion. The binding ratio of thioester: BnDGAT1(1-116)His 6 was based on the molecular mass of 13,278 Da calculated from the amino acid sequence of the N-terminal fragment. BnDGAT1(1-116)His 6 bound more 22:1-CoA then 18:1-CoA at considerably lower concentrations indicating that the N-terminal fragment had a greater affinity for 22:1-CoA. Transformation of the data according to Scatchard [26] revealed plots which rose to a maximum and then decreased suggesting that the interaction between acyl-
37
CoA and BnDGAT1(1-116)His 6 involved positive cooperativity. Substantial binding of thioester occurred in the range of 3 - 6 µM acyl-CoA, which is the estimated physiological range of the acyl-CoA pool concentration in developing zygotic embryos of B. napus [27]. A competition binding study was conducted using BnDGAT1(114 116)His 6 in Lipidex-1000 assays with [1- C]18:1-CoA in the absence or presence of either unlabeled CoA or unlabeled 18:1-CoA (TABLE 1). The same concentration (8 µM) of either free CoA or 18:1-CoA was effective in displacing a similar quantity of radiolabeled acyl-CoA from BnDGAT1(1-116) His 6 suggesting that the CoA component of the thioester had a major role in determining binding. CoA has previously been shown to stimulate microsomal DGAT activity from MD cell suspension cultures of B. napus suggesting that this coenzyme may play a role in regulating cellular DGAT activity [16]. The difference in the binding affinity of 18:1-CoA versus 22:1-CoA shown in Figure 4, however, indicates that the nature of the fatty acyl moiety is important in determining the extent and affinity of binding. Analysis of the BnDGAT1 amino acid sequence using the transmembrane prediction program, TMHMM2.0 [25], indicated that the hydrophilic N-terminal fragment of the polypeptide was probably on the cytosolic side of the ER. The binding of acyl-CoA by BnDGAT1(1-116)His 6 is consistent with the existence of an acyl-CoA binding signature (residues 99-116) in the N-terminal region of BnDGAT1 [10]. BnDGAT2 does not contain the hydrophilic N-terminus present in BnDGAT1. The absence of the acyl-CoA binding site in BnDGAT2 suggests that amino residues involved in catalysis are common to both BnDGAT1 and BnDGAT2 because cDNAs encoding both of these enzymes were functionally expressed in yeast [10]. Different species of cytosolic acyl-CoA, at various concentrations, might differentially affect the performance of BnDGAT1 in the ER. Although BnDGAT1 was cloned from a low-erucic acid cultivar B. napus, BnDGAT1(1-116)His 6 still displayed a relatively strong interaction with 22:1-CoA. Microsomal DGATs from both high- and low-erucic cultivars of B. napus have been shown to exhibit a similar pattern of acyl-CoA specificity suggesting that the breeding process used to produce low-erucic acid cultivars did not affect the genes encoding DGAT [28]. Thus, modification of the acyl-CoA pool via breeding or genetic engineering might have profound effects on the regulation of BnDGAT1.
Ligand bound (mol/mol BnDGAT(1-116) His6)
3.0
2.5
22:1-CoA 2.0
1.5
1.0
0.5
18:1-CoA
0.0
0
5
10
15
20
25
30
Acyl-CoA concentration (µM)
Figure 4. Effect of total acyl- CoA concentration on the binding of acyl- CoA by BnDGAT1(1-116)His6 . Binding assays were conducted in duplicate in 10 mM potassium phosphate buffer, pH 7.4, at 30 o C with [1- 14 C]acyl-CoAs (18:1 and 22:1) and 0.2 µM protein. Incubations were set up without protein to correct for acyl-CoA that was not adsorbed by the Lipidex-1000. Data points represent mean values. TABLE 1. Effect of non-radiolabeled CoA and 18:1 -CoA on binding of [1-14 C]18:1 -CoA by BnDGAT1(1-116)His6 . All incubation mixtures contained 0.2 µM protein and 19 µM radiolabeled acyl-CoA. Following sedimentation of Lipidex-1000, equal aliquots of the supernatants were assayed for radioactivity. Binding condition DPM ± SD _____________________________________________ Control 3559 ± 987 + 8 µM unlabeled CoA 2212 ± 123 + 8 µM unlabeled 18:1-CoA 1912 ± 101
38
Absorbance (280 nm)
3.4. Gel Filtration Chromatography of BnDGAT1 ( 1-116)His6 An apparent molecular mass of about 45 kDa was determined for BnDGAT1(1-116)His 6 by gel filtration chromatography using a Superdex 200 HR 10/30 column (Figure 5). Given the molecular mass of 13,278 Da for the polypeptide, it appears that BnDGAT1(1-116)His 6 behaved as a multimer during gel filtration chromatography. The smaller shoulder of protein preceding this major peak indicated that different states of aggregation may exist for the N-terminal fragment under these conditions. The gel filtration run depicted in Figure 5 was conducted with 20 mM NaCl in the equilibration buffer. Increasing the salt concentration to 500 mM NaCl did not affect the elution profile of BnDGAT1(1-116) His 6 suggesting that ionic interactions were not a major factor in the self-association of the N-terminal fragment.
0.100 0.090 0.080 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000
← 45 kDa
0
5
10
15
Volume (mL)
Figure 5. Gel filtration chromatography of BnDGAT1(1-116)His6 using a column of Superdex 200 HR 10/30. The column was equilibrated with 10 mM HEPES-NaOH, pH 7.4, containing 20 mM NaCl and 1 mM EDTA. The protein sample was dialyzed into column equilibration buffer. Five hundred microliters of a 5 mg/mL solution of BnDGAT1(1-116)His6 were injected into the column operated at a flow rate of 0.5 mL/min using a biocompatible HPLC system. The void and bed volume of the column was 8.2 and 25 mL, respectively. The column was calibrated with the following protein standards: IgG (160 kDa), Vel = 13.0 mL; BSA (67 kDa), Vel = 14.8 mL; β-lactoglobulin (35 kDa), Vel = 15.9 mL; cytochrome c (12.4 kDa), Vel = 17.6 mL. Vel = elution volume.
3.5. Chemical Cross-linking of BnDGAT1 ( 1-116) His6 Cross-linking experiments with dimethyl suberimidate [23] were conducted to further probe the apparent selfassociating properties of BnDGAT1(1-116)His 6 . The effect of cross-linking was monitored using Western blotting of BnDGAT1(1-116)His 6 species resolved by SDS-PAGE using AbA, which recognized an epitope in residues 2135 in the N-terminal region of the polypeptide (Figure 6). The control immunoblot (lane 1) exhibited an apparent monomer (23 kDa) and dimer (44 kDa). The dimer could not be visualized by Coomassie blue staining suggesting that the immunoreactivity of the dimer was considerably greater than that of the monomer. The molecular mass of the monomer on SDS-PAGE was not in agreement with the calculated molecular mass of 13,278 Da suggesting that BnDGAT1(1-116)His 6 displayed an anomalous subunit molecular mass based on SDSPAGE. Treatment with cross-linking agent resulted in the appearance of a larger multimer (72 kDa), possibly a tetramer, and an enhancement in the quantity of dimer (lane 2). The 72 kDa species also appeared to be present at a barely perceptible level in the control (lane 1). The putative tetramer also was not visible in Coomassie blue stained gels. In general, the results of cross-linking exp eriments supported the observations of self-association of BnDGAT1(1-116)His 6 as assessed by gel filtration chromatography. These results are particularly revealing in the light of recent findings, which have indicated that both human DGAT1 [14] and mammalian ACAT1 [15] are tetrameric proteins. The N-terminal regions of these mammalian acyltransferases have been shown to play a critical role in the formation of tetramers.
39
72 kDa 44 kDa 23 kDa
1
2
Figure 6. Western blot of BnDGAT1(1-116)His6 using AbA before (lane 1) and after (lane 2) chemical cross-linking. BnDGAT1(1-116)His6 (500 µg/mL) was cross-linked in the presence of 4 mg dimethyl suberimidate per mL in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 x SDS loading buffer and boiled for 5 min prior to application to an SDS-PAGE gel (10% total monomer concentration with 1.1 % cross-linker concentration). Western blotting conditions were similar to those described by Nykiforuk et al. [10].
4.
Summary
Immunochemical studies with antibodies recognizing two different peptide segments within BnDGAT1 suggested that the hydrophilic N-terminal region of the enzyme and the second cytosolic loop, for a 9 transmembrane model for the enzyme, resided on the cytosolic side of the ER. BnDGAT1(1-116)His 6 purified by immobilized nickel ion affinity chromatography was crystallized under alkaline conditions. The N-terminal fragment displayed positive cooperativity in interacting with 18:1- or 22:1-CoA. The affinity of BnDGAT1(1116)His 6 for 22:1-CoA was considerably greater than for 18:1-CoA. Although the type of acyl-CoA species influenced binding, competition binding assays revealed that CoA had a major role in this process. The Nterminal fragment of BnDGAT1 appeared to self-associate based on gel filtration chromatography and chemical cross-linking experiments. 5.
Acknowledgements
This research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to R. Weselake and by a grant from the Alberta Agricultural Research Institute to R. Weselake and A. Laroche. W. Wiehler was the recipient of a Post-Graduate Scholarship from NSERC. Funding from the Alberta Network for Proteomics Innovation, Genome Prairie and Genome Canada, NSERC and Western Economic Diversification supported the purchase of some equipment used in this research. 6.
References
[1] Weselake, R.J. (2002) Biochemistry and biotechnology of plant triacylglycerol biosynthesis. In T.M. Kuo and H.W. Gardner (eds.), Lipid Biotechnology. Marcel Dekker, Inc., New York, pp. 27-56. [2] Stobart, K., Mancha, M., Lenman, M., Dahlqvist, A. and Stymne, S. (1997) Triacylglycerols are synthesized and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) Planta 203, 58-66. [3] Dahlqvist, A., Ståhl, U., Lenman, M.A., Lee, M., Sandager, L., Ronne, H. and Stymne, S. (2000) Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS 97, 6487-6492. [4] Cases, S., Smith., S.J., Zheng, Y.-W., Myers, H.M., Lear, S.R., Sande, E., Novak, S., Collins, C., Welch, C.B.,, Lusis, A.J., Erickson, S.K. and Farese, Jr., R.V. (1998) Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. PNAS 95, 13018-13023. [5] Hobbs, D.H., Lu, C. and Hills, M.J. (1999) Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett. 452, 145-149. [6] Zou, J., Wei, Y., Jako, C., Kumar, A., Selvaraj, G. and Taylor, D.C. (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J. 19, 645-653. [7] Routaboul, J.-M., Benning, C., Bechtold, N., Caboche, M. and Lepiniec, L. (1999) The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase. Plant Physiol. Biochem. 37, 831-840. [8] Bouvier-Navé, P., Benveniste, P., Oelkers, P., Sturley, L.D. and Schaller, H. (2000) Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase. Eur. J. Biochem. 267, 85-96. [9] Nykiforuk, C.L., Laroche, A. and Weselake, R.J. (1999) Isolation and sequence analysis of a novel cDNA encoding a putative diacylglycerol acyltransfease from a microspore-derived cell suspension culture of Brassica napus L. cv Jet Neuf (PGR99-123). Plant Physiol. 120, 1207. [10] Nykiforuk, C.L., Furukawa-Stoffer, T.L., Huff, P.W., Sarna, M., Laroche, A., Moloney, M.M. and Weselake, R.J. (2002) Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis. Biochim. Biophys. Acta 1580, 95-109.
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[11] Nykiforuk, C.L., Laroche, A. and Weselake, R.J. (1999) Isolation and characterization of a cDNA encoding a second putative diacylglycerol acyltransferase from a microspore-derived cell suspension culture of Brassica napus L. cv. Jet Neuf (PGR99-158). Plant Physiol. 121, 1957. [12] Lardizabal, K.D., Mai, J.T., Wagner, N.W., Wyrick, A., Voelker, T. and Hawkins, D.J. (2001) DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J. Biol. Chem. 276, 38862-38869. [13] Buhman, K.K., Chen, H.C. and Farese, Jr. R.V. (2001) The enzymes of neutral lipid synthesis. J. Biol. Chem. 276:4036940372. [14] Cheng, D., Meegalla, R.L., He, B., Cromley, D.A., Bilheimer, J.T. and Young, P.R. (2001) Human acyl- CoA:diacylglycerol acyltransferase is a tetrameric protein. Biochem. J. 359, 707-714. [15] Yu, C., Zhang, Y., Lu, X., Chen, J., Chang, C.C.Y. and Chang, T.-Y. (2002) Role of the N-terminal hydrophilic domain of acyl-Coenzyme A:cholesterol acyltransferase 1 on the enzyme’s quaternary structure and catalytic efficiency. Biochemistry 41, 3762-3769. [16] Byers, S.D., Laroche, A., Smith, K.C. and Weselake, R.J. (1999) Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape. Lipids 34, 1143-1149. [17] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding. Anal. Biochem. 72, 248-254. [18] Harlow, E. and Lane, D. (1988) Antibodies, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor. [19] Ausubel, F.M., Bent, R., Kingston, R.E., Moore, D.J., Smith, J.A., Silverman, J.G. and Struhl, K. (2004) Current Protocols in Molecular Biology, John Wiley and Sons, New York. [20] Weselake, R.J., Nykiforuk, C.L., Laroche, A., Patterson, N.A., Wiehler, W.B., Szarka, S.J., Moloney, M.M., Tari, L.W. and Derekh, U. (2000) Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape. Biochem. Soc. Trans. 28, 684-686. [21] Taylor, D.C., Weber, N., Hogge, L.R. and Underhill, E.W. (1990) A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry. Anal. Biochem. 184, 311-316. [22] Rasmussen, J.T., Börchers, T. and Knudsen, J. (1990) Comparison of the binding affinities of acyl-CoA-binding protein and fatty-acid binding protein for long-chain acyl-CoA esters. Biochem. J. 265, 849-855. [23] Davies, G.E. and Stark, G.R. (1970) Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. PNAS 66, 651-656. [24] Morimoto, T., Arpin, M. and Gaetani, S. (1983) Use of proteases for the study of membrane insertion. Meth. Enzymol. 96, 121-150. [25] Sonnhammer, E.L., von Heijne, G. and Krogh, A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. in J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff and C. Sensen (eds), Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology. AAAI Press, Menlo Park, CA, pp. 175-182. [26] Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. [27] Larson, T.R. and Graham, I.A. (2001) A novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant J. 25, 115-125. [28] Cao, Y.-Z. and Huang, A.H.C. (1987) Acyl coenzyme A preference of diacylglycerol acyltransferase from the maturing seeds of Cuphea, maize, rapeseed, and canola. Plant Physiol. 84, 762-765.
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PRODUCTION OF CONJUGATED FATTY ACIDS IN PLANTS HORNUNG, E. 1 , SAALBACH, I.2 and FEUSSNER, I.1 1 Georg-August-University Göttingen Albrecht-von-Haller Institute for Plant Sciences Department for Plant Biochemistry Justus-von-Liebig-Weg 11 D-37077 Göttingen, Germany 2 Institute of Plant Genetics and Crop Plant Research (IPK) Corrensstr. 3 D-06466 Gatersleben, Germany
1. Introduction Fatty acids with conjugated double bond systems such as conjugated linoleic acids (CLAs) are already used in functional foods or animal feed whereas conjugated linolenic acids (CLNAs) like a-eleostearic acid are commonly used for industrial applications like tung oil in formulations for inks, dyes, coatings and resins because of their ability to dry to a clear, hard finish (Sonntag, 1979). CLAs as well as CLNAs exist in various positional isoforms depending on their origin: CLNAs are major fatty acids within seed oils of plants such as tung (Aleurites fordii), marigold (Calendula officinalis) and pomegranate (Punica granatum) containing a-eleostearic acid (18:3? 9Z,11E,13E ), calendic acid (18:3 ? 8E,10E,12Z) and punicic acid (18:3 ? 9Z ,11E,13Z), respectively (Tulloch, 1982). CLA in contrast is present in food derived from ruminant animals and is there the product of ruminal bacteria (Belury, 2002). Different CLA isomers are discussed in being involved in reducing cancer (18:2 ? 9Z,11E ; Durgam and Fernandes, 1997; Ip et al., 1994) and having positive effects on arterial sclerosis (Lee et al., 1994) as well as body fat composition (18:2 ? 10E,12Z; Blankson et al., 2000; DeLany et al., 1999; West et al., 1998). There is also growing evidence that dietary supplementation with CLNAs has an effect on body fat composition (Koba et al., 2002) and in cell culture experiments CLNAs proved to be cytotoxic to tumour cells to a even higher extent than CLA (Igarashi and Miyazawa, 2000). Therefore it exist a growing interest in transferring the responsible biosynthetic genes intro crop plants to obtain significant amounts of specific CLA and CLNA isomers in seed oil. cDNAs encoding enzymes that catalyze the formation of conjugated double bonds out of a single cis-double bond have already been identified and functionally characterized by expressing them in Sacharomyces cerevisiae, somatic soy bean embryos and Arabidopsis thaliana (Cahoon et al., 2001; Hornung et al., 2002; Iwabuchi et al., 2003; Qiu et al., 2001). However, all of these enzymes termed “conjugases” use linole ic acid as precursor to produce CLNAs or a-linolenic acid to produce conjugated octadecatetraenoic acids (Cahoon et al., 1999). Most interestingly for one pla nt conjugase the production of CLA beside CLNA has been reported (Qiu et al., 2001) : CoFADX1, a ? 9 -conjugase from C. officinalis (also termed CoFac2) was reported to produce up to 0.09 % (w/w) of the CLA isomer 18:2 ? 8,10 of total fatty acids in yeast cells expressing the enzyme. Although another group did not report on the production of CLA by this approach (Cahoon et al., 2001) , we investigated as well the possibility of the production of CLA in plant seed oils by expressing either a ? 9 -conjugase (CoFADX1) or a ?12 -conjugase (PuFADX from P. granatum; Hornung et al., 2002) in tobacco seeds under the control of the seed specific promoter USP (Pickardt et al., 1998). In order to analyze whether the production of CLA depends on the use of a special seed specific promoter, we also transformed PuFADX into tobacco under the control of CaMV 35S promoter and under the control of the seed specific promoter LeB4 (Bäumlein et al., 1991).
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2. Materials and methods 2.1 Chemicals Standards of fatty acids as well as all other chemicals were from Sigma (Deisenhofen, Germany); methanol, hexane, 2-propanol (all HPLC grade) were from Baker (USA). 2.2 Cloning and Vector Construction The coding regions of PuFADX and CoFADX were amplified by PCR using the Expand High Fidelity System (Roche Diagnostics, Mannheim, Germany) and suitable recognition sites for restriction enzymes were created. As template cDNA from developing seeds of Punica granatum and Calendula officinalis respectively were used isolated as described in (Fritsche et al., 1999; Hornung et al., 2002). PCR products were cloned into pGEM-T vector as described by the manufacturer (Promega, Mannheim, Germany). After digestion with appropriate restriction enzymes PuFADX or CoFADX were ligated behind the CaMV 35S promoter, the LeB4 promoter (Bäumlein et al., 1991) or the USP promoter (Pickardt et al., 1998) and cloned into binary vector pPTV-Bar (Becker et al., 1992). For the plant transformation Agrobacterium strain EHA105 was used. 2.4 Plant transformation Tobacco leaves were used for Agrobacterium-mediated leaf disc infection as described by Horsch (Horsch et al., 1985). Transformants were selected on 10 mg/l phosphinothricin. Transformation was verified by spraying pla nts with 0.5 % Basta (Aventis) 3 days after they have been transferred from agar plates to soil and by applying PCR with transgene-specific primers on genomic DNA isolated from leaves. 2.4 Extraction and Analysis of Lipids Analysis of fatty composition of plant material was done by homogenizing 5 mg of tobacco seeds in 405 µl of a mixture of toluene and methanol (1:2, by vol.) and 150 µl of sodium methoxide. The samples were incubated while shaking at room temperature for 20 min. Fatty acid methyl esters were extracted by adding 0.5 ml of 1 M NaCl and 0.5 ml of hexane and mixing by vortexing. To analyze fatty acid composition in tobacco leaves, fatty acids were extracted using a modification of the method of Weichert et al. (2002), the solvent was removed and 333 µl of a mixture of toluene and methanol (1:1, v/v) and 167 µl of 0.5 mM sodium methoxide were added. After incubation of the samples for 20 min, 0.5 ml of 1 M NaCl and 0.75 ml hexane were added. The extractions were repeated once and the combined organic phases were evaporated to dryness under a nitrogen stream. Samples were then resuspended in about 40 µl acetonitrile and 1 µl of each sample was analyzed by GC/FID as described elsewhere (Hornung et al., 2002). 3. Results and Discussion To investigate the possibility of modification of pla nt seed oil in order to produce conjugated fatty acids by a transgenic approach tobacco was chosen as a model plant since its seed oil contains substantial amounts of only a single polyunsaturated fatty acid that is linoleic acid (about 75 %, Table 1). 3.1. Production of punicic acid in tobacco seeds First the production of trienoic fatty acids was evaluated. Therefore we transformed tobacco with different conjugases. At first a full length cDNA encoding a ? 9 -conjugase from pomegranate (PuFADX; Hornung et al., 2002) was expressed in N. tabacum cv. Samsun NN under control of the seed specific promoters USP or LeB4, respectively, or expressed under the control of the constitutive CaMV 35S promoter. Transformants (T1) were selected by drug resistance and by PCR. Fatty acid methylesters (FAMEs) were analysed by GC from the leaves of tobacco plants transformed with PuFADX under the control of the CaMV 35S promoter. There was no difference in the fatty acid composition of vegetative tissue between untransformed and transgenic plants (data not shown).
43
However, analysis of the FAMEs from transgenic seeds showed the appearance of a new peak in comparison to seeds from untransformed plants (Figure 1).
18:2 ∆9Z,12Z
A
detector response
16:0 18:1 ∆9Z 18:0 18:2 ∆9Z,12Z
16:0
18:1 ∆9Z
B
18:3∆9Z,11E,13Z
18:0 8
10
12 14 retention time [min]
16
18
Figure 1. GC/FID analysis of FAMEs isolated from tobacco seeds. Transmethylation and analysis of esterified fatty acids was done as described in methods. A shows the chromatogram of FAMEs from untransformed tobacco seeds. B shows the GC/FID analysis of FAMEs from transgenic tobacco seeds expressing PuFADX under the control of the USP -promoter.
The newly produced FAME was identified by authentic standard as punicic acid (18:3? 9Z,11E,13Z). In functional analyses of the enzyme in S. cerevisiae the production of punicic acid by PuFADX has already been shown and the identity of the conjugated fatty acid has been verified by GC/MS and NMR (Hornung et al., 2002). Beside punicic acid no further newly produced fatty acids could be detected neither in seeds of plants transformed with PuFADX under the control of a seed specific promoter nor under the control of a constitutive promoter. This was not unexpected as substrate specificity analysis of PuFADX expressed in yeast cells and supplemented with different fatty acids had already shown that only linoleic acid and ?-linolenic acid are converted into new fatty acids by this enzyme (Hornung et al., 2002). As tobacco seeds don’t contain any ?-linolenic acid there is only linoleic as substrate for the conjugase left. In contrast to our results Iwabuchi et al. (Iwabuchi et al., 2003) detected the formation of new peak tentatively identified as a conjugated octadecatetraenoic acid (18:4) in Arabidopsis seeds as well as in yeast cells supplemented with a-linolenic acid when transformed with punicic acid producing conjugases from Trichosanthes kirilowii and Punica granatum. But even if PuFADX would accept a-linolenic acid as substrate when expressed in plants the concentration of a-linolenic in tobacco seeds is only 0.7 % (w/w) of total fatty acids in comparison to 19.4 % in Arabidopsis seeds. The expression of PuFADX under the control of different promoters was investigated and fatty acid composition of different transgenic plant lines as well as of untransformed tobacco seeds was compared (Table 1). Table 1. Fatty acid composition of tobacco seeds from untransformed plants and from transgenic plants expressing PuFADX.
44
Values are presented as relative ratio in % (w/w) of total fatty acids of tobacco seeds. The means + S.D. were obtained from independent analyses (number in parentheses) of seeds from individual plants. ND, not detected; 35S, CaMV 35S promoter; LeB4, V. faba leguminB4 promoter; USP, V. faba unknown seed protein promoter. Untransformed (n = 6) 16:0 18:0 18:1 ?9Z 18:1 ?11Z 18:2 ?9Z,12Z 18:3 ?9Z,12Z,15Z 18:3 ?9Z,11E,13Z * values smaller than 0.05
35S
LeB4
(n = 5)
USP
(n = 10)
(n = 10)
8.9
+
0*
9.4 +
0.1
9.1 +
0.4
8.1 +
0.5
3.5
+
0.4
2.9 +
0.3
2.7 +
0.3
2.6 +
0.2
11.2
+
0.8
11.0 +
0.5
10.1 +
1.0
17.6 +
2.4
0.4
+
0.3
0.4 +
0.2
0.2 +
0.1
0.5 +
0.3
75.2
+
0.7
75.1 +
0.7
76.8 +
1.4
63.2 +
4.1
0.7
+
0.1
0.8 +
0.1
0.9 +
0.2
0.6 +
0.1
0.2 +
0.1
0.1 +
0*
7.2 +
2.2
ND
The highest accumulation of punicic acid was obtained from tobacco seeds of plants transformed with PuFADX under the control of the seed specific USP promoter. Punicic acid accumulated to 7.2 % (w/w) of the total fatty acids in seeds carrying the USP promoter in comparison to 0.1 % (w/w) in seeds carrying the LeB4 promoter, and to 0.3 % in seeds carrying the constitutive promoter. Punicic acid accumulation was accompanied by large alteration in the fatty acid composition especially in seeds of transformants with PuFADX under the control of the USP promoter. Relative amounts of linolenic acid (18:2? 9Z,12Z) decreased from 75 % in untransformed seeds to 63 % while in contrast the amounts of oleic acid (18:1? 9Z) increased from 11 % in untransformed seeds to 18 % in transgenic seeds. In transgenic seeds with more than 10 % (w/w) of punicic acid the amounts of oleic acid even increased to 21 % reaching a 1.9-fold higher amount than in untransformed seeds, while the amount of linoleic acid decreased to 57 % which means 1.1-fold lower concentration compared to the seeds of untransformed plants. A similar but less dramatic effect, suppression of ? 12 -oleate desaturase, had already been described for plants transformed with other ? 12 -acyl-modifying enzymes including expression of a ? 12 -oleate hydroxylase (Broun et al., 1998a; Broun et al., 1998b) and a ? 12 -linoleate epoxygenase (Singh et al. 2001) in Arabidopsis seeds. The same large alterations in fatty acid composition were observed in somatic soybean embryos and in Arabidopsis seeds expressing ? 12 linoleate conjugases (Cahoon et al., 1999; Iwabuchi et al., 2003) , but interestingly not in somatic soybean embryos expressing a ? 9 -linoleate conjugase (Cahoon et al., 2001). Table 2 shows the exact numbers of analysed tobacco seeds from transformed individual plants. Individual plants are grouped in one of five classes according to the amount of punicic acid detected in the seeds. While only 11 % of plants transformed with PuFADX under the control of the USP promoter produced no detectable amounts of punicic acid, 40 % or 58 %, respectively, of plants expressing PuFADX under the control of LeB4 or CaMV 35S promoter, respectively, produced no punicic acid. None of the plants with LeB4 or CaMV 35S promoter produced more than 1 % punicic acid while 83 % of plants transformed with PuFADX under the control of the USP promoter produced more than 1 % punicic acid, 6.4 % even more than 8 %. Table 2. Amount of punicic acid in tobacco seeds from transgenic plants expressing PuFADX. Punicic acid content of transgenic tobacco seeds was determined and individual plants were grouped into 5 different classes. Number and percentage of plants of each class are listed. 35S, CaMV 35S promoter; LeB4, V. faba leguminB4 promoter; USP, V. faba unknown seed protein promoter. 35S LeB4 USP class punicic acid number of % of analysed number of % of analysed number of % of analysed amount* plants plants plants plants plants plants I 8 – 10 0 0 0 0 3 6,4 II 4 – 7.9 0 0 0 0 7 14,9 III 1 – 3.9 0 0 0 0 29 61,7 IV 0.1 – 0.9 5 42 12 60 3 6,4 V 0 7 58 8 40 5 10,6 *Values are presented as relative ratio in % (w/w) of total fatty acids of tobacco seeds
45
Segregation analysis of plant lines producing more than 4 % punicic acid was performed to obtain homozygous plant lines with only one insertion of the PuFADX cDNA and to analyze the production of high amounts of conjugated fatty acid in the seeds is stable over several generations. Only one of 6 analysed plant lines showed a segregation ratio of 3 : 1 selected by drug resistance. T1 seeds of this plant line contained 4% (w/w) of punicic acid. T2 and T3 seeds of the homozygous plant lines, determined by one further segregation analysis with plants derived from the T2 seeds of the plant line harbouring one insert, produced 9.9 + 0.4 % of punicic acid. 3.2 Production of calendic acid in tobacco seeds To compare the expression of a ? 12 -conjugase and ? 9 -conjugase in tobacco seeds we also expressed the full length cDNA encoding CoFADX1 (Cahoon et al., 2001; Qiu et al., 2001) in N. tabacum cv. Samsun NN. In addition to the comparison of the different conjugases we investigated the production of conjugated linoleic acid (CLA) in tobacco seeds. Qui et al. (Qiu et al., 2001) reported the accumulation of CLA (18:2 ? 8,10) in yeast cells expressing CoFac2 (termed CoFADX1 by Cahoon et al., 2001) in amounts of 0.09 % of total fatty acids when no exogenously fatty acids were supplied. However, Cahoon et al. (2001) could not observe the production of CLA neither in yeast cells nor in somatic soybean embryos. In our expression studies of CoFADX1 in yeast we were also not able to detect any CLA isomer. Supplementation of yeast cells expressing CoFADX1 with linoleic acid resulted in the expected production of calendic acid of up to 0.9 % of total fatty acids. As the experiment with transgenic plants expressing PuFADX already showed that the production of conjugated fatty acids in leaves was below the detection limit, we only expressed CoFADX1 under the control of the seed specific promoter USP. After selection of transformants by drug resistance and PCR T1 tobacco seeds of individual transformed plants were harvested and FAMEs were analysed by GC. The analysis showed the appearance of a new peak in comparison to seeds from untransformed plants (Figure 2).
16:0
18:1∆9Z
A 18:2 ∆9Z,12Z
18:0
detector response
18:3 ∆9Z,12Z,15Z
16:0
B
18:1 ∆9Z 18:2 ∆9Z,12Z
18:0
18:3 ∆8E,10E,12Z 18:3 ∆9Z,12Z,15Z
8
10
12 14 retention time [min]
16
18
Figure 2. GC/FID analysis of FAMEs isolated from tobacco seeds. Transmethylation and analysis of esterified fatty acids were done as described in methods. A shows the chromatogram of FAMEs from untransformed tobacco seeds. B shows the GC/FID analysis of FAMEs from transgenic tobacco seeds expressing CoFADX under the control of the USP -promoter.
46
The newly formed fatty acid was identified as calendic acid by authentic standard. Calendic acid accumulated up to 0.2 % (w/w) of total fatty acids in transgenic tobacco seeds. T1 seeds from 51 individual transgenic tobacco plants were analysed. Calendic acid accumulated in 43 % of all analysed plants. Besides calendic acid no other newly produced fatty acids was detected. Alteration in fatty acid composition was not observed. But in tobacco seeds expressing PuFADX and producing punicic acid in comparable amounts there was also no alteration in fatty acid composition detected. So we could not directly compare the effect of ? 12 -conjugases and ? 9 -conjugases on ? 12 -oleate desaturases and fatty acid metabolism. In summary, we were able to establish a stable expression of a conjugase in tobacco which led to the accumulation of about 10 % (w/v) of total fatty acid of CLNA in seeds. As conjugated fatty acids become more and more interesting not only for industrial applications but also in therapeutic medical applications and as nutraceuticals transgenic approaches to accumula te these fatty acids in common oil crops are under way. CLNAs are already used for industrial applications, for example tung oil – a seed oil with up to 80 % of a-eleostearic acid (18:3? 9Z,11E,13E) – is commonly used in quick drying enamels and varnishes (Sonntag, 1979). For CLA, which is synthesized by rumen bacteria therapeutical medical applications have been studied and a large number of beneficial effects of dietary supplementation with these conjugated fatty acids have been reported. CLA has been shown to be cytotoxic to human and mice tumour cells (Durgam and Fernandes, 1997; Ip et al., 1994). In recent studies with CLNAs like punicic or a-eleostearic acid it has been shown that these fatty acids even are more cytotoxic to tumour cells than is CLA and may be thus useful in anticancer applications (Igarashi and Miyazawa, 2000). Studies with CLA also showed that this fatty acids influence body fat and energy metabolism, reducing body fat mass in mice and humans (Blankson et al., 2000; DeLany et al., 1999; West et al., 1998). In addition it has been suggested that CLNAs are also involved in modulation of body fat and triacylglycerol metabolism but in a different way than CLA (Koba et al., 2002). 4. Acknowledgements The authors are indebted to S. Knüpffer, C. Pernstich and M. Perschel for their technical assistance and the BASF Plant Science is acknowledged for their financial support.
5. References Bäumlein, H., Boerjan, W., Nagy, I., Panitz, R., Inze, D., and Wobus, U. (1991). Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol Gen Genet 225, 121-128. Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992). New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20, 1195-1197. Belury, M. A. (2002). Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu Rev Nutr 22, 505531. Blankson, H., Stakkestad, J. A., Fagertun, H., Thom, E., Wadstein, J., and Gudmundsen, O. (2000). Conjugated linoleic acid reduces body fat mass in overweight and obese humans. J Nutr 130, 2943-2948. Broun, P., Boddupalli, S., and Somerville, C. (1998a). A bifunctional oleate 12-hydroxylase: desaturase from Lesquerella fendleri. Plant J 13, 201-210. Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998b). Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids. Science 282, 1315-1317. Cahoon, E. B., Carlson, T. J., Ripp, K. G., Schweiger, B. J., Cook, G. A., Hall, S. E., and Kinney, A. J. (1999). Biosynthetic origin of conjugated double bonds: Production of fatty acid components of high -value drying oils in transgenic soybean embryos. Proc Natl Acad Sci USA 96, 12935-12940. Cahoon, E. B., Ripp, K. G., Hall, S. E., and Kinney, A. J. (2001). Formation of conjugated ∆8, ∆10 double bonds by ∆12 -oleic acid desaturase related enzymes. Biosynthetic origin of calendic acid. J Biol Chem 276, 2083-2087. DeLany, J. P., Blohm, F., Truett, A. A., Scimeca, J. A., and West, D. B. (1999). Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am J Physiol 276, R1172-1179. Durgam, V. R., and Fernandes, G. (1997). The growth inhibitory effect of conjugated linoleic acid on MCF-7 cells is related to estrogen response system. Cancer Lett 116, 121-130. Fritsche, K., Hornung, E., Peitzsch, N., Renz, A., and Feussner, I. (1999). Isolation and characterization of a calendic acid producing (8,11)linoleoyl desaturase. FEBS Lett 462, 249-253. Hornung, E., Pernstich, C., and Feussner, I. (2002). Formation of conjugated ∆11 ∆13 -double bonds by ∆12-linoleic acid (1,4)-acyl-lipiddesaturase in pomegranate seeds. Eur J Biochem 269, 4852-4859. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231. Igarashi, M., and Miyazawa, T. (2000). Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells. Cancer Lett 148, 173-179.
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Ip, C., Singh, M., Thompson, H. J., and Scimeca, J. A. (1994). Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res 54, 1212-1215. Iwabuchi, M., Kohno-Murase, J., and Imamura, J. (2003). ∆12-Oleate desaturase-related enzymes associated with formation of conjugated trans-∆11, cis-∆13 double bonds. J Biol Chem 278, 4603-4610. Koba, K., Akahoshi, A., Yamasaki, M., Tanaka, K., Yamada, K., Iwata, T., Kamegai, T., Tsutsumi, K., and Sugano, M. (2002). Dietary conjugated linolenic acid in relation to CLA differently modifies body fat mass and serum and liver lipid levels in rats. Lipids 37, 343-350. Lee, K. N., Kritchevsky, D., and Pariza, M. W. (1994). Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108, 19-25. Pickardt, T., Ziervogel, B., Schade, V., Ohl, L., Bäumlein, H., and Meixner, M. (1998). Developmental-regulation and Tissue-specific Expression of Two Different Seed Promoter GUS-fusions in Transgenic Lines of Vicia narbonensis. J Plant Physiol 152, 621-329. Qiu, X., Reed, D. W., Hong, H., MacKenzie, S. L., and Covello, P. S. (2001). Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase. Plant Physiol 125, 847-855. Sonntag, N. O. V. (1979). Separation of fatty acids. In Fatty Acids, E. H. Pryde, ed. (Champaign, The American Oil Chemists´ Society), pp. 125-156. Tulloch, A. P. (1982). 13 C Nuclear magnetic resonance spectroscopic analysis of seed oils containing conjugated unsaturated acids. Lipids 17, 544-550. West, D. B., Delany, J. P., Camet, P. M., Blohm, F., Truett, A. A., and Scimeca, J. (1998). Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. Am J Physiol 275, R667-672.
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BIOSYNTHESIS OF MEMBRANE LIPIDS IN CHLAMYDOMONAS REINHARDTII MOORE, Thomas S., MORONEY, James V., YANG, Wenyu Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 U.S.A.
1. Introduction The overall glycerolipid composition of Chlamydomonas reinhardtii membranes is presented in Table I. This composition is characterized by high concentrations of a lipid not commonly found in higher eukaryotes, diacylglyceryltrimethylhomoserine (DGTS); the most abundant phospholipid is phosphatidylglycerol, and phosphatidylethanolamine is second. In addition, C. reinhardtii membrane lipid composition is characterized by a complete lack of phosphatidylserine (PS) and phosphatidylcholine (PC), and the diacylglyceryltrimethylhomoserine (DGTS) substitutes for the missing PC (Giroud, et al, 1988). This provides a unique opportunity to study biosynthesis of phosphatidylethanolamine (PE) and DGTS in the absence of both PS and PC synthesis. Table I. Glycerolipid composition of Chlamydomonas reinhardtii and its chloroplasts as dry weight (calculated from Beck and Levine, 1977; Janero and Barrnett, 1981). LIPID
RELATIVE ABUNDANCE Whole Cell Chloroplast mol % mol % Diacylglyceryltrimethylhomoserine (DGTS) 16 Phosphatidylethanolamine (PE) 5 Phosphatidylinositol (PI) 2 Phosphatidylglycerol (PG) 10 111 Monogalactosyldiacylglycerol (MGDG) 47 63 Digalactosyldiacylglycerol (DGDG) 16 21 Sulfoquinovosyldiacylglycerol (SQDG) 7 5 1 Assumes all phospholipid of the chloroplasts is PtdGro.
2. Diacylglyceryltrimethylhomoserine We previously reported on investigations related to the biosynthesis of DGTS (Moore et al, 2001) by C. reinhardtii. In summary, we have provided evidence that the homoserine moiety of the headgroup is derived from S-adenosyl-L-methionine (SAM) through the pathway: SAM + lipid precursor → Diacylglycerylhomoserine + Adenosyl –S-CH3 Diacylglycerylhomoserine + 3 SAM → Diacylglyceryltrimethylhomoserine + 3 S-Adenosylhomocysteine This activity was localized in the microsomal fraction of these cells. We also found that phosphatidylinositol biosynthesis was localized to the same fraction and was absent from plastids. We were able to measure both the phosphatidyltransferase and exchange reaction in these organisms (Blouin, et al, 2003). 2. Phosphatidylethanolamine biosynthesis We have concentrated our recent efforts on the final two steps of biosynthesis of PE. As mentioned above, although PE comprises only about 5 % of the membrane lipids of C. reinhardtii, it is the second most abundant membrane lipid outside the plastid (Table 1). It appears to be synthesized exclusively by the pathway: Ethanolamine + ATP → Phosphoethanolamine CTP + phosphoethanolamine → CDPethanolamine + PP I CDPethanolamine + diacylglycerol → Phosphatidylethanolamine + CMP
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The first reaction is catalyzed by ethanolamine kinase [EC 2.7.1.82], and involves the phosphorylation of ethanolamine by ATP to produce phosphoethanolamine. This activity generally is cytosolic and is important for starting the synthesis of headgroups for phosphatidylethanolamine and phosphatidylcholine in numerous organisms (Prud’homme and Moore, 1993), but we have not examined this reaction in C. reinhardtii. The second reaction, catalyzed by CTP: phosphoethanolamine cytidylyltransferase (ECT; EC 2.7.7.14) has been proposed to be the regulatory enzyme in the CDP-ethanolamine pathway of PE biosynthesis, although this is primarily based on an analogy to the similar enzyme involved in PC synthesis. Direct evidence has been lacking for this role. This enzyme initially was reported as being soluble in animals and yeast, but more recent evidence indicates it might also be found in the rough endoplasmic reticulum (van Hellemond, et al, 1994; Vermeulen, 1993). In castor bean, this enzyme is found in both the ER and the mitochondria (Tang and Moore, 1997; Wang and Moore, 1991). The third reaction of the pathway is catalyzed by diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT; EC 2.7.8.1). This enzyme has been found to have variable degrees of specificity, as does the similar enzyme for PC synthesis. In some organisms this enzyme is more specific for CDPethanolamine than in others. Determination of the specificity of this enzyme is important for an overall understanding of the block in PC synthesis of C. reinhardtii. 2.1. Cytidylytransferase Cloned cDNA of the ECT protein from C. reinhardtii encodes a protein of 443 amino acid residues, which is longer than the cDNA for the same protein cloned from yeast, rat or human, the organisms for which we have most information. Specifically, the first 70 amino acid residues do not match the N-terminal portions of the cytidylyltransferases from other organisms; indeed, the first 75 amino acid residues collectively appear to include a subcellular targeting sequence, as predicted by ExPASy Proteomics tools from the ExPASy Molecular Biology Server, which we hypothesize to be a subcellular targeting signal to mitochondria. This hypothesis is supported by compartmentalization studies, in which ECT correlated much more with locations of fumarase, a mitochondrial marker enzyme, than with markers for other organelles or compartments. Predictions of structure show that the C. reinhardtii ECT N-terminus has a small, a-helical, hydrophobic region long enough to span the membrane lending further support for it being a subcellular targeting sequence. On the other hand, overall C. reinhardtii ECT contains 38% hydrophobic amino acids, which is comparable to that in other ECTs such as human (34%), rat (34%) and yeast (37%). A hydrophobicity profile shows that the overall sequence of the C. reinhardtii ECT is hydrophilic, with the exception of the N-terminus forms the apparent transmembrane domain (residues 15-40). In addition to this Chou-Fasman algorithm prediction of the N-terminal a-helix, there are two other regions of hydrophobic residues (position 95-120 and 330-370) in the N- and C-terminal portions of the protein. However, they are broken by stretches of hydrophilic residues and therefore are unlikely to form transmembrane domains. In other respects, the ECT protein in C. reinhardtii is similar to the ECTs from these other organisms. The gene for cytidylyltransferase has a 1329 bp open reading frame (ORF) that starts from base 193 with an ATG start codon and ends at base 1521, followed by the stop codon TGA. The 5' UTR is 192 bp and the 3' UTR is 498 bp. The ATGat base 193 is very likely the start codon for translation initiation, since the deduced amino acid sequence indicates no other methionine residue before the motif HXGH, a sequence believed to be in the first catalytic domain. As mentioned above, the encoded protein from C. reinhardtii would be longer than those of previously reported organisms, but the amino acid residues after position 75 begin to align significantly with the other ECTs. The complete ORF codes for a protein which has a molecular mass calculated to be 49.3 kDa. This is similar to the molecular mass of other ECTs in rat (45.2 kDa), human (43.8 kDa) and yeast (36.9 kDa). The predicted C. reinhardtii ECT protein is 41% identical to human and rat ECTs, and 30% to yeast, which is greater than that of yeast ECT to human or rat ECT proteins, but much lower that that of human to rat ECT protein (88%). The 443 residue C. reinhardtii ECT, as indicated above, is somewhat longer than those of human (389 residues), rat (404 residues) and yeast (323 residues). Exclusion of the 75 residue N-terminal region from the comparisons leads to the identity of the C. reinhardtii ECT protein increasing to 50% for human or rat ECT, and 40% for yeast. The similarity between C. reinhardtii, human, rat and yeast ECTs is present across the entire sequences of the proteins, but there are greater similarities found in the N- and C-terminal regions (residues 71-207, residues 259-426) than in the middle (residues 208-278). When the N- and C-terminal halves within the same ECT protein were aligned for each organism, a significant identity of 33% was found in C. reinhardtii, 30% in human and 32% in rat, but only 25% in yeast. Both halves of the internal repeat sequence of ECT in C. reinhardtii, human and rat contain the HXGH motif. However, the C-terminal half of yeast ECT has
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a single amino acid residue change in the HXGH motif, substituting an aspartate in place of a histidine. In addition, the C. reinhardtii has other conserved residues which are considered important for catalytic activity in the cytidylyltransferase family, such as the sequence RTXGVSTT (residues 93-100) and an aspartate residue two amino acid residues before the HXGH motif in the N-terminal half of the protein. In order to test the enzymatic activity of the ECT gene product, an in-frame fusion of ECT to MAL, which codes for maltose binding protein, was expressed in E. coli under control of a promoter inducible by IPTG. The expected overproduction of the fusion protein upon induction was observed. CDP-ethanolamine was produced from phosphoethanolamine and CTP in a reaction catalyzed by both the cellular extract and purified fusion protein from the overexpressed cells. No CDP-ethanolamine was produced by an extract from cells that carried only the vector pMAL-c2X. In addition, we tested the enzyme activity of the fusion protein under different conditions and found the activity to be dependent on the presence of CTP, phosphoethanolamine and Mg2+. These results clearly indicate that the cDNA described here codes for the structural gene for C. reinhardtii ECT. The ECT activity may be regulated during the cell cycle and reflagellation. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for posttranslational regulation. 2.2. Ethanolaminephosphotransferase A cDNA coding for this enzyme was obtained by PCR-based cloning from a C. reinhardtii cDNA library. A yeast mutant deficient in both cholinephosphotransferase and EPT was complemented by the C. reinhardtii EPT gene coding for EPT. This enzyme was capable of catalyzing the final steps of both PE and PC biosynthesis (see below), supporting the argument that the absence of synthesis of PC in C. reinhardtii stems from a lack of ECT, not EPT. A signature sequence, DGKQARRTGTSSPLGQLFDHGCD, was found in the predicted amino acid sequence of cEPT, and this aligns well with a consensus sequence associated with CDP-alcohol phosphatidyltransferases, D-G-x(2)-A-R-x(8)-G-x(3)-D-x(3)-D. Proteins in this family catalyze phospholipid biosynthesis reactions involving the displacement of CMP from a CDP-alcohol by a second alcohol, with formation of a phosphodiester bond and concomitant breaking of a phosphoride anhydride bond (Kodaki et al., 1987; Hjelmstad and Bell, 1991). Similar enzymes include diacylglycerol cholinephosphotransferase (EC 2.7.8.2), phosphatidylglycerophosphate synthase (EC 2.7.8.5), phosphatidylserine synthase (EC 2.7.8.8), and phosphatidylinositol synthase (EC 2.7.8.11). These enzymes all are about the same size, being from 200 to 400 amino acid residues in length. One conserved region which contains three aspartic acid residues is located in the N-terminal region of the sequences. A second conserved sequence, found in C. reinhardtii EPT at residues 76179, is homologous to similar regions in other AAPTases. Similar conserved regions among the AAPTases have been proposed to contribute to the formation of the active site of the enzymes in the family of CDP-alcohol phosphatidyltransferases (Kodaki et al., 1987; Hjelmstad and Bell, 1991). This region is primarily hydrophilic but also has one or two amphipathic structures capable of forming a-helices that could interact with the membrane on the cytoplasmic side of the ER. Overall, these analyses indicate that C. reinhardtii EPT clearly is a member of the CDP-alcohol phosphatidyltransferase family. Protein structure predictions indicate the occurrence of seven apparent transmembrane domains in cEPT polypeptide located at: TM1,45-67; TM2,176-198; TM3,218-240; TM4,261-280; TM5,290-309; TM6,322-341; TM7,351-373. These domains are found in very similar positions among the protein sequences of all three polypeptides. These results indicate a high degree of conservation in the membrane associated topography of the aminoalcoholphosphotransferases of these organisms. Based on these considerations and previous protease experiments (Vance et al., 1977; Coleman and Bell, 1980; Bell et al., 1981), the catalytic site is predicted to be located on the cytoplasmic side of the ER between TM1 and TM2, as initially proposed by Hjelmstad and Bell (1991). An intracellular compartmentalization prediction indicates a 67% probability of EPT being associated with the endoplasmic reticulum, and 33% for the plasma membrane; this was obtained using the k-nearest neighbor (k-NN) algorithm for assessing the probability of being located at each of the candidate sites (Horton and Nakai, 1997). In addition, a KKXX-like motif representative of a type of ER membrane retention signal, TPKR, was found in the C-terminus of the protein (position 379-382). Moreover, the membrane topology of the
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EPT protein was predicted to have the N-terminus of the protein located inside the ER membrane. These results all support the C. reinhardtii EPT as being an integral membrane protein of the endoplasmic reticulum. The two enzymes which catalyze the final steps of PC and PE biosynthesis, CPT and EPT, respectively, seem specific in substrate utilization and product synthesized in most animal systems investigated (Bell and Coleman, 1980; Percy et al., 1984), but it is not clear in higher plants whether either or both of these enzymes could utilize both CDP-choline and CDP-ethanolamine as substrates to synthesize either of the products, PC and PE. A number of studies have indicated such a scenario is possible (Macher and Mudd, 1974; Lord, 1975; Sparace et al., 1981; Justin et al., 1985; Dewey et al., 1994; Goode and Dewey, 1999). In addition, a human choline/ethanolaminephosphotransferase recently was found to be capable of synthesizing both choline- and ethanolamine-containing phospholipids (Henneberry and McMaster, 1999). In order to test the specificity of the C. reinhardtii enzyme, the yeast mutant strain RK-ec, deficient in both EPT1 and CPT1 activities, was complemented in colonies from cells transformed with a pDB-EPT construct, which was found capable of synthesizing PC. An RK-ec strain without transformation and the RK-ec transformed with empty vector pDB20 had no detectable cholinephosphotransferase activity. Cholinephosphotransferase activity was clearly observed, as a positive control, using a wild type strain of yeast, KT1115. TLC assays demonstrated that more than 95% of the radiolabeled product co-migrated with a PC standard. We set out to directly test for dual substrate utilization by examining the effects of unlabeled CDPethanolamine and CDP-choline on the incorporation of radiolabeled CDP-choline into PC by microsomal membranes purified from yeast RK-ec transformed with pDB-EPT. Microsomal membranes prepared from the yeast strains RK1115, RK-ec transformed with pDB-EPT, and C. reinhardtii strain cc406 cw15 (mt -), all were assayed for PC biosynthesis using [14 C]-CDP-choline; reduction of CDP-choline incorporation was measured following addition of unlabeled, competitive substrates. Increasing concentrations of unlabeled substrate, either CDP-choline or CDP-ethanolamine, strongly reduced the formation of radiolabeled PC in microsomes from yeast KT1115, RK-ec transformed with pDB-EPT and C. reinhardtii. The results with C. reinhardtii microsomal membranes were were very similar to those using microsomal membranes from the yeast strain RKec expressing the C. reinhardtii EPT gene. By contrast, in the yeast strain KT1115 a reduction in formation of radiolabeled PC by CDP-ethanolamine was much less than that resulting from addition of CDP-choline, indicating a stronger affinity for CDP-choline. As a control, microsomal membranes from yeast RK-ec and RKec which had been transformed with pDB-20 showed no detectable cholinephosphotransferase activity. Collectively, these data support the hypothesis that the C. reinhardtii EPT is more similar to plant phosphotransferases in substrate preferences, which are less selective, than it is to the enzyme from other sources, particularly yeast (Sparace et al., 1981; Dewey et al., 1994; Goode and Dewey, 1999; Hjelmstad and Bell, 1991). This is particularly intriguing since the sequence is more similar to human and mouns that to putative plant sequences for EPT. Tests of the effects of increasing concentrations of the water-soluble product of the EPT-catalyzed reaction, CMP, on the cholinephosphotransferase activity of microsomes from yeast KT1115, RK-ec transformed with pDB-EPT and C. reinhardtii strain cc406 cw15 (mt -) were conducted. Increasing CMP decreased the cholinephosphotransferase activity of microsomal membranes from yeast RK-ec transformed with pDB-EPT expressing C. reinhardtii EPTI, similar to the result with microsomal membranes isolated directly from C. reinhardtii cells. On the other hand, yeast KT1115 microsomes responded much less to increasing concentrations of CMP than did yeast RK-ec transformed with pDB-EPT or C. reinhardtii EPT. These results show that the algal enzyme is different from that of the yeast, and that the C. reinhardtii EPT was not altered in the yeast cells with respect to its s ubstrate preference and sensitivity to CMP. In summary, the C. reinhardtii EPT is clearly different from the yeast EPT1 or CPT1. Of the two yeast enzymes, the C. reinhardtii EPT appears more closely related to the yeast EPT1, which also has both ethanolaminephosphotransferase and cholinephosphotransferase activities. However, a slight difference between the two enzymes exists, in that the EPT1 of CPT1-deficient yeast demonstrated a higher specificity for CDP-ethanolamine than for CDP-choline, while C. reinhardtii EPT demonstrated about equal specificities. Since C. reinhardtii does not contain PC, this very high native cholinephosphotransferase activity of the EPT gene product may provide interesting information for studying the evolution of C. reinhardtii and other algae, particularly with respect to substitution of diacylglyceryltrimethylhomoserine for PC in many species.
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3. Discussion and Conclusions The current investigation was initiated because C. reinhardtii synthesizes no PC, but does produce PE. This simplifies all interpretations and allows a more direct observation of EPT itself. The search of the available C. reinhardtii genomic database indicates that no genes coding for the three enzymes in the CDP-choline pathway of PC biosynthesis, choline kinase, CTP: phosphocholine cytidylyltransferase and cholinephosphotransferase, are found in the genome of C. reinhardtii. However, the absence of PC synthesis may primarily be due to the absence of a single enzyme, CTP: phosphocholine cytidylyltransferase, which catalyzes the second step of the pathway, since the final step could be catalyzed by EPT. Evidence with other systems indicates that both the first and last steps of both the CDP-choline and the CDP-ethanolamine pathways could be catalyzed by the same enzyme that can utilize substrates from either pathway, thus supporting this hypothesis (Ishidate, 1997; Lord, 1975; Goode and Dewey, 1999). Our overall view of this compartmentalization of these activities in Chlamydomonas reinhardtii is presented in Figure 1. According to our data, the ECT enzyme of the mitochondria would produce the CDPethanolamine, which then would diffuse to the ER where synthesis of PE would be catalyzed by EPT. The significance of this is not clear, but it may reflect a priority for ATP produced in the mitochondria for growth and maintenance of membranes in a phosphorus-poor environment in which C. reinhardtii sometimes may be found.
Figure 1. A model of possible intracellular interactions among enzymes of phosphatidylethanolamine biosynthesis in Chlamydomonas reinhardtii.
5. Acknowledgements This research was supported by National Science Foundation, USA, grants MCB-9603626 to TSM, and IBN-0212093 to JVM. 6. References Beck JC, Levine RP (1977) Synthesis of chloroplast membrane lipids and chlorophyll in synchronous cultures of Chlamydomonas reinhardtii. Biochim Biophys Acta 489: 360-369 Bell RM, Coleman RA (1980) Enzymes of glycerolipid synthesis in eukaryotes. Annu Rev Biochem 49: 459-487 Bell RM, Ballas LM, Coleman RA (1981) Lipid topogenesis. J Lipid Res 22: 391-403 Blouin A, Lavezzi T, Moore TS (2003) Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP diacylglycerol: myo-inositol 3 phosphatidyltransferase. Plant Physiol Biochem 41: 11-16 Chapman KD (2000) Emerging physiological roles for N-acylphosphatidylethanolamine metabolism in plants: signal transduction and membrane protection. Chem Phys Lipids 2000 108: 221-229
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Coleman R, Bell RM (1980) Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles. J Cell Biol 76: 245-253 Dewey RE, Wilson RF, Novitzky WP, Goode JH (1994) The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast. Plant Cell 6: 1495-1507 Giroud C, Gerber A, Eichenberger W (1988) Lipids of Chlamydomonas reinhardtii. Analysis of molecular species and intacellular site(s) of biosynthesis. Plant Cell Physiol 29: 587-595 Goode JH, Dewey RE (1999) Characterization of aminoalcoholphosphotransferases from Arabidopsis thaliana and soybean. Plant Physiol Biochem 37: 445-457 Henneberry AL, McMaster CR (1999) Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine. Biochem J 339: 291-298 Hjelmstad RH, Bell RM (1991) sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Nucleotide sequence of the EPT1 gene and comparison of the CPT1 and EPT1 gene products. J Biol Chem 266: 5094-5103 Horton P, Nakai K (1997) Better prediction of protein cellular localization sites with the k nearest neighbors classifier. Proc Int Conf Intell Syst Mol Biol 5: 147-152 Ishidate K (1997) Choline/ethanolamine kinase from mammalian tissues. Biochim. Biophys. Acta 1348: 70-78 Janero DR, Barrnett R (1981) Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+: Cell cycle variations in the synthesis and assembly of polar glycerolipid. J Cell Biol 91: 126-134 Justin AM, Demandre C, Tremoliers A, Mazliak P (1985) No discrimination by choline- and ethanolaminephosphotransferases from potato tuber microsomes in molecular species of endogenous diacylglycerols. Biochim Biophys Acta 836: 1-7 Kodaki T, Yamashita S, Nikawa JI (1987) Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae. J Biol Chem 262: 4876- 4881 Lord JM (1975) Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor bean endosperm. Biochem J 151: 451-453 Macher BA, Mudd JB (1974) Biosynthesis of phosphatidylethanolamine are synthesized by enzyme preparations from plant tissues. Plant Physiol 53: 171-175 Moore TS, Du ZR, Chen,Z (2001) Membrane lipid biosynthesis in Chlamydomonas reinhardtii. In vitro biosynthesis of diacylglyceryltrimethylhomoserine. Plant Physiol. 125: 423-429 Percy AK, Carson MA, Moore JF, Waechter CJ (1984) Control of phosphatidylethanolamine metabolism in yeast: diacylglycerol ethanolaminephosphotransferase and diacylglycerol cholinephosphotransferase are separate enzymes. Arch Biochem Biophys 230: 69-81 Prud’homme MP, Moore TS (1992) Phosphatidylcholine synthesis in castor bean endosperm: Free base as intermediates. Plant Physiol 100: 1527-1535 Sparace SA, Wagner LK, Moore TS (1981) Phosphatidylethanolamine synthesis in castor bean endosperm. Plant Physiol 67: 922-925 Tang, F-Q. and Moore, T.S. (1997) Enzymes of the primary phosphatidylethanolamine biosynthesis pathway in postgermination cator bean endosperm. Plant Physiol. 115, 1589-1597 Vance DE, Choy PC, Farren SB, Lim P, Schneider W (1977) Asymmetry of phospholipids biosynthesis. Nature (London) 270: 268-269
van Hellemond JJ, Slot JW, Jeelen MJ, van Golde LM, Vermeulen PS (1994) Ultrastructural localization of CTP:phosphoethanolamine cytidylyltransferase in rat liver. J Biol Chem 269, 15415-15418 Vermeulen PS, Tijburg LB, Geelen MJ, van Golde LM (1993) Immunological characterization, lipid dependence, and subcellular localization of CTP: phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP: phosphocholine cytidylyltransferase. J Biol Chem 268: 74587464 Wang, X-M. and Moore, T.S. (1991) Phosphatidylethanolamine synthesis by castor bean endosperm. Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase. J. Biol. Chem. 266: 19981-19987
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THE ROLES OF LIPID BODIES AND LIPID-BODY PROTEINS IN THE ASSEMBLY AND TRAFFICKING OF LIPIDS IN PLANT CELLS DENIS J. MURPHY
Biotechnology Unit, School of Applied Sciences, University of Glamorgan, CF37 1DL, United Kingdom
Introduction In this paper, the newly emerging roles of lipid assemblies and their associated proteins in non-storage processes in cells will be examined. An expanded version of this review will appear in the forthcoming treatise on plant lipids (1). Lipid-associated proteins are a relatively newly discovered class of proteins that are specifically associated with macromolecular lipid assemblies, other than bilayer membranes, in the cells of a wide range of organisms from eubacteria to mammals. The most common type of non-bilayer lipid assembly in cells is a spherical organelle known as a lipid body or lipid droplet. These organelles, which are typically 0.5 – 2µm in diameter, are made up of a neutral lipid core surrounded by an annulus made up of a phospholipid monolayer and a specific population of proteins. Lipid bodies (often called oil bodies in plants) have tended to be regarded as mere storage sites for carbon and energy. However, recent progress in elucidating the functions of intra and extra-cellular lipid bodies, and especially their associated proteins, have revealed hitherto unsuspected dynamic roles for these organelles in processes, such as lipid import/export and in the subcellular trafficking of both lipids and proteins.
Lipid-associated proteins have been particularly well-characterised in plants, most notably in lipidstoring tissues like seeds and fruits where they are exemplified by the ole osins. Oleosins are tightly associated with storage lipid bodies in many, but not all, oil-accumulating plant tissues. Although oleosin genes appear to be ubiquitous components of the genomes of true plants, i.e. the Plantae, the levels of oleosin protein accumulation can vary enormously between different species. Proteins similar to seed oleosins may also be present within the cytosol of in the cells of certain types of pollen grain that store lipid, for example entomophilous pollen like that of many Brassicaceae, including Arabidopsis thaliana (2). A separate class of oleosin-like proteins, or oleo-pollenins, has also been found in floral tissues, including the tapetum and on the external surfaces of pollen grains. So far reports of these proteins have been restricted to the Brassicaceae and it is not known whether similar proteins are found in other plants (3). Oleosins and oleosin-like proteins appear to be structural proteins with no discernible enzymatic motifs. During recent years, several additional classes of lipid-binding proteins have also been described in plants, including caleosins, steroleosins and protein kinases. Unlike oleosins, caleosins are not only associated with lipid bodies: they have also been found on ER membranes. Since caleosins have a single putative membrane-spanning domain, as well as calcium binding and protein kinase domains, they may have a role in signalling as well as in oil-body assembly and mobilisation. Steroleosins are of unknown function, but may be analogous to the sterol-binding proteins that are principal components of fungal lipid bodies. The plastidial lipid-associated proteins (PLAPs) were originally found in the specialised plastids, called chromoplasts, that are found in non-green pigmented tissues like coloured flower petals and fruits. Inside the chromoplasts, lipidic pigments, and their associated PLAPs such as carotenoids are stored in a range of differently shaped structures, from long, thin fibrils to classic globular droplets similar to storage oil bodies. Caleosins Caleosins were so named because they contain a conserved EF-hand, calcium-binding domain and because they were initially believed to be similar to oleosins in being uniquely associated with oil bodies in seeds (4,5). Similar proteins had been discovered earlier as gene products expressed in developing and germinating seeds of rice in response to abscisic acid or osmotic stress (6). Similar proteins or the genes encoding them have now been found in a wide range of plants from maize, rice and barley to soya and sesame. A caleosin-like sequence is also present in the genome of the single celled alga, Auxenochlorella protothecoides, which indicates that caleosins are probably ubiquitous in plants and algae. This is in contrast with oleosins, which are only found in true plants (so far they have not been reported in algae). Caleosin-like sequences are also present in at least two fungi,
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namely the lipid-accumulating fungus, Neurospora crassa, and the cereal pathogen, Magnaporthe grisea. Immunodetection assays indicated that the caleosin protein from rice, OsEFA27, was associated with a cell membrane fraction (6) although the caleosin from sesame was reported to be exclusively associated with lipid bodies (4). More recently, we have reported evidence from immunofluorescence microscopy that caleosins in rapeseed were localised both on lipid bodies and in specific domains of the ER that may be associated with vesicle trafficking (5). Very recent findings have emphasised that, although there are many intriguing similarities between caleosins and oleosins, there also important differences. In particular, analysis of caleosins from Arabidopsis thaliana shows that they are members of a large family of as many as 9 genes. Although one or more caleosin isoforms are tightly bound to lipid bodies during seed development, other isoforms accumulate in vegetative tissues and are probably integral membrane proteins of the ER (5,7,8). The key structural features of caleosins are an N-terminal region with a single Ca2+-binding EF hand domain, a central hydrophobic region able to form a single bilayer span, and a C-terminal region with several putative protein kinase phosphorylation sites, as shown in Figure 1. So far, only two caleosin proteins have been shown experimentally to bind calcium (6,9). Caleosins lack an N-terminal signal peptide, but do have a central, hydrophobic region of more than 30 residues with the potential to form a transmembrane helix and amphipathic ß-sheets (6). This hydrophobic domain is much shorter than the analogous 70-residue domain of oleosins. Like oleosins, caleosins contain a proline-rich region with the potential to form a “proline knot” motif of the type that appears to be so important in the lipid-body targeting of oleosins (10). In addition to the hydrophobic and proline-rich domains, caleosins also possess an immediately adjacent potential amphipathic α-helical domain, which may play a role in their binding both to bilayer membranes and to lipid bodies. These properties have been used as the basis of structural models of the different forms of caleosins (3). Highly resolving Tricine-based SDS-PAGE gels have enabled us to distinguish physically between the caleosin isoforms from Arabidopsis thaliana that bind respectively to lipid-bodies and the ER membrane (5,7). A 25kDa isoform is only synthesised during the mid-late stage of seed development and is exclusively located on the surfaces of lipid-bodies. This 25kDa isoform persists after seed desiccation and dispersal, along with oleosin, as a major lipid-associated protein and is then mobilised concurrently with the storage lipid bodies after seed germination. In contrast, the 27kDa caleosin isoform is ER-associated and appears to be present in many tissues including roots, stems, young leaves and seeds. Using sections of rapeseed root tip cells, immunoblotting and immunolocalisation studies revealed that caleosin co-localised with the ER marker BiP and also with membranes labelled for α-TIP, a marker for protein storage vacuoles (5). Parallel experiments indicated that immunodetectable oleosin is expressed in rapeseed root tip cells, and that caleosin is associated with it on what appeared to be lipid bodies. The presence of lipid bodies has been reported in root tips or root caps of rice, pea and maize and garden cress and these data indicate that root lipid bodies contain two proteins previously believed to be specific for seed lipid bodies, i.e., caleosin and oleosin. It has also been reported that lipid bodies in root tip cells from garden cress concentrate calcium (11), which would be consistent with the presence of caleosin on their surfaces. From the known primary structures of caleosins and the presence of conserved functional motifs, like EF hands and kinase domains, one can speculate about their possible functions in plants. For example, the Ca2+- binding status of caleosins, and perhaps their phosphorylation status, could well modulate aspects of their function. Caleosins may be involved in processes such as membrane and lipid-body fusion. Ca2+-mediated fusion has been shown to be involved in the maturation of microlipid bodies released from the ER to produce the large cytosolic lipid bodies found in milk secreting cells of mammary glands (3). Likewise, in seeds and other storage-lipid accumulating plant tissues, nascent lipid-bodies are probably released as small droplets from the ER and then undergo several rounds of fusion to produce the mature 0.4-2µm diameter lipid bodies characteristic of such tissues. We have also observed that the lipid-body caleosins persisted throughout seed desiccation, dormancy and for at least the first six days of post-germinative development. It is likely that lipid bodies need to dock with glyoxysomes to facilitate the concerted lipolysis, fatty acid oxidation and gluconeogenesis that occur
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during storage lipid mobilisation. Once again, caleosins may play a role in this lipid trafficking process. The microsomal caleosins found in very young embryos and in other plant tissues, such as roots and leaves, may be involved in other membrane-fission and/or fusion events relating to trafficking between the ER and storage or transport vesicles. The association of caleosins with either ER membranes or lipid bodies may be regulated by their binding of Ca2+ in a similar manner to the Ca2+-mediated association of lipocortin-1 with plasma membranes in human cell lines (12). The emerging dynamic role of lipid bodies in the cellular metabolism of most organisms requires a mechanism for trafficking of components between the lipid bodies and other organelles such as the ER, glyoxysomes and plasma membrane. This will involve protein mediators to regulate appropriate targeting of fusion/fission of the lipid bodies. The role, if any, of caleosins in such processes in plant (and perhaps in fungi) promises to be a fascinating topic for future investigations. Plastid lipid-associated proteins (PLAPs) These proteins are localised exclusively in plastids and, like caleosins but unlike oleosins, they are not restricted to the Plantae but are also found in unicellular photosynthetic eukaryotes (algae) and homologues have even been found in cyanobacteria. Originally regarded as having a storage/stabilisation role for lipidic pigments in chromoplasts, PLAPs are now recognised as having several additional role in plastids. For example, PLAPs are implicated in thylakoid membrane assembly/turnover as well as participating in various stress responses. PLAPs were first described 1976 when it was observed that the pigment-containing fibrillar lipoprotein assemblies of nasturtium flower petal chromoplasts had a distinctive protein composition dominated by a polypeptide of about 30kDa (13). Similar lipid-associated proteins of 35-38kDa have also been reported from higher plant chromoplasts and triacylglycerol/carotenoid globules of the alga, Dunaliella bardawil. The algal protein was localised on the surface of plastoglobuli and, as with oleosin in seeds, its cleavage by trypsin led to coalescence of the globules, which suggested that the function of this PLAP might be to stabilise these plastid lipid bodies. In 1994, the gene encoding the 32kDa bell pepper protein was named fibrillin in view of the fibrillar nature of the chromoplast lipoprotein structures from which it was derived (14). However, it is now clear that the plant protein is associated, not only with fibrils and globules in various plastid types, but also with thylakoid membranes (15,16). Therefore, a more general term such as plastid lipid-associated protein (PLAP) is probably more appropriate (16). It is now clear that PLAPs belong to a large class of homologous proteins found throughout oxygenic photosynthetic organisms. The discovery of a PLAP homologue in the cyanobacterium, Synechocystis, indicates the probable ancient origin of this protein in the endosymbiotic precursors of plastids (17). In addition to forming the major protein component of triacylglycerol /carotenoid-rich fibrils and globules in chromoplasts, PLAPs are present in other plastid types such as elaioplasts and chloroplasts (17,18). The PLAPs of elaioplasts are located on globular lipid bodies that resemble the triacylglycerol /carotenoid globules of chromoplasts, except that their lipid components are mainly sterol esters and fatty aldhydes (16). By way of contrast, the PLAPs of chloroplasts are associated both with plastoglobuli and thylakoid membranes (15,18,19). Indeed, it has been reported that plastids from Brassica rapa can contain up to three distinct PLAP isoforms, each of which is associated with globules containing a different mixture of neutral lipids (20). While the presence of PLAPs on neutral lipid bodies, including fibrils and globules, can be rationalised as providing a stabilising surface structure (14), their apparently ubiquitous distribution in plant tissues and their association with thylakoid membranes is more difficult to explain. A possible clue has come from studies of the induction of PLAP gene expression in response to various environmental factors, including drought stress, wounding or application of exogenous ABA. Since one of the primary responses of plants to such stresses is often a re-arrangement of their photosynthetic membranes, it has been proposed that PLAP has a general role in the formation/disassembly/turnover of plastid membrane complexes (21). More recently, it was found that the PLAP homologue from potato was associated with photosystem II, which is one of the major multi-subunit pigment-protein complexes of thylakoid membranes (22). Antisense-mediated reduction 57
of PLAP accumulation in transgenic potato plants led to reduced photosynthetic efficiency and stunted growth, which reinforces the view that PLAPs play important role(s) in both plastid membranes and lipid bodies. It is likely that there are several classes of PLAP-like proteins in plants. Some of these may be expressed ubiquitously while other, like the ChrC protein of cucumber (23) may have a more restricted pattern of expression, e.g. in response to stress. Those PLAPs that are expressed ubiquitously in plants are of particular interest since they appear to associate strongly both with the monolayer surfaces of plastid lipid bodies and fibrils and with the bilayer membrane of the thylakoids. Analysis of the conserved regions of PLPs does not reveal any obvious homology with other plant lipid-body proteins, such as oleosins or caleosins, although there are some interesting motifs in PLAPs that may be significant in their lipid-binding properties. For example, in the middle of the protein there are two non-polar regions of 16 and 22 residues respectively, each of which is flanked by relatively polar regions, which could potentially form transbila yer or monolayer-associating domains. Further structural studies are required to elucidate the mechanism(s) of lipid binding, and hence the biological functions of PLAPs. Finally, like seed lipid bodies, it is now emerging that plastid lipid assemblies probably contain several minor protein components in addition to the dominant oleosin or PLAP classes (24). In view of their lower abundance, these minor proteins are less likely to have structural roles, but may well be involved in other aspects of lipid-body function or possibly in more generalised intra plastidial lipid trafficking. It is possible that some of these PLAPs may play a role in the conversion of thylakoid membranes into the triacylglycerol-rich globules found in senescing leaves (25) and follow ing exposure to a wide range of stresses, including ozone exposure, fungal infection, chilling, freezing and thawing (3).
Minor lipid-associated proteins in plants Oleosins tend to be very abundant when they occur on storage oil bodies and caleosins are moderately abundant, i.e. caleosin bands can be readily discerned on SDS-PAGE gels of many oil-body extracts. However, there are also several relatively minor lipid-associated polypeptides that appear to be enzymes rather than structural proteins. It is still not known whether these are specific oil-body proteins or mere contaminants. Potential candidates include enzymes involved in triacylglycerol biosynthesis and, for many years, there have been reports of the presence of such enzymes on lipid bodies. This is mirrored by the well-documented findings of both triacylglycerol and sterol ester biosynthetic enzymes on fungal lipid bodies (see below). The apparent presence of triacylglycerol biosynthetic enzymes on seed lipid bodies may actually be due to the existence of membranous appendages of the ER remaining attached to the lipid bodies following their budding off from the ER proper. This has been proposed from ultrastructural studies (26,27) and would explain why membrane bilayer proteins can be associated with a non-bilayer structure like an oil body. The membranous appendages may facilitate re-fusion of oil bodies with the ER for the further metabolism of oil-body triacylglycerol, e.g. by desaturases or transacylases, as has been reported during sunflower seed development (28,29). Similar lipid-body appendages have been described in animal cells (1,3). More recently, the apparently specific binding of a sterol-binding dehydrogenase to oil bodies has been reported in sesame (30). This protein, tentatively named steroleosin, is similar in sequence to a protein encoded by a family of 8 genes in Arabidopsis. It is not known whether all steroleosin-like proteins in plants are associated with oil bodies or whether, like caleosins, some isoforms associate instead with other cellular components. Although steroleosins are relatively minor components of oil bodies and are not yet confirmed as being ubiquitous in plants, their discovery is interesting in view of the finding that another sterol-binding protein, a sterol ∆24-methyltransferase, is the major protein associated with lipid bodies in yeast (31). The importance of this class of enzyme in plants is shown by reports that sterol ∆24-methyltransferases in tobacco and rapeseed control the flux of carbon into sterols in seeds (32) and modulate growth in Arabidopsis (33). Another potentially relevant finding is the recent report of the presence of a sterol dehydrogenase on the surfaces of mammalian lipid bodies, with the implication that these organelles might play important roles in sterol metabolism and other aspects of lipid transport and membrane trafficking (34).
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A further putative oil-body protein recently described from developing sesame seeds is a calciumdependent protein kinase of 55kDa (35). This oil-body-associated polypeptide was capable of calcium-dependent autophosphorylation in vitro and cross-reacted with an antibody to a soybean protein kinase. The detection of similar proteins in oil bodies of other plant species suggests that these lipid-associated kinases may be ubiquitous in oilseeds. It can be speculated that these kinases, which are specifically active during seed development, may be involved in oil-body ontogeny. A possible substrate would be the relatively abundant lipid-associated isoforms of caleosin that are also present in developing seeds and which possess several conserved phosphorylation sites. In some species, seed oil bodies have been found to contain proteins that are associated with the mobilisation of the storage triacylglycerol that occurs after germination. An example is the lipoxygenase that is reportedly associated with oil bodies in cucumber seeds (36). Using GST-fusion constructs, it was shown that this protein can be targeted both to oil bodies and to liposomes and that the targeting required the presence of an N-terminal beta-barrel domain (37). Lipoxygenases are only active with polyunsaturated acyl substrates but many seeds do not store such fatty acids, and it is therefore unlikely that these proteins are present on storage oil bodies in all oilseeds. However, another class of cucumber oil-body protein described by the same group could well be ubiquitous in plants. This is a patatin-like protein that has phospholipase A2 activity that was transiently expressed and associated with oil bodies coincidentally with lipid mobilisation (37). Although the authors posit a role for this protein in storage lipid mobilisation, it could also have a signalling function. Phospholipases A2 have been shown to have such roles in both plants and animals (38). Interestingly, a similar patatin-like protein was recently reported in the proteome of lipid bodies from human CHO K2 cells (39). Patatins are the major family of storage proteins in potato tubers and, like some seed storage proteins, they appear to be derived from a group of enzymes that had esterase activities. If this is the case, then it may be misleading to refer to the motif common to the plant and animal lipid-body proteins as a patatin domain. Rather, these are all proteins with esterase-like domains, some of which have secondarily lost their enzymatic activity and become storage proteins in some higher plants. During the past few years, it has become increasingly evident that lipid bodies in many cells may be far more dynamic that was previously assumed according to the stereotypical view that these lipids were simply rather inert carbon stores. Our changing view of the nature and function of these hitherto misunderstood organelles has emerged largely thanks to recent progress in the characterisation of the various classes of lipid-associated proteins that have been described here. Ironically, the first class of these proteins to be studied in detail, the oleosins, appears to be solely involved in lipid storage and mobilisation, especially in seeds. However, even in the case of oleosins there are hints that they may sometimes be present in non-storage, meristematic tissues of shoots and roots, where they may have other novel functions (3,8). Over the past five years, much of the progress in elucidating the nonstorage roles of intracellular lipid bodies has come from studies in animal systems. Here, new and exciting discoveries are being made at a rapid rate. For example, the PAT family of lipid-associated proteins are now known to be present throughout the Metazoa and are also found in slime molds (40,41); caveolins are true lipid-body proteins (42,43); lipid bodies contain other proteins associated with lipid metabolism and trafficking (39) and lipid-body proteins retain their targeting properties in ectopic systems (44). It is also becoming apparent that lipid-associated proteins may be implicated in a host of serious human diseases and pathologies that include hepatitis C, Parkinson’s disease, CHILD syndrome, retinopathy and even skin irritation (1). For the first time, improved imaging techniques, such as multi- photon, laser-assisted confocal microscopy, coupled with the use of more powerful reporter reagents, like high-output fluorophores, have allowed for the real-time analysis of the behaviour of lipid bodies and their associated proteins in living cells (45). This has enabled investigators to begin to dissect out the various populations of lipid bodies; some in rapid flux in cells while others are less dynamic (46). This kind of direct real-time observational study forms a vital link with other more “snapshot” approaches and is beginning to allow us to assert with some confidence that lipid bodies are much more than mere storage entities. There is, therefore, an emerging consensus that cytosolic lipid bodies in animals and fungi are complex multifunctional organelles that participate in a host of cellular processes including membrane trafficking, lipid-based signalling, sterol homeostasis, (34,39,45).
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Figure 1 Primary sequences and domain organisation of caleosins from plants and fungi. A, domain organisation of a typical caleosin isoform that shows the major putative functional regions, namely the calcium-binding EF hand, membrane spanning region (Memb), proline-rich motif (Pro), tyrosine kinase site (Tyr) and three casein kinase II phosphorylation sites (CK). B, comparison of the 23 amino acid sequences of caleosins from plants and fungi that have been described to date. CLO1-9, the nine caleosin -like sequences from Arabidopsis; BARLEY1-3, three barley sequences; RICE1, 2, EFA27, three rice sequences; FAGUS, one sequence from fig; SOYA, one sequence from soya; SESAME, one sequence from sesame; NEUROSPORA, one sequence from the fungus Neurospora crassa ; 1-, 2-MAGNAPORTHE, two sequence from the fungus Magnaporthe grisea, AUXENOCHLORELLA, one sequence from the microalga Auxenochlorella protothecoides. Data were obtained from BLAST searches and aligned using the PRODOM database.
A 1
245 AtClo-1 EF hand Memb Pro Tyr
2CK
CK
61
B
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AMELIORATION OF SENSITIVITY TO UV-B IN ARABIDOPSIS BY SUPPRESSION OF A PUTATIVE PHOSPHOLIPASE J.E. THOMPSON, M. LO, C. TAYLOR, L. WANG, L. NOWACK, T.W. WANG Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
1. Introduction Phospholipases have been implicated in the catabolism of membrane lipids during senescence as well as in membrane turnover and signal transduction (Thompson et al 1998; Wang, 2001). They are classified according to their sites of hydrolysis (Fig. 1). Thus, the actions of phospholipase D and phospholipase C on phospholipids generate phosphatidic acid and diacylglycerol, respectively, whereas phospholipase A1 and phospholipase A2 deesterify fatty acids from the Sn 1 and Sn 2 positions, respectively, of phospholipids forming the corresponding lysophospholipids.
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CH2- O - C- R 1 A2 • || ↓ • O R2 - C - O - CH || • O O • | CH2- O - P - O - X ↑
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Figure 1. Diagram illustrating the catalytic sites of phospholipase A1 , phospoholipase A2 , phospholipase C and phospholipase D
There is increasing evidence that some phospholipases are encoded by multigene families. For example, in Arabidopsis, there are at least five isoforms of phospholipase D, designated a, ß, ?, d and e (Wang, 2001). Each isoform contains two active site regions encoded by the consensus sequence, HXKXXXD, that are essential for catalytic activity (Xie et al., 2000). In keeping with the fact that it is encoded by a multigene family, phospholipase D is involved in a broad range of cell functions including signaling (Wang, 2001). Suppression of phospholipase D in transgenic plants delays the onset of abscisic acid- and ethylene- induced yellowing of detached leaves indicating that the enzyme is also involved in senescence (Fan et al., 1997). Lipases that deesterify fatty acids from complex lipids contain the consensus sequence [LIV]-X-[LIVAFY][LIAMVST]-G-[HYWV]-S-X-G-[GSTAC], which encodes the esterase motif (Derewenda and Derewenda, 1991). Phospholipase A1 and A 2 as well as lipolytic acyl hydrolase feature this motif. Wax esters are among the many substrates deesterified by lipolytic acyl hydrolase (Galliard, 1971). In addition, unlike phospholipase A1
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and A2 , lipolytic acyl hydrolase does not exhibit positional specificity and is capable of cleaving both Sn 1 and Sn 2 fatty acids from phospholipids as well as triacylglycerides (Galliard, 1971). For example, the well characterized protein, patatin, which is abundant in potato tubers, rapidly deesterifies both fatty acids from phospholipid substrates indicating that it is a lipolytic acyl hydrolase (Senda et al, 1996). Recently, a lipase gene encoding a senescence-associated lipolytic acyl hydrolase was isolated from Arabidopsis (He and Gan, 2002). Transgenic plants in which the expression of this lipolytic acyl hydrolase was suppressed exhibited delayed leaf senescence, and over-expression of the gene resulted in early leaf senescence (He and Gan, 2002). Similarly, a cDNA encoding an ethylene-inducible lipase that is up-regulated in senescing petals and appears to be a lipolytic acyl hydrolase has been isolated from the petals of carnation (Dianthus caryophyllus) flowers (Hong et al., 2000). 2. Results and Discussion A full-length cDNA corresponding to the Arabidopsis sequence, GenBank accession number At2g42690, has been found to encode a phospholipase that is localized in the cytosol and expressed in all organs of the plant. The lipase is up-regulated in rosette leaves when Arabidopsis plants are exposed to sublethal doses of UV-B. Transgenic plants in which expression of the lipase is suppressed show enhanced tolerance to UV-B stress and are unable to up-regulate pathogenesis -related protein-1 (PR-1) when irradiated with UV-B. 2.1 cDNA Corresponding to At2g42690 Encodes a Phospholipase Full-length cDNA for At2g42690 was obtained by reverse transcription-PCR using RNA isolated from the rosette leaves of 4-week-old Arabidopsis plants, ecotype Columbia, as template. The plants were grown at 23°C under 150 µmol m-2 .s -1 photosynthetically active radiation in 16-h-light/8-h-dark photoperiods. The inferred amino acid sequence contains the lipase consensus motif, [LIV]-X-[LIVAFY]-[LIAMVST]-G-[HYWV]-S-XG-[GSTAC], indicating that the protein is a lipase capable of deesterfiying fatty acids from complex lipids (Fig. 2). Measurements of fatty acid deesterification catalyzed by corresponding recombinant protein confirmed that the translation product of At2g42690 is capable of cleaving fatty acids from complex lipid substrates and also indicated that it is a phospholipase. To obtain recombinant protein, the full-length cDNA was ligated into the expression vector, pTrc 99a, which is inducible in the presence of isopropylthio-ß-galactosidase, and overexpressed in E. coli. Fatty acid deesterification was measured using the NEFA colorimetric kit (Wako Chemicals, Neuss, Germany) as described by Ishiguro et al. (2001). Isolated recombinant protein proved capable of deesterifying fatty acids from soybean phosphatidylcholine, but was much less active when monogalactosyldiacylglycerol or trilinolein were used as substrates (Table 1). Thus the enzyme appears to be a phospholipase. 2.2 UV-B Inducibility and Expression Patterns of the Phospholipase The expression patterns of the phospholipase were determined by Western blotting of protein extracts using a polyclonal antibody raised in rabbits against a synthetic peptide corresponding to amino acids 386 to 407 of the phospholipase protein. A 47-kD band corresponding to the expected size of the protein was detectable in rosette leaves, stems, flowers, siliques and seeds, although the abundance of the protein was much lower in seeds (Fig. 3). Western blot analysis of isolated subcellular fractions indicate that the phospholipase is localized in the cytosol. Specifically, the protein is clearly discernible in isolated cytosolic and broken chloroplast fractions, but it is either not detectable or present in very small amounts in purified stromal, intact chloroplast or chloroplastic membrane fractions (Fig. 4). Nor is it detectable in microsomes (data not shown). In addition, Northern blot analysis of total RNA preparations isolated from rosette leaves of 3.5-week-old plants indicate that transcript levels for the phospholipase are strongly up-regulated upon exposure of plants to sublethal UV-B (Fig. 5).
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V L A P V E E E P V P E F * Figure 2. Amino acid sequence of the coding region for the Arabidopsis sequence, GenBank accession number At2g42690. The amino acid sequence was inferred from the corresponding cDNA sequence. The lipase consensus sequence is highlighted. The asterisk denotes the stop codon.
2.3 Suppression of the Phospholipase in Transgenic Plants Phospholipase protein was suppressed in transgenic Arabidopsis plants by constitutive expression of antisense At2g42690 cDNA. The cDNA was subcloned into the binary vector, pKYLX71 (Schardl et al., 1987), under the regulation of two consecutive copies of the cauliflower mosaic virus promoter, and the resultant construct, which also contained a kanamycin resistance gene, was introduced into Agrobacterium tumefaciens. Four-weekold Arabidopsis plants were transformed with this Agrobacterium culture by vacuum infiltration (Bechtold et al., 1993). Three homozygous transgenic lines, 4-2A-5, 1-4C-8 and 3-2C-5, obtained by kanamycin screening were found to have reduced expression of the phospholipase protein relative to wild-type plants. From densitometer scans of Western blots, it was apparent that levels of rosette leaf phospholipase were 40%, 80% and 30% lower for the transgenic lines 4-2A-5, 1-4C-8 and 3-2C-5, respectively, than for corresponding wildtype plants.
TABLE 1. Deesterification of fatty acids by At2g42690-encoded recombinant protein Relative Activity _______________________________________ PC* MGDG** TAG*** _____________________________________________________________________________________ Recombinant protein
1.0
0.58
0.21
C. rugosa lipase
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0.55
R. miehei lipase
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Catalytic activity was measured in vitro. The data are expressed as relative activities compared to the maximum activity (set at a value of 1) for each lipase. C. rugosa lipase, a known phospholipase; R. miehei lipase, a known galactolipase. * soybean phosphatidylcholine ** monogalactosyldiacylglycerol ***triolein
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Figure 3. Relative levels of phospholipase protein in total protein extracts of tissues from 6-week-old wild-type Arabidopsis plants. The data are band intensity measurements obtained by densitometric scanning of Western blots probed with phospholipase antibody. The band intensity for leaf tissue was set at 100.
Lane
Figure 4. Relative levels of phospholipase protein in subcellular fractions isolated from the rosette leaves of 3.5-week-old Arabidopsis plants. The data are band intensity measurements obtained by densitometric scanning of Western blots probed with phospholipase antibody. The band intensity for cytosol was set at 100.
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Figure 5. Relative levels of phospholipase transcript in total RNA preparations from rosette leaves of 3.5-week-old Arabidopsis plants exposed to varying doses of sublethal UV-B irradiation. The plants were treated for up to 4 consecutive days. The daily treatment consisted of 16 h of exposure to photosynthetically active radiation plus UV-B followed by 8 h of darkness. Cumulative hours of exposure to UV-B are indicated. The data are band intensities obtained by densitometric scanning of Northern blots probed with full-length At2g42690 cDNA The band intensity for 56 h of cumulative exposure to UV-B was set at 100.
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Transgenic plants for all three lines were indistinguishable from wild-type plants when grown to maturity under normal conditions. This was evident both from the appearance of the plants and also from quantitative measurements of biomass. However, when subjected to sublethal doses of UV-B irradiation, the transgenic plants showed symptoms of stress that were not evident in the wild-type plants. Sublethal UV-B stress was applied when the plants were 3.5 weeks of age. They were placed in a growth chamber fitted with UV lamps that produced 187.94 µmol m-2 .s -1 UV-B and 15.04 µmol m-2 .s -1 UV-A during a 24 h cycle consisting of 16 h of irradiation followed by 8 h of darkness. The irradiation from the UV lamps was passed through a cellulose acetate filter to screen out any incidental UV-C irradiation. Wild-type and transgenic plants were maintained under these treatment conditions from 3.5 weeks of age until they reached maturity and produced seed. Within 2 days, and particularly by 4 days, of the initiation of the treatment, growth of the wild -type plants had fallen behind that for the transgenic plants (Figs. 6 and 7). Within 4 to 6 days of treatment, anthocyanin accumulation, a symptom of stress in Arabidopsis, was evident in the rosette leaves of wild -type plants stressed with UV-B, but not in the leaves of transgenic plants exposed to UV-B (Figs. 7 and 8).
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Figure 6. Photograph of 3.5 week-old wild-type and transgenic (line 4-2A-5) plants exposed for 2 days to control light conditions or to UVB stress. The plants were placed in growth chambers operating on a 24 h cycle of 16 h of irradiation and 8 h of darkness. The control plants were irradiated with 187.94 µmol m -2 . s-1 photosynthetically active radiation. The UV-B-treated plants were irradiated with 187.94 µmol m 2 . s-1 photosynthetically active radiation, 2.46 µmol m -2 . s-1 UV-B and 15.04 µmol m-2 . s-1 UV-A.
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Figure 7. Photograph of 3.5 week-old wild-type and transgenic (line 4-2A-5) plants exposed for 4 subsequent days to control light conditions or to UV-B stress. The plants were placed in growth chambers operating on a 24 h cycle of 16 h of irradiation and 8 h of darkness. The control plants were irradiated with 187.94 µmol m -2 . s-1 photsynthetically active radiation. The UV-B-treated plants were irradiated with 187.94 µmol m -2 . s-1 photsynthetically active radiation, 2.46 µmol m -2 . s-1 UV-B and 15.04 µmol m -2 . s-1 UV-A.
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Figure 8. Photograph of 3.5 week-old wild-type and transgenic (line 4-2A-5) plants exposed for 6 subsequent days to UV-B stress. The plants were placed in a growth chamber operating on a 24 h cycle of 16 h of irradiation and 8 h of darkness. The irradiation consisted of 187.94 µmol m-2 . s-1 photsynthetically active radiation, 2.46 µmol m -2 . s-1 UV-B and 15.04 µmol m -2 . s-1 UV-A.
A further indication of the reduced sensitivity of the transgenic plants to UV-B is the finding that they were unable to up-regulate pathogenesis -related protein-1 (PR-1) when subjected to UV-B stress, whereas this protein was up-regulated in UV-B-stressed wild-type plants (Fig. 9). UV-B radiation is known to simulate some of the
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effects of pathogen ingression on gene expression, including the up-regulation of PR-1 (AH-Mackerness et al., 1999). The finding that suppression of the putative phospholipase encoded by At2g42690 prevents this upregulation of PR-1 suggests that this lipase may participate in the octadecanoid pathway, releasing fatty acid substrate for subsequent oxidation by lipoxygenase.
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A B Figure 9. Northern blot illustrating the effects of UV-B treatment on pathogenesis protein -1 (PR-1) expression in the rosette leaves of 3.5 week old wild-type (A) and transgenic (line 2-3A-5) (B) plants. Blots of total RNA were probed with full-length PR-1 cDNA. Each lane contained 10 µg RNA.
Literature Cited AH-Mackerness S, Surplus SL, Blake P, John CF, Buchanan-Wollaston V, Jordan BR, Thomas B (1999) Ultraviolet-B-induced stress and changes in gene expression in Arabiodpsis thaliana: role of signaling pathways controlled by jasmonic acid, ethylene and reactive oxygen species. Pl. Cell Environ. 22: 1413-1423. Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Paris Life Sciences 316: 1194-1199 Derewenda ZS, Derewenda U (1991) Relationships among serine hydrolases; evidence for a common structural motif in triacylglyceride lipases and esterases. Biochem Cell Biol 69: 842-851 Fan L, Sheng S, Wang X (1997) Antisense suppression of phospholipase D retards abscisic acid- and ethylene-promoted senescence of postharvest Arabiodopsis leaves. Plant Cell 9: 2183-2196 Galliard T (1971) The enzymatic deacylation of phospholipids and galactolipids in plants. Purification and properties of a lipolytic acyl hydrolase from potato tubers. Biochem J 121: 379-390 He Y, Gan SA (2002) Gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis. Plant Cell 14: 805-815 Hong Y, Wang TW, Hudak KA, Schade F, Froese CD, Thompson JE (2000) An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence. Proc Natl Acad Sci USA 97: 8717-8722 Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K (2001) The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence and flower opening in Arabidopsis. Plant Cell 13: 2191-2209 Schardl CL, Byrd AD, Benzio G, Altschuler MA, Hildebrand DF, Hunt AG (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61: 1-11 Senda K, Yoshioka H, Doke N, Kawakita K (1996) A cytosolic phospholipase A2 from potato tissues appears to be patatin. Plant Cell Physiol 37: 347-353 Thompson JE, Froese CD, Madey E, Smith MD, Hong Y (1998) Lipid metabolism during plant senescence. Prog Lipid Res 372: 119-141 Wang X (2001) Plant phospholipases. Annu Rev Plant Physiol Plant Mol Biol 52: 211-231 Xie Z, Ho WT, Exton JH (2000) Association of the N- and C-terminal domains of phospholipase D: contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme. J Biol Chem 275: 2496224969
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IMPLICATION OF MEMBRANE LIPIDS IN PLANT RESPONSE TO A COLD SHOCK, FROM SIGNALLING TO ADAPTATION
G. TASSEVA*, M.-N. VAULTIER*, C. CANTREL, F. COCHET, J. DAVY de VIRVILLE, C. DEMANDRE, A.-M. JUSTIN, J.-C. KADER, F. MOREAU, E. RUELLAND and A. ZACHOWSKI Plant Cellular and Molecular Physiology, UMR 7632 CNRS, Université Pierre et Marie Curie, case 154, 4 place Jussieu, 75252 Paris cedex 05, France
1. Introduction
Temperature is one of the main environmental factors that alter plant development. One mechanism of adaptation to growth temperature in plants, like in many prokaryotes and animals, corresponds to remodelling of membrane lipid composition in order to maintain membrane integrity and properties and membrane-associated protein functions under a wide range of environmental temperatures. Among changes, cold acclimation involves an increase in the fatty acid unsaturation degree, supposed to be a major factor in membrane fluidity, i.e. the extent of disorder and molecular motion in the lipid bilayer. On the basis of mechanisms of thermal adaptation characterized in bacteria, it has been proposed that the modifications in the fluidity were such that they led to homeoviscous adaptation of the membrane [5, 10, 19]. Most of the analyses focused on total cellular membranes while little is known about the responses of specific cellular compartments, with some exceptions such as mitochondrial membranes [3, 6, 13], tonoplast [1], and plasma membrane [27]. While endoplasmic reticulum (ER) is the site of the eukaryotic pathway of fatty acid and lipid biosynthesis, changes in its membrane composition in response to cold stress have not yet been fully characterized in plants. Two glycerolipid biosynthesis pathways, namely the prokaryotic (plastidial) and the eukaryotic (cytoplasmic) pathways, are active in plants [16]. The 16:0, 18:0 and 18:1 fatty acids are synthesized in the plastidial compartment. The 18:1 is further desaturated by the plastidial acyl- lipid desaturases, or alternatively exported to the ER for desaturation by the microsomal acyl-lipid desaturases. For these reasons, it is expected that changes which may occur in fatty acid and lipid composition of the ER membrane in response to cold would affect all cellular membranes, including plastidial membranes, since a reversible exchange of lipids exists between the eukaryotic and the prokaryotic pathways [2, 14].
While many genes [20] and transcription factors [21, 22] have been shown to be induced by a cold treatment, the perception mechanism(s) and the signalling pathway(s) of the temperature are poorly known. In Synechocystis, Vigh and his collaborators [26] proposed that rigidification of the plasma membrane might be the event that initiates the signalling cascade. Suzuki and collaborators [23, 24] identified a histidine kinase that would act as the cold sensor. In higher plants, a drop in temperature is known to trigger an early rise in cytosolic free calcium concentration [11, 17] and protein phosphorylation through the MAP kinase cascade [8, 9, 15]. On the other hand, phospholipid signalling seems to be also an element of the response to the change in environmental temperature. De Nisi and Zocchi [7] showed that in roots of maize plantlets a cold exposure induced a decrease in the level of membrane polyphosphoinositides. Knight and collaborators [11] showed that in Arabidopsis thaliana seedlings, the cold-induced cytoplasmic calcium rise could be disturbed by altering inositol trisphosphate (InsP3 ) metabolism. These findings arose the question whether some membrane enzymes of phospholipid metabolism, such as phospholipases C and D, could be implicated in the cold signal transduction pathway. Here we will report on changes in membrane lipids triggered by low temperature at these two levels. First, a long-term response representing the ability of ER membranes of Brassica napus hypocotyls to adjust their lipid composition and dynamics during cold acclimation. Then, a short-term response of plant cells to a drop in temperature that is the activation of phospholipases C and D, leading to an increase in phosphatidic acid content in the cell membranes. 2- Lowering the temperature of growth induces an increase in membrane lipid unsaturation We followed changes in lipid composition of endoplasmic reticulum (ER) membranes of rapeseeds when plantlets are cultivated at different temperatures or when they are transferred at a low temperature of growth. In order to prevent plastidial contamination of the ER-enriched cell fractions, this study was performed on etiolated hypocotyls. Two cultivars of rape were used, a freezing-tolerant, cv. Tradition, and a freezing-sensitive one, cv. ISL/97/2/P. Seeds were germinated on moist vermiculite in the dark at 22°C for 4 days. At that stage, some etiolated hypocotyls were transferred to 4°C for further growth in the dark for 3 or 13 days. Other hypocotyls were grown continuously at 4°C for 30 days. These conditions are not challenging the freezing tolerance or sensitivity of the cultivars but are representative of hardening conditions which are preparing plants to sub-zero temperature survival and increasing their freezing tolerance [25]. At the desired time, hypocotyls were harvested, cleaned from roots and cotyledons, ground in a blender and ER-enriched membrane fraction prepared by differential centrifugations and density flotation. Assessment of fraction purity was based on specific activity
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assays of enzymes either markers of ER (e.g. NADH cytochrome c reductase) or of other cellular membranes (e.g. ATPases). Membrane lipids were extracted by organic solvents, separated by thin layer chromatography on silica plates and the fatty acid content was analyzed by gas chromatography after methanolysis of any fraction. Comparisons of composition of samples purified from plantlets grown at 22°C and at 4°C allows one to infer the adaptative capability of the cultivar to low temperature. Comparison of data obtained with samples at various times after a temperature shift is indicative of the kinetics of lipid modification induced by the new conditions. An extensive analysis (fatty acid composition of the whole fraction and of each individual phospholipids and their relative amounts) of ER-enriched fractions purified from both cultivars has been obtained (Tasseva et al., in preparation). Here, we will mainly consider changes in the overall amount of poly-unsaturated fatty acids, i.e. linoleic (18:2) and linolenic (18:3) acids (figure 1).
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Figure 1: Content (in percent of total fatty acids) in linoleic and linolenic acids of ER membranes isolated from rapeseed hypocotyls grown for 4 days at 22°C (black bars) or 30 days at 4°C (hatched bars), or transferred to 4°C for 3 days (medium grey bars) or 13 days (light grey bars) after 4 days of growth at 22°C. After purification of ER fractions, lipids were extracted by organic solvents and fatty acids separated and quantified by gas chromatography. A freezing-tolerant (Tradition or T) and a freezing-sensitive (ISL or I) cultivar have been utilised.
It is striking that the poly-unsaturated fatty acid composition of ER membranes in each cultivar was comparable when hypocotyls were grown at constant temperature, i.e. at 22 or 4°C. At the higher temperature, 18:2 fatty acids accounted for approximately 40% of total fatty acids while 18:3 one represented 15-20% of this population. At the lower temperature, the percentages were ca. 25% and 40%, respectively. In that sense, no striking differences exist between the two cultivars whatever their agronomical sensitivity to freezing temperature is. A distinctive behaviour appeared when the kinetics of changes in fatty acid composition were studied. It is obvious that significant modifications in 18:2 and 18:3 contents have already occurred three days after the temperature shift in membranes from Tradition cultivar, which was not the case in membranes from ISL cultivar where it took almost 13 days to reach these changes. It has to be noted that after a period of 13 days at 4°C, a great part of the potential changes, with respect to the composition obtained in hypocotyls grown continuously at low temperature, has been matched for Tradition variety. If the experimental conditions are considered as simulating cold acclimation and preparation of plants to freezing temperature, it appears that a freezing tolerant variety (Tradition) will require a much shorter acclimation period to engage the necessary changes than a freezing sensitive one (ISL). However, it is premature to generalize this behaviour and to relate these observed changes to the agronomical character of the variety. In fact, such observations would have to be repeated with many other cultivars, keeping in mind that membrane lipid composition is not the only parameter affected by temperature and necessary to resistance. The overall changes in poly-unsaturated fatty acid content could be equally found at the level of each phospholipid present in the ER membranes. Other changes concerned the phosphatidylcholine-tophosphatidylethanolamine mass ratio and the protein-to-lipid mass ratio, which decreased when the growth temperature was lower. All these changes are thought to favour a higher membrane fluidity, which could eventually lead to homeoviscous adaptation: ideally, after all changes were completed, the fluidity of the bilayer measured under the new temperature conditions would be close or identical to that measured under the initial conditions in the unmodified membrane [5]. But a membrane bilayer is an anisotropic medium, where motions in various directions are not equivalent and present various characteristic times. As a consequence, each of the movements has its own dependence on the membrane composition [4]. Checking fluidity adaptation in modified membranes thus requires assaying different parameters. In the ER membranes, we looked for the evolution of
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local microviscosity (as inferred from the fluorescence depolarization of anthroyloxy -labelled fatty acids) and lipid lateral diffusion in the membrane plane (using the excimer-forming probe, 1-pyrenedodecanoic acid). No changes have been detected in local microviscosity between the different samples from a given cultivar (not shown). As to lateral diffusion, even if it was faster in membranes isolated from hypocotyls grown at low temperature, homeoviscous adaptation was far from being achieved. Overall, these membranes remained rigid at low temperature, even if profound changes in the lipid composition have occurred. Such a lack of adaptation to lower temperature of growth has also been encountered with soybean hypocotyls [6] and, at that time, has been tentatively attributed to the known inability of this plant to grow even at fresh temperatures. Now, considering data obtained with rapeseed, this characteristic cannot be put forward. In fact, one can wonder whether homeoviscous adaptation is an absolute requirement for adaptation to low temp erature knowing that any enzymatic reaction, thus the whole metabolism, will be slowed down. 3- Lowering temperature immediately turns on lipid signalling; activation of phospholipases Alterations in membrane lipid composition also occur right after the environmental conditions are changed. Here, studies were carried out with Arabidopsis thaliana cells grown in suspension, conditions allowing one to easily label phospholipids and favouring an identical change in temperature to the whole sample. Briefly, suspension were labelled with 33 P phosphate at 22°C for a given period of time (generally 2 hours) then vials were transferred into water-baths of given temperatures (between 20°C and 0°C). Cells were sampled at different times and the phospholipids composition determined as above. If cold shock activates some phospholipases, new lipid species should appear with time. Figure 2 shows what happened after cells grown at 22°C were transferred to 0°C.
Figure 2: Autoradiograph of a plate showing the turnover of phospholipids during an exposure at 0°C. Phospholipids were labeled in presence of 53 MBq/L [33P]-PO43- for two hours, at 22°C. The cold treatment was then performed by transferring the culture flasks into a water bath at 0°C. Lipids were extracted at different times after the temperature treatment and separated by thin layer chromatography using an alkaline solvent. The plate was analyzed with a Storm system. An autoradiograph of a plate representative from a typical experiment is shown. Unlabeled phospholipid standards were run in parallel to identify the spots and were revealed by iodine vapor. Taken from [18].
When the temperature was set at 0°C, the quantity of phosphatidic acid (PtdOH), a minor phospholipid in nontreated cells (less than 1% of the total labeled lipids), rose up to reach about 9% of total labeled phospholipids after 10 min, then the level of PtdOH decreased slowly and still represented about 6% of total labeled phospholipids after 140 min [18]. Phosphatidylinositol bis -phosphate (PtdInsP2 ) became undetectable within 1 min. Phosphatidylinositol monophosphate (PtdInsP) level dramatically decreased shortly after temperature drop and remained low. Undoubtly, some phospholipase C (PLC) activity has been activated following temperature drop. This has been verified by assaying the appearance of inositol-trisphosphate (InsP3 ), one of the hydrolysis products of PtdInsP2 by PLC. Indeed, while no InsP3 could be detected in non-treated cells, it was rapidly produced during a 0°C treatment and maximu m of InsP3 accumulation was attained after 2 min of exposure, before level decreased. Involvement of PLC activity was also ascertained by use of inhibitors such as neomycine or edelfosine which both decreased the production of PtdOH and InsP3 according to the dose used. However, PLC does not produce directly PtdOH from poly-phosphoinositide, but rather di-acyl-glycerides (DAG), which are phosphorylated from ATP into PtdOH by a DAG-kinase activity. The existence of this pathway was shown
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by preincubating cells with R59022, a DAG kinase inhibitor [12], prior to the temperature exposure. The decrease in PtdOH formed was dependent on the concentration of the inhibitor but a fraction of PtdOH appeared to be resistant even at high concentrations. In fact, PtdOH can be produced by a different pathway implying hydrolysis of phosphatidyl-choline (PtdCho) or –ethanolamine (PtdEtn) by a phospholipase D (PLD) activity. We demonstrated that an activation of that type of activity occurred right after temperature drop. First, it is known that PLD is able to perform a transphosphatidylation reaction between PtdCho or PtdEtn and a primary alcohol present in the incubation medium. The final product will be phosphatidylbutanol or phosphatidylethanol if 1-butanol or ethanol are added to the incubation, respectively. Under our experimental conditions, such artificial phospholipids appeared as a function of time after cells were transferred to 0°C. Moreover, the phosphate of PtdOH species formed by PLD action arose from the phosphate present on PtdCho, for instance. On the other hand, the phosphate of PtdOH species formed by action of the PLC and DAG-kinase system came from the γ-phosphate of ATP. ATP having a very fast turn-over, its labeling is sensitive to the presence of an excess of cold phosphate in the incubation. Again, under these conditions, some radioactive PtdOH still appeared, proving the involvement of a PLD pathway. Another clue for the simultaneous activation of the two phospholipase pathways results from the analysis of the molecular species of PtdOH. In fact, PtdCho and PtdEtn, on the one hand, and PtdIns, on the other hand, differ strongly in their fatty acid distribution, and, hence, in the molecular species they contain. Namely, phosphoinositides have a higher content in saturated fatty acids than the other two lipids. Analysis can be performed on labeled PtdOH obtained either after a very short 33 P labeling or after a prolonged one followed by addition of an excess of unlabelled phosphate. In the first case, only ATP is radioactive, thus only PtdOH formed by the PLC / DAG-kinase pathway is labeled. In the second one, only PtdOH resulting from PLD activation is labeled. Such assays confirmed that two categories of PtdOH exist at the same time, one with molecular species related to PtdIns, the other one with molecular species resembling those of PtdCho (article in preparation). 4- Is membrane rigidification the temperature sensor? Vigh and collaborators [26] have reported evidence that rigidification of plasma membrane might be the first step in temperature transduction in the cell. To test the hypothesis in our conditions, we designed experiments where we measured the onset of phospholipase activation when lowering incubation temperature. In other words, we measured PtdOH formation in suspension transferred to decreasing lower temperatures and compared data obtained with cells whose fatty acid composition, hence fluidity was different. A prediction would be that more fluid membranes would begin to react at a lower temp erature than more rigid membranes. Thus, experiments were carried out either with A. thaliana suspensions previously grown at 22°C or with suspension grown for 5 days at 15°C. We first verified that a growth at a lower temperature induced a change in fatty acid composition leading to the presence of more poly-unsaturated species. As expected, a marked increase in 18:3 species and a decrease in 18:2 species could be noticed in cells grown at 15°C (figure 3). According to results previously given for rapeseed hypocotyls, such a change should be accompanied by a higher fluidity of these membranes.
50 40 30 22°C 15°C
20 10 0 18:2 (PtdCho)
18:3 (PtdCho)
18:2 (PtdEtn)
18:3 (PtdEtn)
Figure 3: Poly -unsaturated fatty acid composition of membranes from suspension cells grown at 22°C (black bars) or at 15°C (grey bars). Lipids were extracted and analyzed as previously described. Composition is shown for the two main phospholipids found in cell membranes, namely phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn).
Under these conditions, appearance of PtdOH within the cell lipids as a function of the incubation temperature showed some differences between the two populations (figure 4).
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Figure 4: Rate of PtdOH formation as a function of final temperature in cell suspension. After 5 days of growth either at 22°C or at 15°C, cells were labelled by 33P (see above) then transferred into water baths equilibrated at the given temperature. PtdOH amounts formed were measured after one minute of incubation and expressed related to the amount of total labelled phospholipids.
Clearly, there was a temperature limit above which no PtdOH was produced at a pronounced level. For suspension previously grown at 22°C, this onset could be estimated at 12°C, while for suspension grown at 15°C, it was around 8°C. These results are thus strongly in favour of membrane rigidification being an essential signal in temperature perception. More data are required to reinforce this point of view. We have performed similar assays with A. thaliana cells mutated in the ER desaturases and whose fatty acid composition is strongly modified: the fad2 mutant, where oleate desaturase is absent, contains almost no poly-unsaturated fatty acids [3] while the fad3+ mutant, over-expressing the linoleate desaturase, contains an elevated amount of 18:3 species. Again, data seemed to confirm the above hypothesis (Vaultier et al., in preparation). Finally one could wonder which could be the first relay of membrane rigidification in the signalling cascade. A lot remains to be done to answer this question, but rather to be at the phospholipase level, it could be localized at the level of membrane ionic channels, as a calcium influx precedes and is required by phospholipase activation. 5. References
[1] Behzadipour M., Ratajczak R., Faist K., Pawlitschek P., Tremolières A. and Kluge M. (1998) Phenotypic adaptation of tonoplast fluidity to growth temperature in the CAM plant Kalanchoe daigremontiana Ham. et Per. is accompagnied by changes in the membrane phospholipid and protein composition. J. Memb. Biol. 166, 61-70 [2] Browse J. and Somerville C.R. (1991) Glycerolipid synthesis: Biochemistry and regulation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42, 467-506 [3] Caiveau O., Fortune D., Cantrel C., Zachowski A. and Moreau F. (2001) Consequences of omega-6-oleate desaturase deficiency on lip id dynamics and functional properties of mitochondrial membranes of Arabidopsis thaliana. J. Biol. Chem. 276, 5788-5794 [4] Cantrel C., Caiveau O., Moreau F. and Zachowski A. (2000) Lipid lateral diffusion and local microviscosity in plant mitochondrial membranes with various length and unsaturation of fatty acids. Physiol. Plant. 110, 443-449. [5] Cossins A.R. (1994) Homeoviscous adaptation of biological membranes and its functional significance. In: Temperature adaptation of biological membranes (ed A.R. Cossins), pp. 63-76. Portland Press, London. [6] Davy de Virville J., Cantrel C., Bousquet A.-L., Hoffelt M., Tenreiro A.-M., Vaz Pinto V., Arrabaça J.D., Caiveau O., Moreau F. and Zachowski A. (2002) Homeoviscous and functional adaptations of mitochondrial membranes to growth temperature in soybean seedlings. Plant Cell Environ. 25, 1289-1297 [7] De Nisi P. and Zocchi G. (1996) The role of calcium in the cold shock responses. Plant Sci. 121, 161-166 [8] Ichimura K., Mizoguchi T., Yoshida R., Yuasa T. and Shinozaki K. (2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J. 24, 655-665 [9] Jonak C., Kiergerl S., Ligterink W., Barker P.J., Huskisson N.S. and Hirt H. (1996) Stress signaling in plants: a mitogen-activ ated protein kinase pathway is activated by cold and drought. Proc. Natl. Acad. Sci. USA 93, 11274-11279 [10] Kates M., Pugh E.L. and Ferrante G. (1984) Regulation of membrane fluidity by lipid desaturases. Biomembranes 12, 379-395 [11] Knight H., Trewavas A.J. and Knight M. (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8, 489-503 [12] Lundberg G.A. and Sommarin M. (1992) Diacylglycerol kinase in plasma membranes from wheat. Biochim. Biophys. Acta 1123, 177183 [13] Lyons J.M., Wheaton T.A. and Pratt H.K. (1964) Relationship between the physical nature of mitochondrial membranes and chilling sensitivity in plants. Plant Physiol. 39, 262-268 [14] Miquel M. and Browse J. (1992) Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase. J. Biol. Chem. 267, 1502-1509 [15] Mizoguchi T., Irie K., Hirayama T., Hayashida N., Yamaguchi-Shinozaki K., Matsumoto K. and Shinozaki K. (1996) A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 93, 765-789 [16] Ohlrogge J. and Browse J. (1995) Lipid biosynthesis. Plant Cell 7, 957-70 [17] Plieth C., Hansen U.P., Knight H. and Knight M. (1999) Temperature sensing by plants: the prim ary characteristics of signal perception and calcium response. Plant J. 18: 491-497 [18] Ruelland E., Cantrel C., Gawer M., Kader J.C. and Zachowski A. (2002) Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis thaliana suspension cells. Plant Physiol. 130, 999-1007 [19] Russel N.J. (1984) Mechanisms for thermal adaptation in bacteria: blueprint for survival. TIBS 9, 108-112
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[20] Seki M., Narusaka M., Abe H., Kasuga M., Yamaguchi- Shinozaki K., Carninci P., Hayashizaki Y. and Shinozaki K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold Stresses by using a full-length cDNA microarray. Plant Cell 13, 61-72 [21] Shinwari Z.K., Nakashima K., Miura S., Kasuga M., Seki M., Yamaguchi- Shinozaki K. and Shinozaki K. (1998) An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochem. Biophys. Res. Com. 250, 161-170 [22] Stockinger E.J., Gilmour S.J. and Thomashow M.F. (1997) Protein Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. U S A 94, 1035-40 [23] Suzuki I., Kanesaki Y., Mikami K., Kanehisa M. and Murata N. (2001) Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis. Mol. Microbiol. 401, 235-244 [24] Suzuki I., Los D.A., Kanesaki Y., Mikami K. and Murata N. (2000) The pathway for perception and transduction of low-temperature signals in Synechocystis. EMBO J. 19, 1327-1334 [25] Thomashow M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 571-599 [26] Vigh L., Los D.A., Horvath I. and Murata N. (1993) The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 90, 369-374 [27] Yoshida S. (1984) Chemical and biophysical changes in the plasma membrane during cold acclimation of mulberry bark cells (Morus bombycis Koidz. cv Goroji). Plant Physiol. 76, 257-265 6- Acknowledgements G. Tasseva and M.N. Vaultier are recipients of a fellowship from the Ministère de la Recherche et de la Technologie. They have equally contributed to the work reported here, and have to be considered as first author as well. Research was supported by grants from the CNRS and the UPMC.
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GENETIC ENGINEERING FOR MODIFYING FATTY ACID COMPOSITION OF PALM OIL G.K.A PARVEEZ *; O.ABRIZAH, A.M.Y.MASANI, A. SITI NOR AKMAR A., R. UMI SALAMAH, S. RAVIGADEVI, B. BAHARIAH, A.H. TARMIZI, I., ZAMZURI, A.D. KUSHAIRI, S.C. CHEAH, AND M.W. BASRI. Advanced Biotechnology and Breeding Centre, Biological Research Division, Malaysian Palm Oil Board (MPOB), P.O. Box 10620, 50720 Kuala Lumpur, Malaysia. (*Corresponding author:
[email protected])
1. Abstract Malaysia is the largest producer of palm oil in the world and contributing around half of the world's palm oil production. The major problems faced by the oil palm industry are labor and arable land shortages which have forced the industry to increase the return per unit area. Genetic engineering, as a promising approach to overcome the above problem; has been initiated at MPOB since late 1980’s. Since the first successful production of transgenic oil palm and followed by the isolation of useful genes and tissuespecific promoters, efforts to add value to oil palm via genetic engineering was actively carried out. Two main targets for genetic engineering of oil palm are increasing oleic and stearic acid contents. Constructions of transformation vectors carrying different genes and promoters and in sense or antisense orientation have been achieved successfully. Transformation of oil palm calli with the above constructs has resulted in production of Basta resistant colonies which were later regenerated to produce full-grown transgenic palms. Some transgenic palms have been transferred onto soil in contained greenhouse. Molecular and fatty acid analyses are being carried out to verify their transgenic status of the oil palm tissues and plantlets. The progress made to date and problem faced during the experimental period will be elaborated. 2. Introduction Palm oil is the second largest source of edible oil in the world, which is produced in the tropical countries (Scowcroft, 1990). It is used as the most price competitive liquid cooking oil in many parts of the world. It is also used in the making of other food products like shortenings, margarines (Sudin, et al., 1993) and spreads (Pantzaris, 1993). The challenge that the oil palm industry will face in the 21st century is the ability to maintain profitability in the face of labor shortage and limited land resources. At present, palm oil is contributing around 20% of world oil and fat production. It is prospected that the demand for oil will grow faster then the rise in supply (Oil World Annual, 2001). By the year 2020, it is expected that nearly 26% of the world's oil and fat demand will have to be met by palm oil and it will capture approximately 50% of the world's oil and fat trade (Rajanaidu and Jalani, 1995). Due to this projected demand, it is important to increase the yield of oil palm as well as to improve the palm oil quality at a better rate than that has been achieved by conventional breeding (Parveez, 1998). Present oil palm planting materials are derived from a narrow gene pool, which restricts the introduction of new traits via conventional breeding techniques. The long generation time (approximately 7-10 years), and the open pollinated behaviour of oil palm contribute to the slowness of conventional genetic improvement methods besides requiring large amounts of planting material. All the above limitations make oil palm an ideal crop for the use of genetic engineering tools in its improvement. Developments of oil palm tissue culture techniques, combined with the ability to transfer genes of interest into elite germplasm are attractive tools techniques with which to overcome the slowness of genetic improvement in this perennial crop. Additionally, genetic engineering can source genes from any plant, animal, bacteria, fungus or virus (Gasser and Fraley, 1992). Approximately, four to five years are required to produce transgenic plantlets carrying a new trait from initial date of explants culture. Taking into account the requirement of back-crossing in conventional breeding, genetic engineering could save 80 90% of the time required for introducing a new gene/trait into oil palm (Parveez, 1998).
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The ultimate goal of the MPOB oil palm genetic engineering programme is to increase oleic acid content at the expense of palmitic acid. A number of other transgenic products have also been targeted such as stearic acid, palmitoleic acid, ricinoleic acid and biodegradable plastics (Parveez, 2003). In order to tailor fatty acid composition of palm oil, the regulation of fatty acid biosynthesis in the oil palm has to be clearly defined. Since the of this project is to decrease palmitic acid and increase oleic acid, a pertinent question to be answered was why palmitic acid accumulates in the oil palm resulting in 44% palmitic acid content in palm oil. Based on the fatty acid composition of palm oil and the fatty acid biosynthesis pathway common to all plants (Figure 1), the following postulation was made: I. $-ketoacyl ACP synthase II (KAS II) activity is rate-limiting in the oil palm mesocarp resulting in a “bottleneck” of palmitic acid II. Thioesterase activity towards palmitoyl ACP is very high resulting in the release of palmitic acid III. Oil palm mesocarp contains an active stearoyl ACP desaturase. Thus most of the stearoyl ACP formed is effectively desaturated to oleic acid. Increasing stearoyl ACP desaturase is unlikely to increase oleic acid (unless the stearoyl ACP pool is increased) {Cheah et al., 1995}. Therefore, two approaches were considered for channeling palmitic acid further along the pathway to produce more oleic acid: i) stimulate KAS II activity, and ii) reduce thioesterase activity towards palmitoyl ACP. The antisense copy of the palmitoyl ACP thioesterase gene will cause the activity of palmitoyl ACP thioesterase enzyme to be down regulated and the accumulation of palmitic acid would be reduced. P a l m i t o l e i c Acid C16:1 ↑ d esaturase B-ketoacyl -A CP | Stearoyl -A C P Syhthase II C2 F A S I | Desaturase + ------------ → 1 6 : 0 -ACP --------- → C18:0 -ACP ---------→ C18:1 -ACP 7C3 | | | PalmitoylACP Stearoyl -ACP Oleoyl -ACP Thioesterase Thioesterase Thioesterase ↓ ↓ ↓ C16:0 C18:0 C18:1 Oleic Acid | Palmitic Acid Stearic A cid Oleate-1 2 Hydroxylase ↓ 1 2 -OH, 18:1 Ricinoleic Acid
Figure 1 : Possible reactions involved in the modification of products of fatty acid synthetase Biochemical studies have demonstrated that oil palm contains an active stearoyl-ACP desaturase, therefore down-regulating the activity of stearoyl-ACP desaturase could reduce the conversion of stearoyl-ACP into oleoyl-ACP. Using a similar approach, Knutzon and colleagues (1992) have for the first time, transformed rapeseed with an antisense copy of a stearoyl-ACP desaturase. High stearic acid transgenic oil palm is expected to give rise to new applications such as cocoa butter substitute and personal care products such as lotions, shaving cream and rubbing oils (Parveez et al., 1999). In this paper, the progress made in transforming an antisense sequence of the palmitoyl ACP thioesterase and antisense sequence of stearoyl-ACP desaturase genes under the control of CaMV35S or ubiquitin promoters, into embryogenic calli of oil palm and finally producing transgenic plants, will be discussed
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3. Materials And Methods 3.1 Maintenance of embryogenic calli. Embryogenic callus was maintain on agar-solidified medium containing MS macro and micronutrients supplemented with 2.2 mg/l 2,4-D and 30 gm/l sucrose. The medium was adjusted to pH 5.7 with KOH prior to autoclaving. Embryogenic callus was cultured at 28o C, in the dark, and subcultured every 30 days onto fresh medium. 3.2 Bombardment of Embryogenic calli: Five microlitres of DNA solution (1µg/µl), 50µl of CaCl2 (2.5M) and 20µl spermidine (0.1M, free base form) were added sequentially to the 50µl particles suspension. The mixture was vortexed for 3 minutes, spun for 10 second at 10,000 rpm and the supernatant discarded. The pellet was washed with 250µl of absolute ethanol. The final pellet was resuspended in 60µl of absolute ethanol. Six microlitre of the solution was loaded onto the centre of the macrocarrier and was air dried. Bombardments were carried out once at the following conditions; 1100 Psi rupture disc pressure; 6mm rupture disc to macrocarrier dis tance; 11mm macrocarrier to stopping plate distance, 75mm stopping plate to target tissue distance and 67.5 mmHg vacuum pressure (Parveez, 1998). 3.3 Selection of transformed embryogenic callus: Minimal inhibitory concentrations of selection agents for oil palm have been determined previously (Parveez et al., 1996). Embryogenic callus was exposed to medium containing either Basta concentrations of 50mg/l, at one weeks after bombardment. Tissues were subcultured to fresh medium under selection at monthly intervals. 3.4 Production of Oil Palm Polyembryogenic Cultures: Embryogenic cultures were maintained on media containing MS macro and micronutrients and Y3 vitamins supplemented with 100mg/l each of myo-inositol, L-glutamine, L-arginine and L-asparagine, 5µM IBA, 0.7% agar and 30gm/l sucrose to form polyembryogenic cultures. The medium was adjusted to pH 5.7 with KOH prior to autoclaving. Embryogenic calli were incubated at 28o C in the presence of light and were subcultured every 30 days onto fresh medium (Parveez, 1998). 3.5 Small Plantlets Production from Polyembryogenic Cultures: Small platelets were produced from polyembryogenic cultures on media containing MS macro and micronutrients and Y3 vitamins supplemented with 100mg/l each of myo-inositol, L-glutamine, L-arginine and L-asparagine, 0.1µM NAA, 0.4% agar and 30gm/l sucrose. The medium was adjusted to pH 5.7 with KOH prior to autoclaving. Polyembryogenic calli were incubated at 28o C in light until sufficient shoots were produced (Parveez, 1998). 3.6 Root Initiation from Oil Palm Cultures : Roots were initiated from small plantlets on media containing MS macro and micronutrients and Y3 vitamins supplemented with 300mg/l L-glutamine, 100mg/l myo-inositol, 10µM 2,4-D, 70µM NAA, 0.15% activated charcoal and 60gm/l sucrose. The medium was adjusted to pH 5.7 with KOH prior to autoclaving. Small plantlets were incubated at 28o C in light until roots formed. The full regenerated plantlets were later transferred into polybags and grown in a biosafety greenhouse (Parveez, 1998). 3.7 Preparation of total DNA from embryoids and small plantlest:. Resistant embryoids and leaflets were selected randomly and subjected to total DNA isolationd according to the method of Ellis (1993). Tissues (10-50 mg) were placed in a 1.5 ml microfuge tube and immersed in liquid nitrogen. Frozen tissues was ground to a fine powder in the presence of 400 Fl EB2 buffer (500 mM NaCl, 100 mM Tris -Cl {pH 8.0} and 50 mM EDTA {pH 8.0}) and 20 Fl 20% SDS. Four hundred Fl of phenol mix (1:1; phenol:chlorofom) were then added, thoroughly mixed and centrifuged (12,000 rpm, 2 min, RT). The aqueous phase was transferred to a new tube and mixed with 800 Fl absolute ethanol. DNA was recovered by centrifugation (12,000 rpm, 5 min, RT). The pellet was washed with 70% ethanol and dissolved in 50 Fl TE (10 mM Tris -Cl and 1mM EDTA, pH 8.0). 3.8 Polymerase chain reaction (PCR) . Amplification of bar and palmitoyl-ACP thioesterase genes was carried out using standard and touch-down PCR protocols (Sambrook et al., 1989). Fifty ng of oil palm DNA and one ng of transforming plasmid DNA were used in PCR reactions. In the standard procedure the following condition was used: 30 cycles at 92o C (50 sec), 60o C (50 sec) and 72o C (60 sec). For the touch down procedure, 10 cycles 92o C (45 sec), 70o C (45 sec; 0.5o C per cycle), 72o C (60 sec) and 20 cycles 92o C (45 sec), 65o C (45 sec) and 72o C (60 sec) were used.
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Amplified DNA fragments were checked by electrophoresis on 1.4% agarose gels in 0.5X TBE (45 mM Tris Borate; 1mM EDTA, pH 8.0) buffer (Parveez, 1998). 4. Results And Discussion 4.1 Selection of Transformed Callus: Oil palm embryogenic calli were bombarded with plasmids carrying bar gene and antisense palmitoyl-ACP thioesterase or antisense stearoyl-ACP desaturase genes driven by constitutive promoters (Figure 2), respectively, using the conditions as described in Materials and Methods. Selection of transformants was carried out using BastaTM at concentration of 50 mg/l one week after bombardment. Bombarded embryogenic calli were cultured on medium free of the selective agent for one week. Upon transferring to medium containing selective agents, untransformed cells began to die and only resistant cells proliferated. Resistant colonies normally appear after 5-6 months on selection medium (subcultured to fresh medium once a month) [Figure 3]. After 2-3 subcultures, the transgenic embryogenic callus was transferred onto polyembryogenic induction medium. The transgenic embryogenic callus started to regenerate. Whitish embryoids and greenish polyembryogenic callus started to develop after three to five months of culture on polyembryogenesis inducing medium. After one to two months, some of these polyembryogenic cultures started to produce shoots (Figure 4). Once these shoots were big enough, they were isolated individually and transferred onto shooting medium for shoot elongation. Finally, the shoots were transferred into test tubes containing liquid medium for further development and root initiation. At this time, transgenic embryogenic callus cultures at various stages of development were obtained, i.e. from embryogenesis to rooting. The plants have been transferred onto soil and grown in a biosafety greenhouse (Figure 5). Embryoids or leaves were harvested from the obtained plantlets (depend on availability) for DNA extraction and subsequent molecular analysis. 4.2 Molecular Analysis of Transgenic Tissues: DNA from few embryoids or plantlets (originating from few different resistant embryogenic callus clumps) was isolated and subjected to PCR analyses. DNA from untransformed plants was also isolated and used as negative controls. Wherever applicable, DNA isolated from transgenic embryogenic callus (previously proven by PCR) was also used as a positive transformed control. DNA concentration and purity were determined by using a spectrophotometer. DNA from putative transformed embryoids and plantlets and one untransformed plant was subjected to amplification of an oil palm internal control sequence for reliable PCR analysis of the transgene. Since all the transgenic samples were derived from embryogenic calli bombarded with a plasmid carrying bar gene and selected on herbicide BastaTM, the amplification of the bar gene was used for verification of transformation. After amplification using a touchdown protocol, almost all the putative transformants, and the positive controls showed amplification of the bar gene (Figure 6). The same 460bp size band was also amplified when the transforming plasmid was used. No amplification of the bar gene was observed for negative and water controls. The amplification of the 460 bp fragment was obtained due to the amplification part of bar gene. The samples showing positive amplification of bar gene were later subjected to amplification of the antisense palmitoyl ACP thioesterase gene. Again PCR analysis of the antisense palmitoyl ACP thioesterase gene revealed that most of the samples were also positive for the antisense gene (Figure 7). Primers were designed in such a way to contain portion of the CaMV35S promoter which will only amplify the antisense gene used for transformation and not the endogenous oil palm antisense palmitoyl ACP thioesterase gene. 5. Concluding Remarks In this paper we have demonstrated the transformation of oil palm embryogenic calli with an antisense palmitoyl ACP thioesterase and Basta resistant genes. The integration of stearoyl-ACP desaturase gene is yet to be determined as the cultures available is not enough for any molecular analysis. The stable integration of the genes was proved via PCR analysis. The positive samples will later be subjected to Southern blot and gas chromatography analysis to further confirm the integration and copy number of the transferred gene and determine for possible changes in fatty acid composition in the tissues, respectively.
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35S promoter - full-length antisense desaturase.
35S - antisense palmitoyl ACP TE. BAR NOS
BAR
NOS
UBI
35S2E
NOS 35S
MAR AsDES
AsC18T LB
LB
NOS MAR
NOS
RB
RB
Ubi promoter - full-length antisense desaturase BAR
NOS
UBI
UBI
MAR AsDES
. LB
NOS MAR RB
Figure 2: Schematic diagram of plasmids used for transformation. Bar gene was used as selectable marker. Antisense palmitoyl- ACP thioesterase gene (left) and antisense stearoyl-ACP desaturase gene (middle and right) driven CaMV35S or ubiquitin promoters, were used for increasing oleic acid and stearic acid content, respectively.
Figure 3: Production of resistant embryogenic callus. Resistant embryogenic callus proliferating on medium containing basta.
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Figure production from polyembryogenic cultures derived from transgenic embryogenic calli .
Figure 5: Normal looking transgenic oil palm plantlets in the biosafety nursery.
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4:
Shoot
M
U <-------------------------------------------T----------------------------------->.
Figure 6 : PCR analysis of oil palm tissues using bar gene primers. Line M = 1Kb DNA marker (BRL), U: untransformed control, T: putative transgenic samples. Expected bar gene amplified fragment sized 460 bp.
M U W <--------------------------------T------------------------------->.
Figure 7: PCR analysis of oil palm tissues using antisense palmitoyl ACP thioesterase gene primers. Line M = 1Kb DNA marker (BRL), U: untransformed control, W = water control, T: putative transgenic samples. Expected antisense palmitoyl ACP thioesterase gene amplified fragment sized 900 bp.
6. Acknowledgements The authors thank the Director -General of MPOB for permission to publish this paper. We would like to acknowledge Mr. Abdul Masani Mat Yunus, Pn. Fatimah Tahir, Mr. Mohd Ali Abu Hanafiah Idris, Ms. Siti Marlia Silong, Mrs. Nik Rafeah Nik, Ms. Zurina Mohd Nor, Ms. Zuliana Dali, Ms. Norlinda Yazid, Pn. Fazurini Zakaria, Ms. Noraida Jusoh, Ms. Hairulaili Roslan, Mr. Hasnurol Jalil, and Ms Nor Salasiah Abdul Wahab, for their technical assistance. This research was funded by Malaysian Pam Oil Board under budget No: R000398000-RB02.
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7. References Cheah, SC; Sambanthamurthi, R; Siti Nor Akmar, A; Abrizah, O; Manaf, MAA, Umi Salamah, R and Parveez, GKA (1995). Towards genetic engineering of oil palm (Elaeis guineensis Jacq.). in J.C. Kader and P. Mazliak (eds.) Plant Lipid Metabolism . Kluwer Academic Publishers, Netherlands, pp. 570-572. Ellis THN (1993) Approaches to the genetic mapping of pea. Modern methods of plant analysis. Vegetables and Vegetable Prod 16:117160. Gasser, CS and Fraley RT (1992). Transgenic crops. Scientific American. June :62-69. Knutzon, D.S.; Thompson, G.A.; Radke, S.E.; Johnson, W.B.; Knauf, V.C. Kridl, J.C. (1992) Modification of Brassica seed oil by antisense expression of a stearoyl-ACP desaturase gene. Proceedings National Academy of Science USA. 89, 2624-2628. Oil World Annual (2001). ISTA Meilke GmbH, Germany. Pantzaris, TP (1993). Trends in yellow fats consumption in EEC. Palm Oil Dev. 18:3-7. Parveez GKA, Chowdhury MKU. and Saleh NM (1996) Determination of minimal inhibitory concentration of selection agents for oil palm (Elaeis guineensis Jacq.) transformation. AsPac J Mol Biol Biotechnology 4:219-228. Parveez, GKA (1998). Optimization of parameters involved in the transformation of oil palm using the biolistic method. Ph.D thesis, Universiti Putra Malaysia. Parveez, GKA (2003). Novel products from transgenic oil palm. AgBiotechNet. Vol. 5. July, ABN 113, 1-8. Pryde, EH (1983). Utilization of commercial oilseed crops. Econ. Bot. 37: 459-477. Rajanaidu, N and Jalani, BS (1995). World-wide performance of DXP planting material and future prospects. In Proceedings of 1995 PORIM National Oil Palm Conference. - T echnologies in Plantation, The Way Forward. 11-12 July 1995. Kuala Lumpur: Palm Oil Research Institute of Malaysia, pp.1-29. Sambrook J, Fritsch EF. and Maniatis T (1989) Molecular Cloning: A laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Scowcroft, WR (1990). New fats and oils through biotechnology. INFORM. 1:945-951 Sudin, N; Sahri, MM, Kun, TY and Oh, F (1993). Modification of palm kernel oil and their fractions for margarine. Palm Oil Dev. 18:1 -3.
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CURRENT STATUS IN GENETIC ALTERATION OF FATTY ACID COMPOSITION IN OIL PALM SHAH, FARIDA H. 1, 2 ; BHORE, SUBHASH J1, 2; CHA THYE SAN 3 ; and TAN CHYE LING1 1
School of Bioscience and Biotechnology, Faculty of Science and Technology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia. 2 Current address: Melaka Institute of Biotechnology, Pejabat Pos Ayer Keroh, 75450 Melaka, Malaysia. 3 Department of Biological Sciences, Faculty of Science and Technology, KUSTEM, Mengabang Telipot, 21030 Kuala Terengganu, Malaysia. Email:
[email protected].
1. Abstract The ability to manipulate fatty acid biosynthesis pathway especially in oil palm will enable the oil to be used in much wider applications in the edible as well as non-edible sectors. Palmitic acid (C16:0 ) usually represents 5 to 10 % of the total fatty acids in most of the seed oils, even though it can be as high as 77 %. Palm oil obtained from mesocarp of oil palm, Elaeis guineensis Jacq. Tenera contains = 44 % C16:0 while in the South American oil palm species, E. oleifera the C16:0 is much lower (25 %) with the oleic content being as high as 70 %. The ultimate goal of our research program is to be able to manipulate the fatty acid composition in oil palm so that its applications are widened. We have successfully isolated 6 key genes namely, ∆- 9- Stearoyl-ACP- Desaturase, ß-Ketoacyl-ACP Synthase I (KAS I), KAS II, KAS III, Palmitoyl-ACP Thioesterase (PATE), and Oleoyl-ACP Thioesterase (OTE) involved in oil palm fatty acid biosynthesis. Mesocarp, Kernel and leaf specific gene promoters were also isolated. To manipulate fatty acid composition of palm oil by post-transcriptional regulation of ∆- 9-Stearoyl-ACP- Desaturase, and PATE, we have successfully transformed oil palm with antisense constructs of PATE and desaturase genes. PCR, nucleotide sequencing, and Southern hybridization confirmed the successful integration of antisense constructs in the oil palm. This paper represents all the work carried out to date and the strategy employed to attain the objective. 2. Introduction Oil palm is an important and major commodity worldwide. Oil palm (Elaeis guineensis Jacq) is the second largest supplier of fats and oils to the world market of fats and oils, and Malaysia is the number one producer and exporter of the palm oil. Oil quality improvement of oil palm by traditional plant breeding methods is very time consuming process because of its long generation time (7-10 years). The fatty acid composition of the vegetable oil determines its qualities and utilities. Palmitic acid (C16:0 ) usually represents 5 to 10 % of the total fatty acids in most of the seed oils, even though it can be as high as 77 %. Palm oil obtained from mesocarp of oil palm E. guineensis Jacq. Tenera contains = 44 % C16:0 (Rajanaidu et al., 1997). Research indicates that relatively high percentage of C16:0 in mesocarp of oil palm is in part due to PATE enzyme, which is known to terminate fatty acyl group extension via hydrolyzing an acyl group on palmitic acid. Like soybean and / or cotton, high-stearate, high-oleic, high-linoleic, or low palmitate oil palms can be obtained by employing DNA recombinant technology (Liu et al., 2002; Thelen and Ohlrogge, 2002). Different key genes involved in oil palm fatty acid biosynthesis were isolated, which could be used for the genetic manipulation of oil palm, E. guineensis Jacq. Tenera (and E. oleifera) fatty acid biosynthesis at different levels (Shah and Rashid, 2000; Shah et al., 2000; Shah and Asemota, 2000; Shah and Hanafi, 2000; Shah and Cha, 2000; Shah and Cha, 2003; Cha, 2001). In addition to this, tissue specific gene promoters such as kernel, and mesocarp-specific gene promoters were isolated after isolation of tissue–specific genes (Cha and Shah, 2001; Shah and Cha, 2000; Cha, 2001). In our previous experiments, we found that embryogenic callus and IZEs are the most suitable target tissues for the biolistic mediated genetic transformation of the oil palm (Shah et al., 2003). However, we found that more than 30 % plantlets regenerated from transformed, and selected embryogenic calli displayed the phenotypic abnormalities (Tan et al., 2004). Therefore, IZEs were used as target tissue to avoid phenotypic abnormalities and to develop a quicker protocol for the genetic transformation of oil palm, E. guineensis Jacq. Tenera, and E.
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oleifera. In this paper, we report a summary of our oil palm project work carried out to date and the strategies employed to attain the objectives. 3. Materials and Methods 3.1 Plant Materials Twelve weeks old [weeks after pollination (WAP)] fresh fruit bunches (FFB) of field grown E. guineensis Jacq. Tenera (palm No 245, 253, 250, 353, and 368), and E. oleifera were harvested, and IZEs were removed aseptically from the fru its separated from FFB. The IZEs were used as target tissues. The Plant Breeding Department, United Plantation Berhad, Perak, Malaysia, generously supplied fruits. 3.2 Isolation and characterization of key genes involved in the fatty acid biosynthesis pathway Six different key genes namely, ? -9-Stearoyl-ACP-Desaturase, KAS I, KAS II, KAS III, PATE, and OTE were isolated by employing different techniques. Isolated genes were characterized by Southern and Northern hybridization. Using differential display method, tissue specific genes were identified and their promoters were isolated by inverse PCR and genome walking method. 3.3 Construction of antisense ∆ -9-Stearoyl-ACP-Desaturase, and Palmitoyl -ACP Thioesterase gene constructs Using constitutive promoter and oil palm mesocarp specific promoter (OPMSP), antisense expression cassettes were constructed for the PATE and desaturase gene (Shah and Cha, 2000). The cDNA clones of the PATE, and desaturase gene were used as a template to synthesize their antisense sequence. 3.4 Transformation, selection and regeneration By using biolistic method of DNA delivery, E. guineensis Jacq. Tenera and E. oleifera IZEs were bombarded with the two antisense constructs separately by employing the combination of all optimized physical parameters reported by Parveez et al (1997) with some minor modifications. After 7 days of bombardment IZEs were shifted onto selection medium. After selection, plantlets were regenerated and analyzed by PCR, nucleotide sequencing of the PCR products, and southern hybridization. 4. Results and Discussion In order to manipulate the fatty acid biosynthesis pathway in oil palm, it is necessary to characterize the key genes in terms of expression and regulation. We have successfully isolated 6 key genes namely, ∆ - 9- StearoylACP-Desaturase, KAS I, KAS II, KAS III, PATE, and OTE involved in the oil palm fatty acid biosynthesis. In all experiments the gene expression was compared between mesocarp and kernel within and between two Elaeis species to unders tand the differences or similarities in their expression during their fruit development process. Our results showed that the key genes are differential expressed with some being developmentally regulated and some are more highly expressed than others. Northern analysis showed that the E. guineensis Jacq. Tenera ? -9Stearoyl-ACP Desaturase (both isoforms pTD7, and pTD 8) is developmentally regulated (Shah et al., 2000). In mesocarp tissue, both isoforms of the desaturase are highly expressed during 17-20 WAP of fruit development (Figure 1). Therefore, ∆ -9-Stearoyl-ACP desaturase enzyme will not be a limiting when excess amount of C16:0ACP is available as a result of PATE gene silencing. MT 5 ? -9-Stearoyl-ACP Desaturase-I (pTD7)
1.8 kb
? -9-Stearoyl-ACP Desaturase-II (pTD8)
1.8 kb
MT 12
MT MT 15 17
MT 20
MO MO 5 15
KT 15
KO 15
RT
LT
RO
LO
Figure 1. Autoradiograms of Northern hybridization showing temporal regulation of the ? -9-Stearoyl-ACP -Desaturase. M, K, L, and R represents mesocarp, kernel, leaf, and root respectively. T and O stands for, Elaeis guineensis Jacq. Tenera, and E. oleifera respectively.
An interesting observation was that the KAS II and PATE genes are not regulated developmentally, and their level of expression was more or less the same in both Elaeis species (Figure 2). Sequencing of the genes for both in both Elaeis species was completed and their comparison between two species showed a very high (96
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%) homology (Cha and Shah, 2003). However, the genome organization of PATE genes in E. guineensis and E. oleifera differs with the former having an extra intron in the 5’ region. This may suggest a difference in the posttranscriptional and /or translational regulation of PATE, and might account for the difference in the C16:0 levels. In addition to this, genome analysis of PATE suggests that there may be two isoforms for PATE gene in oil palm. Our work suggested that the high levels of C16:0 in E. guineensis may be due to high activity of PATE compared to E. oleifera. Similarly we have isolated the gene for KAS III form both Elaeis species. MT 5
MT 12
MT 15
MT 17
MT 20
KT 12
KT 15
KT 17
MO MO MO MO MO 5 12 15 17 20
KO KO KO 12 15 17
RT LT
Figure 2. Northern blot analysis for PATE (FatB1) gene of Elaeis guineensis Jacq Tenera, and E. oleifera. Numbers, 5,12, 15, 17, and 20 represents the age in weeks (WAP) of developing mesocarp (M), and kernel (K) tissues. R and L stands for Root and Leaf respectively.
To obtain efficient promoters, tissue specific genes were identified by differential display method. Mesocarp tissue specific genes those are highly regulated during the active fatty acid biosynthesis in developing fruit mesocarp were selected for promoter isolation. In the production of low-palmitate oil palm, two strategies will be used 1) where the PATE is down-regulated or switched off, and 2) where the KAS II is over-expressed and PATE is switched off simultaneously. By post-transcriptional silencing of the ∆ - 9- Stearoyl-ACP- Desaturase, high stearic (HS) oil palm can be obtained. We successfully integrated the antisense ∆-9-Stearoyl-ACPDesaturase into E. guineensis Jacq. Tenera, and Dura (Shah et al., 2003). To minimize the C16:0 in oil palm, we successfully transformed IZEs of both Elaeis species with antisense PATE driven by OPMSP. Analysis of the E. oleifera plantlets revealed that 5 out of 56 selection marker (HYG) resistant plantlets were successfully transformed with antisense PATE gene driven by OPMSP. It was reconfirmed by PCR (Figure 3A), nucleotide sequencing of the PCR products and southern hybridiM 1 2 3 4 5 6 7
1
2 3 4 5 6 Figure 3. (A) PCR amplification of the regenerated E. oleifera plantlets. Lane M Marker, lane 1 plasmid DNA (+ve control), lane 2 non-transformed control (-ve control), lanes 3-7 transgenic plantlets. (B) Autoradiogram of southern hybridization of the transgenic plantlets. DNA was digested with EcoRI. Lane 1 non-transformed control, lane 2-6 trangenic plantlets.
A
B
-zation (Figure 3B) further confirmed the successful integration of antisense PATE. Work is underway to analyze the transformants in E. guineensis Jacq. Tenera. Oil palm takes long time to complete organogenesis in vitro and has very long generation time. Therefore, a lot more has to be done to get homozygous transgenic oil palm plants which can be marketed for commercial planting.
Acknowledgements The authors are grateful to the Ministry of Science and Technology of Malaysian Government for funding (Grant No IRPA: 09-02-02-0161), to the United Plantation Berhad, Perak, Malaysia for supplying fresh fruit bunches of Elaeis guineensis Jacq. Tenera and E. oleifera for this study.
5. References 1. 2. 3. 4. 5. 6. 7. 8.
Cha,T.S. and Shah,F.H. (2003) Elaeis guineensis beta-ketoacyl-ACP synthase II mRNA, complete cds. GenBank Accession No AF220453. Cha TS, and Shah F. H. (2001) Kernel-specific cDNA clones encoding three different isoforms of seed storage protein glutelin from oil palm Elaeis guineensis. Plant Sci. 160: 913-923. Cha T.S. (2001) Characterization of key genes and mesocarp-specific promoter for genetic manipulation of oil palm. PhD Thesis. National University of Malaysia. Liu Qing, Surinder P. Singh, and Allan G. Green. 2002. High -stearic and high-oleic cotton seed oils produced by hairpin RNAmediated post-transcriptional gene silncing. Plant Physiol. 129:1732-1743. Rajanaidu N., B.S. Jalani and A. Kushairi. (1997) Genetic improvement of oil palm, in: M. S. Kang (Ed.), Crop improvement for the 21st century, Published by Research signpost, India, pp 127-137. Shah,F.H. and Rashid,O. (2000) Elaeis guineensis stearoyl- Acyl-carrier protein desaturase mRNA, partial cds. GenBank Accession No U68756. Shah,F.H., Rashid,O. and Cha T.S. (2000) Temporal regulation of two isoforms of cDNA clones encoding delta 9-stearoyl- ACP desaturase from oil palm (Elaeis guineensis). Plant Science. 152: 27-33. Shah,F.H. and Asemota,O. (2000) Elaeis guineensis oleoyl thioesterase mRNA, partial cds. GenBank Accession No AF143095.
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9. Shah,F.H. and Hanafi,S. (2000) Elaeis guineensis ketoacyl synthase III mRNA, partial cds. GenBank Accession No AF143502. 10. Shah F.H., and Cha T.S. (2000) A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase., Plant Science., 154:153-160. 11. Shah,F.H. and Cha,T.S. (2003) Elaeis guineensis Jacq Tenera palmitoyl-acyl carrier protein thioesterase (PATE) mRNA, complete cds. GenBank Accession No AF147879. 12. Shah F. H., Tan C. L.., Cha T. S. and Fathurrahman. (2003) Genetic manipulation of fatty acids in oil palm using biolistics: Strategies to determine the most efficient target tissues. In: N. Murata et al., (Eds), Advanced Research on Plant Lipids. Kluwer Academic Publishers. PP 419-422. 13. Tan C.L., Bhore S.J, and Shah F.H. (2004) Quantitative and qualitative assessment of phenotypic variation in oil palm (Elaeis guineensis Jacq. Tenera) plantlets regenerated from transformed and non-transformed calli. Indian J. Plant Physiology, in press. 14. Thelen Jay J. and John B. Ohlrogge. (2002) Metabolic Engineering of Fatty Acid Biosynthesis in Plants. Metabolic Engineering. 4: 12-21.
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MICROSOMAL OLEATE DESATURASE (FAD2) FROM OILSEEDS: NEW INSIGHTS INTO THE MECHANISMS OF REGULATION BY TEMPERATURE AND OXYGEN J.M. MARTÍNEZ-RIVAS, A. SÁNCHEZ-GARCÍA, M.D. SICARDO, A.B. ESTEBAN AND M. MANCHA Instituto de la Grasa, CSIC. Avda. Padre García Tejero 4, 41012-Sevilla, Spain
1. Introduction The characteristics of fats and oils are highly dependent on their fatty acid composition. Oleic and linoleic acids are the major fatty acids of vegetable oils, therefore, their relative proportions in oil crops determine relevant technological and nutritional properties of edible oils (Vos, 2003). It is very well known that environmental temperature during oilseed development modifies the proportion of linoleic acid depending on the geographical area and year, resulting in an unwanted variation of the oleic to linoleic ratio in the final composition of the oil (Canvin et al., 1965). High temperatures decrease the linoleic acid content of oilseed oils. However, the extension of this temperature effect varies depending on the plant species. While in sunflower seeds the percentage of linoleic acid is highly influenced by growth temperature (Harris et al., 1978; Lajara et al., 1990), in safflower seeds it is much less temperature dependent (Knowles, 1972), with oilseed rape (Trémolière et al., 1982) and soybean (Wolf et al., 1982) seeds being moderately affected. Currently, the market demands oils with a constant degree of unsaturation, and, for that reason, it is an important goal to obtain oilseed varieties with fatty acid composition not affected by the environmental temperature. To accomplish this aim it is essential to completely elucidate the mechanisms by which temperature influences the linoleate content of oilseeds. The enzyme responsible for the synthesis of linoleic acid from oleic acid is the microsomal oleoyl phosphatidylcholine desaturase (FAD2; EC 1.3.1.35) (Shanklin and Cahoon, 1998). This is a membrane-bound enzyme that catalyzes the first extra -plastidial desaturation in plants, converting oleic acid, preferentially esterified in the sn-2 position of phosphatidylcholine, to linoleic acid. The reaction involves the concomitant reduction of molecular oxygen to water and requires the presence of NADH, NADH-cyt b 5 reductase and cyt b5 as electron donor system (Smith et al., 1990). Several mechanisms have been proposed to explain how temperature regulates FAD2 activity. Firstly, substrate availability was thought to limit desaturase activity. Being oxygen the common substrate for all the desaturases, Harris and James (1969) suggested that the higher solubility of this gas in water at low temperature might increase the total desaturase activity by increasing the availability of oxygen as substrate in non-photosynthetic tissues. Although varying oxygen concentrations in vitro have not been considered relevant to affect FAD2 activity in developing safflower seeds (Browse and Slack, 1983), recently, this hypothesis has been given more credence in cultured cell systems such as sycamore cells, where oxygen availability could be limiting for the ∆12-desaturase (Rebeille et al., 1980). A second method of regulating FAD2 activity by temperature is the activation-inactivation of the enzyme. No evidence for post-translational modification or allosteric activation has been reported so far. Although for non-plant organisms it has been described that a change in membrane fluidity caused by low temperature could produce a conformational change of the enzyme that improves the interaction with the substrates or the electron transfer components (Skriver and Thompson, 1979), no evidence was observed which would indicate that the FAD2 activity from developing safflower seeds were regulated by a change in the fluidity of the microsomal membranes (Stymne and Stobart, 1985). A third response is to increase or decrease the amount of desaturase protein. This change could be caused by a change in mRNA stability and/or induction or repression of the FAD2 gene. However, no significant change caused by low temperature has been detected in the transcript level of the soybean FAD2 genes (Heppard et al., 1996).
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Our group has been studied this phenomenon in sunflower seeds showing that temperature affects the de novo oleate biosynthesis (García-Díaz et al., 2002) and its mobilization from pre-formed triacylglycerols (Garcés et al., 1994; Sarmiento et al., 1998), thus modifying the available amount of oleate for desaturation. In addition, temperature regulates the FAD2 enzyme. Two separate mechanisms were proposed to be involved in the temperature regulation of the FAD2 activity: a direct effect and an indirect effect by which temperature determines the availability of oxygen, which, in turn, regulates the FAD2 activity (García-Díaz et al., 2002). In order to further investigate this hypothesis in oilseeds, we have studied the changes in the level of FAD2 activity under different temperature conditions and oxygen availability in sunflower and safflower seeds, which represent two opposite models with regard to the temperature effect on the linoleate content in oilseeds. In addition, both developing seeds are characterized by a very low level of linolenic acid, avoiding the contribution of the linoleate desaturase (FAD3) to this phenomenon, and facilitating the study of the temperature regulation specifically for the FAD2 activity. The results explain the differences in the temperature effect on the seed linoleate content between the two species. 2. Materials and methods 2.1. Plant material Normal-type sunflower (Helianthus annuus L. cv. HA-89) and high linoleate safflower (Carthamus tinctorius L. cv. Rancho) seeds were provided by Dr. J. M. Fernández-Martínez, IAS, CSIC, Córdoba, Spain. Plants were cultivated in a growth chamber with a 16 h photoperiod, photon flux density of 300 µmol m-2 s -1 , at 25/15 ºC (day/night). Although the fruit of sunflower and safflower are referred to as a cypsela or achene, for the purposes of this study the more familiar term seed will be used. Sunflower seeds of 18-20 days after flowering (DAF), or safflower seeds of 14-18 DAF, which corresponds to the period of active triacylglycerol biosynthesis, were collected for the experiments. Lots of twenty peeled seeds (achenes without hull and seed membrane), corresponding to approximately 0.5 g, were collected from different capitula and plants to assure homogeneity. For details see legends to figures. 2.2. Subcellular fractionation Twenty peeled seeds (approx. 0.5 g) were ground in a pre-cooled mortar with 10 ml of 50 mM HEPES buffer (pH 7.2) containing 0.6 M sorbitol, 40 mM Na-ascorbate, 1 mM Na 2 EDTA and 1 mM MgSO4 . All manipulations were done at 4 ºC. The homogenate was centrifuged at 10,000 g for 5 min. The fat layer was discarded and the supernatant was centrifuged for 1 h at 100,000 g. The pellet containing the microsomal fraction was resuspended in 1 ml grinding medium and stored at –80 ºC. 2.3. In vitro assay of FAD2 activity The in vitro assay of microsomal oleate desaturase activity was carried out as described by García-Díaz et al. (2002) using 50 µl of sunflower seed microsomal suspension (corresponding to approximately 25 mg fresh seed tissue and 100 µg microsomal protein) or 5 µl of safflower seed microsomal suspension (corresponding to approximately 2.5 mg fresh seed tissue and 30 µg microsomal protein) and 30 min as incubation time in both cases. Incubation temperature was 20 or 25 ºC for sunflower or safflower microsomes, respectively, except when indicated. 3. Results and discussion 3.1. Direct temperature effect on the FAD2 activity from oilseeds To investigate the direct temperature regulation without the influence of oxygen availability, peeled seeds were used to assure that oxygen concentration is saturating for oleate desaturation. After 6 h of incubation at temperatures between 10 and 40 ºC, the microsomal fraction was isolated and the FAD2 activity determined. This in vivo heat-resistance profile (Figure 1) showed that the sunflower FAD2 was unstable at temperatures higher than 25 ºC, whereas the safflower enzyme retained its maxima l activity level until 30 ºC, exhibiting a higher thermal resistance.
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FAD2 activity (%)
100
Safflower
80 60
Sunflower
40 20 0 10
15
20
25
30
35
40
Temperature (ºC) Figure 1. Effect of temperature on the FAD2 activity in peeled developing sunflower (?) or safflower (?) seeds. Peeled seeds were incubated at the indicated temperatures in a stream of water-saturated air for 6 h, homogeneized, and stored at –20 ºC. The homogenate was used to isolate the microsomal fraction and the FAD2 activity was measured as described in Materials and methods (100% was 45 and 48 nmol linoleate (g FW)-1 h –1 for sunflower and safflower seeds, respectively).
The thermal properties of the FAD2 enzyme were also characterized in vitro using microsomal membranes isolated from both developing seeds. Optimal temperature was 20 and 25 ºC for the sunflower and safflower FAD2 enzyme, respectively (Martínez-Rivas et al., 2003a; Esteban et al., 2004). The in vitro heat-resistance profile (Figure 2) was also obtained preincubating the microsomes at different temperatures for 30 min and then assaying the activity under standard conditions.
100 Safflower
FAD2 activity (%)
80 Sunflower
60 40 20 0 10
15
20
25
30
35
40
Temperature (ºC) Figure 2. Heat-resistance profile of FAD2 enzyme from developing sunflower (?) or safflower (?) seeds. Microsomal samples isolated from developing seeds homogeneized immediately after dehulling were incubated at the indicated temperatures. After 30 min, the samples were cooled rapidly on ice and assayed for FAD2 activity as indicated in Materials and methods (100% was 19 and 46 nmol linoleate (g FW)-1 h –1 for sunflower and safflower seeds, respectively).
The sunflower enzyme showed maximal activity until 25 ºC, and the temperature at which 50 % of the enzyme activity was recovered was 30 ºC, whereas no activity was detected at 40 ºC. In contrast, the safflower FAD2 exhibited its maximal activity level until 30 ºC, and the temperature at which 50 % of the enzyme activity was
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recovered was 34 ºC, while 10% activity was still observed at 40 ºC. These results confirm the lower thermal stability of the sunflower enzyme compared to the safflower FAD2. Moreover, results obtained from experiments using peeled seeds from both plants subjected to temperature changes (Martínez-Rivas et al., 2003a; Esteban et al., 2004) also support the hypothesis that the thermal stability of the FAD2 enzyme should be the main factor responsible for the direct temperature control of the FAD2 activity. Low temperatures preserved a high activity level, while high temperatures produced the non-reversible heat denaturation of the enzyme, decreasing its activity level. This mechanism could be enough to explain the observed direct temperature regulation of the FAD2 enzyme in vivo, as suggested by the high correlation obtained between in vivo and in vitro thermal properties. In addition, the lower thermal stability of the sunflower FAD2 enzyme, compared with that of safflower, could explain at least partially, why the linoleate content of sunflower seeds is more dependent on growth temperature than that of safflower seeds. Interestingly, the thermal resistance of the FAD2 enzyme from soybean, an oilseed with a significant temperature dependence of the linoleate content, was similar to sunflower FAD2, and therefore, much lower than the safflower enzyme (Cheesbrough, 1989). 3.2. Indirect temperature effect on the FAD2 activity from oilseeds affecting oxygen availability Further research has been done to confirm if different conditions of oxy gen availability regulate the FAD2 activity level in developing oilseeds. Peeled seeds were incubated immediately after dehulling at 20 ºC for different times, and the FAD2 activity was determined in the corresponding microsomal fractions (Figure 3).
Safflower
50
FAD2 activity
40
Sunflower
30 20 10
0
2
4
6
8
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Time (h) Figure 3. Effect of hull removing on FAD2 activity in developing sunflower (?) or safflower (?) seeds. Peeled seeds were incubated at 20 ºC in a stream of water-saturated air. At the indicated times, the seeds were homogeneized and stored at –20 ºC. The homogenate was used to isolate the microsomal fraction, and the FAD2 activity was measured as described in Materials and methods and expressed as nmol linoleate (g FW)-1 h –1 .
Hull removing of sunflower seeds produced a dramatic increase of the FAD2 activity that could be due to an increase in oxygen availability caused by the absence of the hull. On the contrary, in the same experiment safflower seeds exhibited a high and constant FAD2 activity level after dehulling. In addition, if both peeled oilseeds were shifted to anoxia once they had reached their maximum activity levels, a rapid and strong decrease was observed, while subsequent air replacement produced the fast recovery of the maximal FAD2 activity for seeds of both plants (Martínez-Rivas et al., 2003b; Esteban et al., 2004). These data confirm that oxygen is the responsible for the fast changes of FAD2 activity in both oilseeds, and show that it is a reversible mechanism. Similar rapid reversible changes in the FAD2 activity were obtained when sunflower detached achenes were subjected to temperature shifts, demonstrating that temperature indirectly control oxygen availability inside the achene (Martínez-Rivas et al., 2003a). The high speed of the process also indicates that an activationinactivation mechanism could be involved. Furthermore, when sunflower microsomes isolated from a homogenate obtained immediately after dehulling, were incubated for 2 h at 20 ºC instead of peeled seeds, no effect on FAD2 activity was detected, suggesting that oxygen could initiate a signal transduction cascade in vivo, instead of being the direct responsible for the reversible change in the activity level (Martínez-Rivas et al., 2003b).
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To determine the oxygen level that caused a decrease in the FAD2 activity, peeled seeds from both plants were incubated for 30 min at different concentrations of the gas (Figure 4).
Safflower
FAD2 activity
50 40
Sunflower 30 20 10 0
2
4
6
8
10
12
14
16
18
20
[O2 ] (%) Figure 4. Effect of oxygen concentration on FAD2 activity in peeled developing sunflower (?) or safflower (?) seeds. Peeled seeds were incubated at 20 ºC in a stream of water-saturated nitrogen with different oxygen concentrations. After 30 min, the seeds were homogeneized and stored at –20 ºC. The homogenate was used to isolate the microsomal fraction, and the FAD2 activity was measured as described in Materials and methods and expressed as nmol linoleate (g FW) -1 h –1 .
In sunflower seeds, the FAD2 activity was partially inactivated at oxygen concentration lower than 3%. According with these data, the characteristic low FAD2 activity level detected in intact sunflower seeds (Figure 3) indicates a hypoxic environment (in the range of 1-4%) inside the sunflower achene. This hypoxic situation inside developing seeds has been reported in soybean (Shelp et al., 1995) and Brassica rapa (Porterfield et al., 1999). Unlike the sunflower enzyme, at an oxygen concentration of 3% the safflower FAD2 kept its maximal activity level, pointing out that is active at lower oxygen concentrations than the sunflower FAD2. However, the high FAD2 activity level found in hull enclosed safflower seeds, in comparison with the low level detected in intact sunflower seeds (Figure 3), should be explained not only by the small difference in the sensitivity to low oxygen concentrations between the safflower and sunflower FAD2 enzyme (Figure 4), but also by a higher oxygen concentration present inside the safflower achenes. This differential oxygen availability between sunflower and safflower seeds could be due to differences in its diffusion through the hull and in the level of respiration that competes with desaturation for a limited supply of oxygen in the seed inside the achene (GarcíaDíaz et al., 2002). Thus, in plants cultivated under the same physiological conditions, safflower seeds show a higher FAD2 activity level than sunflower seeds. In conclusion, two different mechanisms are involved in the temperature regulation of FAD2 activity in oilseeds (Figure 5): a long-term direct effect mostly related to the low thermal stability of the enzyme (Martínez-Rivas et al., 2003a) and, secondly, a short-term indirect effect by which temperature affects the availability of oxygen, that, in turn, regulates the reversible changes of the enzyme activity level (Martínez-Rivas et al., 2003b).
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Direct temperature effect
Thermal denaturation of the enzyme
Decrease
FAD 2
High temperature Decrease oxygen availability
Reversible inactivation of the enzyme
Decrease linoleic content
Decrease FAD2 activity
Figure 5. Mechanisms involved in the temperature regulation of FAD2 activity in oilseeds.
Therefore, the higher thermal stability of the safflower FAD2 enzyme and the lower dependence on oxygen availability could explain why the linoleate content in safflower seeds is less affected by temperature and oxygen than in sunflower seeds, where the FAD2 enzyme is less thermally resistant and more subjected to oxygen shortage. These results indicate that sunflower and safflower represents two models for the temperature and oxygen regulation of oleate desaturation in oilseeds (Esteban et al., 2004). Both temperature regulation mechanisms are of particular significance as they act during field growth conditions of oilseed plants. Molecular characterization of the proposed mechanisms is currently under way in our laboratory.
4. Acknowledgments This research was supported by grant AGL2001-1060 from MCYT (Spain). J.M.M.-R. is the recipient of a postdoctoral contract within the “Ramón y Cajal” Program and A.S.-G. and M.D.S. are the recipient of predoctoral fellowships, all from MCYT (Spain). 5. References Browse J, Slack R (1983) The effects of temperature and oxygen on the rates of fatty acid synthesis and oleate desaturation in safflower (Carthamus tinctorius) seed. Biochim Biophys Acta 753, 145-152. Canvin DT (1965) The effect of temperature on the oil content and fatty acid composition of the oils from several oil seed crops. Can J Bot 43, 63-69. Cheesbrough TM (1989) Changes in the enzymes for fatty acid synthesis and desaturation during acclimation of developing soybean seeds to altered growth temperature. Plant Physiol 90, 760-764. Esteban AB, Sicardo MD, Mancha M, Martínez-Rivas JM (2004) Growth temperature control of the linoleic acid content in safflower (Carthamus tinctorius) seed oil. J Agric Food Chem 52, 332-336. Garcés R, Sarmiento C, Mancha M (1994) Oleate from triacylglycerols is desaturated in cold-induced developing sunflower (Helianthus annuus L.) seeds. Planta 193, 473-477. García-Díaz MT, Martínez-Rivas JM, Mancha M (2002) Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds. Physiol Plant 114, 13-20. Harris P, James AT (1969) The effect of low temperatures on fatty acid biosynthesis in plants. Biochem J 112, 325-330. Harris HM, McWilliam JR, Mason WK (1978) Influence of temperature on oil content and composition of sunflower seed. Aust J Agric Res 29, 1203-1212. Heppard EM, Kinney AJ, Stecca KL, Miao G-H (1996) Developmental and growth temperature regulation of two different microsomal ω-6 desaturase genes in soybeans. Plant Physiol 110, 311-319. Knowles PF (1972) The plant geneticist´s contribution toward changing lipid and amino acid composition of safflower. J Am Oil Chem Soc 49, 27-29. Lajara JR, Díaz U, Quidiello DR (1990) Definite influence of location and climatic conditions on the fatty acid composition of sunflower seed oil. J Am Oil Chem So c 67, 618-623.
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Martínez-Rivas JM, Sánchez-García A, Sicardo MD, García-Díaz MT, Mancha M (2003a) Oxygen-independent temperature regulation of the microsomal oleate desaturase (FAD2) activity in developing sunflower (Helianthus annuus) seeds. Physiol Plant 117, 179-185. Martínez-Rivas JM, Sánchez-García A, Sicardo MD, Mancha M (2003b) Oxygen availability regulates microsomal oleate desaturase (FAD2) in sunflower developing seeds by two different mechanisms. In Murata, N., Yamada, M., Nishida, I., Okuyama, H., Sekiya, J., Wada, H., (eds.), Advanced Research on Plant Lipids. Kluwer Academic Publishers, Dordrecht, Netherlands, pp 109-112. Porterfield DM, Kuang A, Smith PJS, Crispi ML, Musgrave ME (1999) Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development. Can J Bot 77, 1439-1446. Rebeille F, Bligny R, Douce R (1980) Oxygen and temperature effects on the fatty acid composition in sycamore cells (Acer pseudoplatanus L.) Biochim Biophys Acta 620, 1-9. Sarmiento C, Garcés R, Mancha M (1998) Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation. Planta 205, 595-600. Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Annu Rev Plant Physiol Plant Mol Biol 49, 611-641. Shelp BJ, Walton CS, Snedden WA, Tuin LG, Oresnik IJ, Layzell DB (1995) Gaba shunt in developing soybean seeds is associated with hypoxia. Physiol Plant 94, 219-228. Skriver L, Thompson GA Jr. (1979) Temperature-induced changes in fatty acid unsaturation of Tetrahymena membranes do not require induced fatty acid desaturase synthesis. Biochim Biophys Acta 572, 376-381. Smith MA, Cross AR, Jones OTG, Griffiths WT, Stymne S, Stobart K (1990) Electron-transport components of the 1-acyl-2-oleoyl-snglycerol-3-phosphocholine ∆12-desaturase (∆12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons. Biochem J 272, 23-29. Stymne S, Stobart K (1985) The effect of temperature on the activity of 2-oleoyl-sn-phosphatidylcholine desaturase in modified microsomal membranes from the cotyledons of maturating safflower seed. Physiol Veg 24, 45-51. Trémolières A, Dubacq JP, Drapier D (1982) Unsaturated fatty acids in maturing seeds of sunflower and rape: regulation by temperature and light intensity. Phytochemistry 21, 41-45. Vos E (2003) Linoleic acid, ‘vitamin F6’– is the Western World getting too much? Probably. Lipid Technology 15, 81-84. Wolf RB, Cavins JF, Kleiman R, Black LT (1982) Effect of temperature on soybean seed constituents: oil, protein, moisture, fatty acids, amino acids and sugars. J Am Oil Chem Soc 59, 230-232.
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OXIDATION STRESS INDUCE LEAF LIPID CHANGES. TARAN Nataliya, BATSMANOVA Ludmila, OKANENKO Alexander. National Taras Shevchenko University of Kyiv, Ukraine. The results of studies devoted to lipid involvement in adaptation processes show that just galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are among the most susceptible polar lipids. MGDG and DGDG occur in all higher plants and are the predominant lipid components of chloroplast membranes. The third glycolipid is sulfolipid sulfoquinovosyl diacylglycerol (SQDG) with a sulfonic acid derivative of glucose. They are thought to be of major importance to chloroplast morphology and physiology, although direct experimental evidence is still lacking. (Dormann et al., 1995). The glycerolipid DGDG is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus (Hartel et al., 1997). Bearing in mind that oxidative stress is a component part of the reaction of plants to many other stresses, any changes in lipid composition are of special significance. Data available evidence that oxidative processes induced by high concentration of ozone affect glycolipid composition. Sakaki et al. (1985) observed marked decreases in these galactolipids, which started within 2 hours of the onset of ozone exposure. Loss of MGDG was more rapid than that of DGDG, resulting in a significant reduction of MGDG/DGDG ratio in spinach (Sakaki et al. 1985) and snapbean (Whitaker et al. 1990) at least. T.Sakaki considers the first phase of the injury development to continue for the initial 8 h of exposure. A little loss of pigments and lipids (MGDG significantly and DGDG slightly) accompanied by slight increase of MDA content take place while this period. However, marked oxidation of ascorbate and inactivation superoxidismutase (SOD) and ascorbate peroxidase (AP) have already occurred during this period. The second phase characterised by massive destruction of pigments and lipids starts with drastic fall of MGDG and less sharp decrease of DGDG accompanied by significant increase of TG, 1,2-DG and MDA (Sakaki, 1998). But it is interesting that anionic lipid - SQDG and PI - amounts were stable while the time of exposure (in spinach leaves, at least). Lipid changes similar to those in spinach were also observed in several plant species, and in broad bean leaves the SQDG increase took place. Oxidative stress induced fall of both GL (MGDG especially drastic) content while SQDG level was stable in a number of plants. As a result SQDG content relative to glycolipid quantity increased by 7-45% (depending upon species) (Sakaki et al., 1985, 1994). But results obtained by Carlsson et al. (1994) with garden pea evidence that moderately enhanced ozone level caused large decreases not only in the contents of MGDG and DGDG, but in SQDG also. Compared with charcoalfiltered air, fumigation with ozone resulted in decreased 18:3 and increased 18:2 in MGDG and SQDG, while the fatty acid composition of DGDG was unaffected. Concerning the molecular bases of these structural changes Sakaki et al. (1990, 1994) suggested that the primary reaction of ozone is the stimulation of galactolipase activity resulting in the enhanced production of free fatty acid in chloroplasts. It is considered that an increase in galactolipase activity is a general feature in response to ozone. Hellgren et al. (1995) demonstrated that ozone stimulated degradation of galactolipids in garden pea leaves probably by galactolipase without effects on the de nova lipid synthesis. Besides, it is well-known that galactolipids as unsaturated compounds are good substrate for forming peroxidation products observed at ozone action (Maccarrone et al., 1997). Therefore it seems to be worthwhile to explore various tension oxidative stress affect upon wheat glycolipid composition and accompany processes. As factor causing oxidative stress we used hydrogen peroxide being known to induce ROS formation. But several studies on stress-acclimation revealed the dual role of H2 O2 as a mediator of oxidative injury causing direct damage to the membrane via formation of hydroxyl radicals and as a signal molecule in the systemic acquired resistance (Lamb, Dixon, 1997) and in the acclimation to the photooxidative stress (Karpinski et al., 1999). Series of laboratory and field experiments were performed.
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Materials and Methods . In laboratory experiments winter wheat plants were grown as hydroponic cultures at illumination 2,5 kLx at plant level and temperature +24? ?. Oxidative stress was created by adding 50, 100, 150, 200 µ? hydrogen peroxide solution in root ambient 4 hours at the age of 7 days. In order to investigate ? 2 ? 2 affect at exposition elongation the other plant group was treated by 500 µ? by spraying their upper part and keep them in the condition for 4, 24, 48, 72 hours. In field experiments we studied glycolipid composition changes in plant leaves of winter wheat plants while oxidation stress action. Oxidative stress was created by spraying of upper plant part hydrogen peroxide solution (1mM) in tillering stage. Glycolipid composition determination was performed in 24 and 96 hours- after treatment (first treatment). Then plants were sprayed again (second treatment) and in 24 and 96 hours- after this treatment and at following tillering stage glycolipid composition was determined. Lipids were extracted according to L.Zill and E.Harmon (1962). Gala ctolipid quantity was determined densitometring TLC plates against standards (Yamamoto, 1980), SQDG – according to Kean (1968) and POL as hydroperoxides (Droillard, Paulin, Massot, 1987) and MDA accumulation (Heath, Packer, 1968). Results and Discussion. Data obtained showed that hydrogen peroxide affect pigment and glycolipid composition with increasing lipid peroxidation activity in dose and time dependent manner. So treatment with various hydrogen peroxide concentration caused only slight MGDG decrease and significant SQDG content increase in all variants (Fig. 1a). But in variant with higher hydrogen peroxide concentration treatment (500µM) the gradual decrease of SQDG content accompanied by gradual MGDG content increase during the exposure time was observed. It was accompanied by SOD activity increase at 24 hour following by decrease at 48 hour (Fig. 2a)and the CAT increase at the first hours with falling down at 24 hour (Fig. 2b). MDA quantity increased significantly in dose dependent manner (Fig.3a). But higher dose (500 µM) caused drastic fall for the first 24 hours and changed by double increase (comparing to the meaning at 24 hour) at 48 hour (Fig.3b). It agreed with the SOD activity splash at 24 hour. Comparison of the glycolipid content changes and enzyme activities during 72 hours of exposition have not revealed any correlation. Results of field experiments showed slight increase of MGDG content and drastic fall of SQDG level in 24 hours with the next MGDG decrease and significant SQDG accumulation. But this compound quantity was stable after the second treatment when light MGDG decease was noted. DGDG level was stable while the whole experiment. Concerning peroxidation products one could note growth of hydroperoxides level as in 24 so and in 96 hour accompanied by stable MDA content after the first treatment (Fig. 5a). SOD activity was severe inhibited, but CAT activity was raised by 20% at 24 hour with next return down to control. Hydroperoxide content splash was similar in 24 hours of second treatment, but fell lower the control meaning (-20%) in 96 hours and stay being low control at flowering stage (Fig. 5b). MDA level was similar the meaning in 24 hours after the first treatment but fell twice in 96 hours and at flowering stage.
96
Exp 4
49,0
40,8
10,2
Exp 72
59,83
38,69 1,47
Exp 3
51,1
35,6
13,3
Exp 48
59,98
36,59
Exp 2
49,6
39,7
10,7
Exp 24
53,17
39,63
7,19
34,7
12,2
Exp 4
53,75
40,6
5,65
Exp 1
53,1
Contr
54,6 MGDG
37,3 DGDG
46,58
Con
8,1
SQDG
44,29
MGDG
DGDG
a
3,44
9,13
SQDG
b
Fig.1a. Hydrogen peroxide action upon glycolipid composition depending upon concentration: exp 1 – 50µM; exp 2 - 100µM; exp 3 - 150µM; exp 4 - 200µM Fig.1b. Hydrogen peroxide treatment (spraying with 500µM) action upon glycolipid composition depending upon exposure: exp 1 – 4 h; exp 2 - 24 h; exp 3 - for 48 h; exp 4 - for 72 h
40
40 M H 2O 2 /g-min
SOD activity units
50
30 20 10 0 1
2
3
Contr
4 Exp
5
30 20 10 0 1
24
48
72
2
Contr
a
3
4
5
Exp
24 48
72
b
Fig.2. Hydrogen peroxide treatment (spraying with 500 µM) action upon SQD (a) and CAT (b) activities depending upon exposure:exposure from 1 up to 72 hour is shown on the x axis.
These events agreed with the SOD activity spla sh in 96 hours and CAT activity increase in 24 and 96 hours. Thus, results of the field experiments showed that second treatment caused less disturbance in glycolipid composition and induced deep hydroperoxide decrease in 96 hours and flowering stage. And the main conclusion from these experiments could be following – the main stable compound of the glycolipid fraction in the condition of hydrogene peroxide impact is DGDG; most labile was SQDG and MGDG stayed between them. And results obtained in field experiments after the second spraying seem to show that adaptation processes alleviated glycolipid disturbance caused by oxidation owing to SOD and CAT activation. And result of this “hardening” one could see in drastic fall of lipid hydroperoxide level in 96 hours and following flowering stage.
97
Difference from the control, %
Difference from the control, %
70 60 50 40 30 20 10 0 Exp 1
Exp 2
Exp 3
60 50 40 30 20 10 0 Exp 1
Exp 4
a
Exp 2
Exp 3
b
Fig.3. Hydrogen peroxide treatment action upon MDA content depending upon concentration (a): exp 1 – 50µM; exp 2 - 100µM; exp 3 - 150µM; exp 4 - 200µM and upon exposure: exp 1 – 4 h; exp 2 – 24 h; exp 3 – 48 h.
Exp 96 Con 96 Exp 24 Con 24
56,34
22,43
66,15 59,2 54,3 MGDG
21,23
50,8
36,36
12,83
21,2
12,65
Con 96
52,8
34,95
12,24
28,18
10,87
Exp 24
53,62
33,94
12,43
28,38 DGDG
Exp 96
17,27
Con 24
58,38 MGDG
SQDG
a
29,36 DGDG
12,26
SQDG
b
Fig.4a. Hydrogen peroxide treatment (spraying with 1 mM) action upon glycolipid composition of field growing wheat plants (mol% after the first treatment in tubing stage ): con 24 - control in 24h –; exp 24 – in 24 h after treatment; con 96 - control in 96h; exp 96 – in 96h after treatment Fig.4b. Hydrogen peroxide treatment (spraying with 1 mM in 96h after the first treatment) action upon glycolipid composition of field growing wheat plants (mol% after the second treatment in tubing stage ): con 24 – control in 24h; exp 24 – in 24 h after treatment; con 96 - control in 96h; exp 96 – in 96h after treatment
Thus, our data are some different from those presented in literature – we observed beside MGDG decrease slight it increase in laboratory experiment and stable DGDG level almost in all experiment variants. MGDG is known to be responsible for membrane monolayer structure whereas DGDG stabilises membrane belayer configuration (Murphy 1982, 1986). Besides, MGDG content changes could be connected with signal function. As it was said earlier, MGDG as most unsaturated lipid is good substrate for oxylipin formation (Blee, Joyard, 1996) and regulate violaxanthin de epoxidase activity (because of epoxidation take place inside rich with MGDG thylakoid domens (Latowski et al., 2000). MGDG level decrease seems to be a result of increase in galactolipase activity considered as a general feature in response to oxidative stress (Hellgren, 1995). Concerning the nature of this increase we suppose that possible activation of MGDG synthase take place and consequent preceding MGDG synthesis for compensation losses as most susceptible to destruction glycolipid and substrate for signal agent forming. In common events observed could be directed perhaps to chloroplast membrane configuration and stability conservation. Besides, it could be connected with processes of protein synthesized transport. In processes of protein transport the transit peptide inserts most efficiently in monolayers of PG, SQDG and MGDG suggesting that these lipid classes are
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mainly responsible for insertion into the target lipid extract (de Kruijff et al. 1998). It is important because of well-known forced synthesis a wide set of specific proteins while stress action.
50 Difference from the control, %
Difference from the control, %
80 60 40 20 0 -20
HPO
MDA
SOD
CAT
-40 -60 -80
24 h
96 h
40 30 20 10 0 -10
HPO
MDA
CAT
-20 -30
24 h
a
SOD
96 h
flowering
b
Fig. 5. Hydrogen peroxide treatment action upon glycolipid composition of field growing wheat plants: a - first spraying with 1 mM in tubing stage; b - second spraying with 500 µM in 96h after the first treatment; HPO hydroperoxides; MDA malone dialdehide; SOD – superoxidesmutase activity; CAT – catalase activity
Considering the possible functions of SQDG content deviations we should accent that significant it content changes were observed while various stress factor action – heat, drought (Okanenko, Taran, 1998), heavy metals (Kosyk et al., 2003) and invasion (Senchugova, Taran, Okanenko, 1999). In this experiment SQDG decrease in some variants could be stipulated by two reasons – competentive using of sulphur for synthesis of specific peptides – phytochelatins defending cell while oxidation inducing factor action. It could take place because of catching APS (common sulphur-supplying precursor) for cystein synthesis (Schmidt, Jäger, 1992). It is not excluded that SQDG could be the source of sulphur supplying for cystein synthesis. Taking into account that at growing plants in water culture the sulphur pool could be limited the suggestions like this could be quite possible. In photosynthetising tissues it seems putative to assume availability of all structural and functional SQDG molecules peculiarities known for today to supply their taking part in adaptation reaction as cytochrome oxidase, CF1 , F1 , ATPase regulators, protectors and stabilising agents for D1/D2 dimers and LHCII (Livn and Racker 1969, Pick et al. 1985). Taking into account the SQDG localisation on the native heterodimer D1/D2 surface (Vijayan et al. 1998), one could assume that it might hold monomers together as dimer (de Kruijff et al. 1998). Therefore it is not excluded that SQDG certain molecular specie accumulation can prevent RC PSII degradation. Function of the compound in non-photosynthesising tissues could be connected with negative charge domination requirement for univalent cation (Na+ and K+) being necessary for lipoprotein complex stabilisation. Besides, this substance can realize ATP-ase and PL A2 activity regulation (Vishwanath et al. 1996) both in photosynthetic and non- photosynthetic tissues. SQDG could inhibit the nonbilayer structure forming by means of making bilayer MGDG structure organisation and takes part in the MGDG synthesis via regulation UDP- galactoso:diacylglycerol galactosyl transferase activity thus correcting MGDG/DGDG ratio in membrane (Coves et al. 1988, Li et al. 1997). Certain role SQDG plays in processes of protein synthesized transport. It is important because of forced synthesis a wide set of specific proteins while stress action. The transit peptide inserts most efficiently in monolayers of PG, SQDG and MGDG suggesting that these lipid classes are mainly responsible for insertion into the target lipid of membrane (de Kruijff et al. 1998). The anionic lipids are the strongest determinants for lipid insertion and MGDG contributes most to the specific insertion into the target lipid extract. Acid lipids like SQDG can also cause vde-independent violoxanthin transformation perhaps because of acid lipid unspesific action (Latowski et al., 2000) Besides, it was shown that charged lipids could suppress the formation of nonbilayer structures by imposing a bilayer arrangement on MGDG (Quinn, 1998). The balance between the charged SQDG, PG and GL is probably controlled by the regulation
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of MGDG synthesis. The activity of UDP-galactose: diacylglycerol galactosyltransferase is dependent upon negatively charged lipids (Coves, Joyard, Douce, 1988; Li, Karlsson, Wieslander, 1997). Thus one could see that SQDG could be a chain of general adaptation mechanism of plant organisms. Summing up we could conclude, that oxidation stress impact upon glycolipid composition in dose and time dependent manner and main changes touch mainly MGDG and, espessially, SQDG level. The character of this changes could be interpreted as directed to support chloroplast membrane configuration and support functional activity RC PS II, CF1 and F1 . References Blee, E., Joyard J. 1996. Envelope Membranes from Spinach Chloroplasts Are a Site ofMetabolism of Fatty Acid Hydroperoxides. J. Plant Physiol. 1996. 110: 445–454. Carlsson AS, Hellgren LI, Sellden G, Sandelius AS. 1994. Effects of moderately enhanced levels of ozone on the acyl lipid composition of leaves of garden pea (Pisum sativum) Physiol. Plant. 91: 754-763 Coves J., Joyard J., Douce R. 1988. Lipid requirement and kinetic studies of solubilized UDPgalactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes. Proc. Natl. Acad. Sci USA. 85: 4966-4970 Dormann P, Hoffmann-Benning S, Balbo I, Benning C. 1995. Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell.;7: 1801-10 Droillard M.J., Paulin A., Massot J.C. 1987. Free radical production, catalase and superoxide dismutase activities and membrane integrity during senescence of petals of cut carnations (Dianthus caryophyllus). Physiol. Plant. 71: 197-202. Hartel H, Lokstein H, Dormann P, Grimm B, Benning C. 1997. Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana. Plant Physiol.115: 1175-84 Heath R.L., Packer L. 1968. Photoperoxidation in isolated chloroplasts. I Kinetic and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys.125: 189-198. Hellgren, L.I., Carlsson, A.S., Sellden, G. and Sandelius, A.S. 1995. In situ leaflipid metabolism in garden pea (Pisum sativum L.) exposed to moderately enhanced levels of ozone. J. Exp.Bot. 6: 221-230. Karpinski, S-, Reynolds, H., Karpinska, B., Wingsle, G., Creissen,G. and Mullineaux, P. 1999. Systemic signaling and acclimation in response to excess excitation energy in Arabidopsls. Science. 284: 654-657. Kean E.L. 1968. Rapid sensitive spectrophotometric method for quantitative determination of sulfatides. Journal of lipid research. 9: 314-327 Kosyk O, Okanenko A, Taran N. 2003. The Effect of Lead on Sulphoquinovosyl- diacylglycerol Content in Leaves and Roots of Wheat Seedlings. In:. Su lfur Transport and Assimilation in Plants. Ed.Davidian J-C et al., Buckhuys Publ. Leiden, The Netherlands: 255-256. Kruijff B. de, Pilon R., Hof R., van’t., Demel R. 1998. Lipid-protein interactions in chloroplast protein import. In: Lipids in photosynthes is: structure, function and genetics. Advances in photosynthesis 6, Siegenthaler P.-A., Murata N., eds. Pp. 191-208, The Netherlands: Kluwer Acad. Publ. Lamb, C. and Dixon, R.A. 1997. The oxidative burst in plant disease resistance. Annu. Rev. Plant PhysioL Plant Mol. Biol. 48: 251-275. Latowski, D., Kostecka, A. and Strzalka, K. 2000. Effect of monogalactosyldiacylglycerol and other thylakoid lipids on violaxanthin de-epoxidation in liposomes. Bioch. Soc. Trans. 28 Part. 6: 810-812. Li L., Karlsson O.P., Wieslander A. 1997. Activating amphiphiles cause a conformational changes of the 1,2diacylglycerol transferase from Acholeplasma laidlavii membranes according to proteolitic digestion. J. Biol. Chem. 272: 29602-29606. Livn A., Racker E. 1969. Partial resolution of the enzymes catalyzing photophosphorylation. V. Interaction of coupling factor I from chloroplasts with ribonucleic acid and lipids. – J. of Biol. Chem. 244: 1332-1338. Maccarrone M., Veldink G.A., Vliegenthart J.F.G., Agroé A.F. 1997. Ozone stress modulates amine oxidase and lipoxygenase expression in lentil (Lens culinaris) seedlings. FEBS Letters 408: 241-244 Murphy D. 1982. The importance of non-planar bilayer regions in photosynthetic membranes and their stabilisation by galactolipids. FEBS Lett 150: 19-26. Murphy D. 1986. The molecular organisation of the photosynthetic membranes of higher plants. Biochim.Biophys.Acta864: 33-94. Okanenko A., Taran N. 1998. Impact of heat stress on cereal lipid composition. In: Responses of Plant to Air Pollution and Global Change. Ed.: L.J.De Kok, I.Stulen. Backhuys Publishers, Leiden, The Netherlands. 1998.P.391-394
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Pick U., Gounaris K., Weiss M., Barber J. 1985. Tightly bound sulfolipids in chloroplast CF0 -CF1 . Biochim. Biophys. Acta 808: 415-420. Quinn P.J. 1998. The role of lipids in stability of plant membranes. In: Advances in plant lipid research, Sanches J., Gerda-Olmedo E., Martinez-Force E., eds. pp. 361-366, Spain. Quinn P.J., Williams J.P. 1983. The structural role of lipids in photosynthetic membranes. Biochim. Biophys. Acta. 737: 223-266. Sakaki, T., Saito, K., Kawaguchi, A., Kondo, N. and Yamada, M. 1990. Conversion of monogalactosyldiacylglycerols to triacylglycerols in ozone-fumigated spinach leaves. Plant Physiol. 94: 766-772. Sakaki T. 1998. Photochemical oxidants: toxicity. In: de Kok LJ and Stulen I (eds) Responses of Plant Metabolism to Air Pollution and Global Change, pp 117—129. Backhuys Publishers, Leiden, The Netherlands Sakaki T, Ohnishi J, Kondo N and Yamada M. 1985. Polar and neutral lipid changes in spinach leaves with ozone fumigation. Triacylglycerol synthesis from polar lipids. Plant Cell Physiol 26: 253—262 Sakaki T, Tanaka K and Yamada M (1994) General metabolic changes in leaf lipids in response to ozone. Plant Cell Physiol 35: 53-62 Schmidt A., Jager K. 1992. Open questions about sulfur metabolism in plants. In: Annual Review of Plant Physiology and Plant Molecular Biology 43: 325-349. Senchugova N, Taran N and Okanenko A. 1999. Virus impact upon bean photo-synthesising tissue lipid composition. Arch Phytopath Pflanz 32: 471--477 Taran N, Okanenko A ,. Musienko N. 2000. Sulpholipid reflects plant resistance to stress-factor action. Biochem. Soc. Trans. 28, part 6: 922-924. Vishwanath B.S., Eichenberger W., Frey F.J., Frey B.M. 1996. Interaction of plant lipids with 14 kDa phospholipase A 2 enzymes. Biochem. Journ. 320: 93-99. Whitaker BD, Lee EH, Rowland RA. 1990. EDU and ozone protection: Foliar glycerolipids and steryl lipids in snapbean exposed to O3 Physiol. Plant. 80: 286-293 Wingsle, G., Mattson, A., Ekblad, A., Hallgren, J.-E. and Selstam, E. 1992. Activities of glutathione reductase and superoxide dismutase in relation to changes of lipids and pigments due to ozone in seedlings of Pinus sylvestris (L.). Plant Sci. 82: 167-178. Yamamoto H. 1980. High speed quantitative assey on TLC/HPTLC plates. In: Instrumental HPTLC. Ed. W. Bertch and Raser R. New York. 367-384 Zill L., Harmon E. 1962. Lipids of photosynthetic tissue. I.Salicilic acid chromatography of the lipids from whole leaves and chloroplasts. Biochem.Biophys.Acta. 57: 573-575.
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OCCURRENCE OF LIPOPHILIC MICELLAE FORMED BY FATTY ALCOHOLS AND FATTY-ACID SODIUM SALTS IN JOJOBA-WAX AQUEOUS HYDROLYZATE
A.G. VERESHCHAGIN Institute of Plant Physiology, Russian Academy of Sciences, 127276 Moscow, Russia
Introduction
Jojoba (Simmondsia chinensis [Link] Schneider, Buxaceae) is cultivated in certain warm countries for obtaining its seed oil comprising 45–60% of dry wt. The difference between jojoba oil and all other plant lipids is that this oil is not a triglyceride but a wax ester between very-long-chain fatty acids and higher fatty alcohols without the intervention of glycerol. This wax has numerous applicatio ns related to its physical and/or chemical properties. As a substitute for a hard-to-get sperm-whale wax, it is widely used as a component of skin-care, cosmetic, and pharmaceutical products, as well as an unmetabolized noncaloric “fat”, and finds many industrial applications in preparing surfactants, highpressure lubricants, protective coatings etc. (Wisniak, 1977). Therefore, several studies were devoted to the composition of jojoba wax (Hamilton and Raie, 1975; Spencer et al., 1977; Wisniak, 1977), but results of some of them have been rather contradictory (see below). Results and Discussion In our work, while investigating the biosynthesis of this wax, it became essential to prepare tens-ofmg quantities of its fatty-acid and fatty-alcohol components. To this end, 170 mg of wax (Jojoba Bean Oil, Sigma-Aldrich Co., Cat. No. J-1375) was first purified by placing it on the 10 × 0.8 cm Woelm Silica Gel (Woelm, CCM, France) column and eluting with 8 ml of hexane. As a result, 165 mg of 99.6% pure wax was obtained. It could be assumed that the most immediate approach to obtaining acids and alcohols from jojoba wax would consist of its direct saponification in an alkaline medium. For this purpose, we followed the instructions given in the standard textbook by Kates (1972), who recommended boiling of 30 mg of wax in a 0.3 N NaOH in MeOH-water (9:1) mixture for 1–2 h. However, in our experiments, refluxing of the jojoba wax in a 4% NaOH for up to 6 h failed to bring about complete hydrolysis of the ester bonds. Such results were consistent with those described earlier, when an exhaustive saponification of this wax was not achieved by using a 30% KOH solution or a 7-day-long hydrolysis with 1 M EtONa in EtOH (Miwa, 1971). It seems that the difficulties in performing alkaline hydrolysis of S. sinensis wax were caused by the fact that it almost totally consists of very-long-chain (C38–C46 ) aliphatic esters (Spencer et al., 1977). Wax splitting can also be performed via its acid-catalyzed alcoholysis (Miwa, 1971) and some authors used to this end mild ethanolysis in the presence of HCl (Miwa, 1984). Subsequently, however, it was found that the products of this reaction, regardless of its duration and HCl concentration, always contained considerable amounts (8% or more) of wax. In its ester composition, this wax notably differed from the initial one, and therefore it was concluded that it was synthesized in the reaction mixture itself, due to the setting-up of an equilibrium between wax esters, fatty acid ethyl esters, and fatty alcohols according to thermodynamic rules, which brought about wax resynthesis (Graille et al., 1986).
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We suggested that such resynthesis could be abolished by replacing EtOH with a more polar alcohol, MeOH: their dielectric constants directly proportional to the polarity are equal to 24.3 and 32.6, respectively (Reichardt, 1973). Indeed, refluxing of the mixture of purified wax (150 mg), dry MeOH (10 ml), and AcCl (0.5 ml) for 120 min brought about, as shown by TLC, a complete conversion of the wax into fatty alcohols and fatty acid methyl esters. Earlier, the saponification of very-long-chain fatty acid ethyl esters derived from jojoba wax required an overnight boiling with 1 N aqueous-alcoholic KOH solution (Miwa, 1971; Hamilton and Raie, 1975). Meanwhile, in our work, exhaustive saponification of their methyl esters with NaOH in MeOH : water (99 : 1) was achieved in only 60 min. In order to separate wax unsaponifiables, including fatty alcohols, from the fatty-acid Na salts, Kates (1972) recommended the extraction of the alkaline hydrolyzate (see above) with 4 x 5 ml of petroleum ether, bp 30–60o C. However, as regards jojoba wax, our results showed that treatment of its hydrolyzate with either hexane or Et2 O brought about a transfer in the organic phase not only of fatty alcohols, but also the bulk of fatty acids as their Na salts. As a result, this phase formed a stable opaque emulsion, which did not break for a long time. Thus, a commonly used technique of separating unsaponifiables by the extraction of lipidsaponification products with nonpolar solvents proved inadequate with respect to jojoba wax. For establishing the reason of this phenomenon, it must be recognized that this wax differs from other plant lipids in the composition of its hydrolyzate, which consists of equal amounts of unsaponifiable (fatty alcohol) and fatty acid fractions; both of them include solely very-long-chain monounsaturated aliphatic moieties. Moreover, it has long been known that soap micellae have the power of holding oil-soluble materials in what is an equivalent to hydrocarbon solution between their long hydrocarbon chains (Markley, 1961). Therefore, we conclude that, in the aqueous-alkaline hydrolyzate of jojoba wax, fatty alcohols and fatty-acid Na salts formed stable lipophilic micellae highly soluble in hexane and Et2 O. In order to break these micellae, we employed the technique of solid-phase extraction rather than the liquid-phase one. The products of alkaline hydrolysis of the jojoba wax methanolyzate were exhaustively dried in vacuo with heating, and the fatty alcohols were repeatedly washed out from the solid phase with dry Et2 O. Subsequently, this phase was acidified, and the fatty acids were recovered with hexane. The purity of both preparation was assessed on the basis of the fact that a minimal weight of lipids, which could be visualized on a TLC plate with phosphomolybdic acid, was equal to 0.1 µg. This test showed that the preparations of fatty acids and fatty alcohols were 99.4 and 99.2% pure, respectively. The yield of their sum estimated gravimetrically amounted to 98.4% of the theoretical one. Table 1. Fatty acid and fatty alcohol composition of jojoba wax Acids Palmitic Palmitoleic Stearic Oleic Arachidic Eicos-11-enoic Docosanoic Docos-13-enoic Tetracos-15-enoic
% 1.2 0.3 0.1 11.4 0.1 71.5 0.2 13.8 1.4
Alcohols Hexadecanol Hexadec-7 enol Octadecanol Octadec-9-enol Eicosanol Eicos-11-enol Docosanol Docos-13-enol Tetracos-15-enol
% 0.1 – 0.2 1.1 trace 43.8 1.0 44.9 8.9
The composition of fatty acids as their methyl esters. and fatty alcohols determined by capillary GLC as described earlier (Pchelkin et al., 2001) is shown in Table 1. The results obtained were close to those found elsewhere (Miwa, 1971, 1984; Spencer et al.., 1977; Wisniak, 1977). Moreover, they were consistent with the evidence that all monounsaturated moieties of jojoba wax are characterized
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by ω9 structure (Spencer et al., 1977; Wisniak, 1977) and did not support the claims of Hamilton and Raie (1975), according to which the fatty alcohols of this wax contained double bonds at the ω4-, ω5-, ω6-, ω7-, ω8-, and ω9-positions. Finally, our preliminary data suggest that fatty alcohols can be quantitatively separated from fatty acid methyl esters by single -solvent dry-column chromatography. References Graille, J., Pina, M. and Pioch, D. (1986) Routine analysis of jojoba wax fatty acids and alcohols by single column capillary GC. J. Amer. Oil Chem. Soc. 63, 111-116 Hamilton, R.J. and Raie, M.J. (1975) Structure of the alcohols derived from wax esters in jojoba oil. Chem. and Phys. Lipids 14, 92-96. Kates, M. (1972) Techniques in Lipidology. North-Holland Publ. Co. Amsterdam. Markley, K. (1961) Fatty Acids. Their Chemistry, Properties, Production, and Uses. Interscience Publishers, New York and London. Miwa, T.K. (1971) Jojoba oil wax esters and derived fatty acids and alcohols. J. Amer. Oil Chem. Soc. 48, 299-306. Miwa, T.K. (1984) Structural determination and uses of jojoba oil. J. Amer. Oil Chem. Soc. 61, 407-410. Pchelkin, V.P., Kuznetsova, E.I., Tsydendambaev, V.D., and Vereshchagin, A.G. (2001) Determination of the positionalspecies composition of plant reserve triacylglycerols by partial chemical degradation. Russian J. Plant Physiol. 48, 701-707. Reichardt, C. (1969) Loesungsmittel-Effekte in der organischer Chemie. Verlag Chemie, Berlin. Spencer, G.F., Plattner, R.D. and Miwa, T.K. (1977) Jojoba wax analysis by HPLC and GC/MS. J. Amer. Oil Chem. Soc. 54, 187-189. Wisniak, J. (1977) Jojoba oil and derivatives. Progr. Chem. Fats Lipids 15, 167-218.
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EFFECT OF TOBACCO MOSAIC VIRUS INFECTION ON PHOSPHOLIPID CONTENT, PHOSPHOLIPASE D ACTIVITY AND REACTIVE OXYGEN SPECIES PRODUCTION IN TOBACCO LEAVES I.M. KOTEL’NIKOVA1 , E.V. NEKRASOV2 , A. V. KRYLOV2 1 Amur Research Integrated Institute of Amur Research Centre, Far East Branch, Russian Academy of Sciences 1, Relochny Lane, Blagoveshchensk, 675000 Russia 2 Botanical Garden of Amur Research Centre, Far East Branch, Russian Academy of Sciences 1, Relochny Lane, Blagoveshchensk, 675000 Russia
1. Introduction Phospholipids play multiple roles in cells. They do not have only a static function, serving as the matrix for proteins involved in different cellular processes. Phospholipids are active participants that influence on the properties of the proteins associated with the membrane and serve as signal precursors or signalling molecules themselves [1]. Phospholipids signalling is mediated by phospholipases, which hydrolyse them. The major plant phospholipase family is the phospholipase D with the greatest molecular variety [2, 3]. Phospholipase D is involved in wide variety of cellular and physiological processes [3, 4, 5]. Therefore Wang (2002) has raised a question, whether plants use phospholipase D more than other organisms as part of the regulatory machinery in cellular functions [3].
Phospholipase D (EC 3.1.4.4) hydrolyses phospholipids at the terminal phosphodiesteric bond, producing phosphatidic acid and a free hydrophilic head group [6]. It also can transfer the phosphatidyl moiety to primary alcohols to form phosphatidylalcohol [6]. Hydrolysis of membrane phospholipids by phospholipase D occurs either during physiological changes in plants, or during responses to a variety of stress factors. Phosphatidic acid accumulation was induced by wounding, osmotic stress, as well as treatment with abscisic acid, ethylene and with variety of pathogen elicitors [3, 7]. In plant cells, phosphatidic acid can trigger several signalling cascades and act on several targets, such as small G-proteins, protein kinases, MAP kinases, phospholipases C and A2 , and phosphatidylinositol-4-kinase [7]. The physiological role of phospholipase D in the defence to plant pathogen is still poorly understood. The activation of phospholipase D was occurred in plants infected with bacteria, fungi, and dodder [811]. However facts concerning a participation phospholipase D in plant-pathogen interaction have a contradictory character. It has been shown that in rice leaves undergoing with bacteria, that phospholipase D molecules can be distributed along the plasma membrane by different manner, which depends on type of interaction. During undergoing susceptible interaction phospholipase D was distributed evenly along the plasma membrane but during undergoing resistant interaction phospholipase D became clustered at the site where the pathogen attached [11]. However some authors have not observed involvement of phospholipase D in cellular signal transduction in plantpathogen interaction. Phospholipase D activity did not change in tobacco leaves infected with Erwinia amylovora [8], and in cultured soybean cells treated by elicitors - bacterial harpin, oligogalacturonic acid and extract of Verticillium dahliae, which activated oxidative burst in plants by different signalling ways [12]. One possible mechanism by which phospholipase D is involved in defence responses is by affecting on reactive oxygen species generation via activation NADPH oxidase [2, 3,
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4]. Another possible mechanism of action is by regulation the traffickin g and secretion of defence compounds [2, 3, 4]. An activation defence response of resistant plants may involve separate signalling pathways and to vary from plant-pathogen combination [13]. The defence signal transduction pathways that induce resistance to bacteria and fungi differ from the pathways, which trigger resistance to viruses [14]. Therefore it is necessary to investigate more models of plant-pathogen interactions. We have been investigated phospholipid content and phospholipase D activity in the leaves of two tobacco (Nicotiana tabacum L.) cultivars infected with virus. Induction of plant resistance to viral infection is associated with the appearance of hypersensitive response. Burst of active oxygen is an early event which occurred after contacts many resistant plants with viruses [15-17]. Furthermore, chemical treatments of the infected leaves with inhibitors of generation or scavengers of active oxygen species caused a reduction or an inhibition in necrotic lesion formation [16, 17]. An increase of lipoxygenase activity and a decrease in polyunsaturated fatty acids [15, 18, 19] and an appearance of lipid peroxidation process [15, 17] cause the changes in cell membrane permeability and electrolytes leakage from cells [15, 17, 18], that occurs during some hours after inoculation plants with viruses. As a result, a local cell death at the infection site is initiated. The death of host cells during hypersensitive response is not sufficient in itself to limit the spread of a virus. Cell death is a result of induction of defence response causing pathogen localisation as it considered [13, 14]. The susceptibility of plants to viruses leads to their infection and development of various symptoms of disease. Virus multiplication in plants brings various changes such as morphological, physiological and biochemical ones. The changes are result both the intracellular viral parasitism, and the activation of plant defensive mechanisms. The work compares the phospholipid composition and phospholipase D activ ity in the susceptible and resistant plants infected with viruses. 2. Plant material and method 2.1. Plant material Two tobacco cultivars - Xanthi necrotic (Xanthi nc.) and Samsun was used. Tobacco plants cv. Samsun are susceptible to tobacco mosaic virus (TMV), plants cv. Xanthi nc. are resistant and evolve hypersensitive response. Tobacco (Nicotiana tabacum L.) plants were grown in ? greenhouse. The infection was performed using mechanical inoculation leaves with the common TMV strain. Control plant leaves were rubbed with water. Six leaves of each plant, starting from an uppermost, fully expanded leaf, were treated. For analyses the disks were cut out with a cork borer from the entire leaf area devoid of a midrib. 2.2. Lipid analysis Before lipid extraction, the leaves were boiled for 3 min. The lipids were extracted according to Bligh and Dyer technique [20]. The phospholipids were separated by two-dimensional TLC [21]. Phospholipid classes were identified according to the positioning of their spots on the chromatograms, as well as by using specific reagents [22]. The phospholipid amount was determined as lipid phosphorus content [23]. 2.2. Determination of phospholipase D activity The phospholipase D activity was determined by its transferase activity as the amount of phosphatidylmethanol (PM) produced. To this end, we incubated an aqueous enzyme extract in 50% (v/v) aqueous methanol in the presence of phosphatidylcholine using the technique described in [24]. Phosphatidylcholine and PM were visualised and quantified as described in [24].
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Detection of superoxide radicals was based on its ability to reduce nitroblue tetrazolium, as described in [25]. 3. The phospholipid content and phospholipase D activity in the infected tobacco leaves during the appearance of the disease symptoms 3.1. The phospholipid content and phospholipase D activity in tobacco leaves The phospholipids characteristic of photosynthesizing tissues of higher plants, viz., phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, and phosphatidylserine were identified in tobacco leaves. The phospholipase D activity in leaves of healthy cv. Xanthy nc. plants exceed than in cv. Samsun leaves (Fig. 2). In leaves of plants cv. Xanthi nc. activity of enzyme varied from 9,2 to 15,1 nmol / (mg 15 min), and in leaves of plants cv. Samsun - from 5,8 to 11,4 nmol / (mg of 15 min). In our experiments, within one or two days after infection, numerous spots of dead tissue, i.e., the necrotic zones, appeared on the cv. Xanthy nc. leaves. During subsequent days, these zones increased in size, and by the fourth day after infection, they became brown. The first disease symptoms in the inoculated and young cv. Samsun leaves appeared after 7 days and were manifested as a faint yellowgreen mosaic. Sampling time was chosen on a basis of differences in the time of appearance of infection symptoms for the two cultivars. For cv. Xanthy nc., the time of sampling was 0.1 day (2--2.5 h after infection), 0.5 day (12 h), as well as within 1, 2, 3, and 4 days after infection; for cv. Samsun, these time points were 0.1, 0.5, 1, 2, 4, and 7 days after infection. Sampling was performed in such a way that various leaf stories were represented. 3.2. The phospholipid content and phospholipase D activity in infected leaves of resistant tobacco plants In the course of development of the hypersensitive response in cv. Xanthy nc. substantial changes in the content of separate phospholipid classes in leaves took place (Fig. 1). As regards the content of major phospholipid classes, the greatest changes were experienced by phosphatidylglycerol. The occurrence of necrotic lesions within 2 days after infection was accompanied by a subsequent fall in the proportion of this phospholipid. As a result, the most substantial differences between infected and control plants were observed after 4 days (Fig. 1a). During the same period, the contents of phosphatidic acid and diphosphatidylglycerol gradually increased (Fig. 1b). There was no change in the total phospholipid content in Xanthi nc. leaves during 4 days after infection TMV. a 50,0
5,0
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Phospholipid content, %
b
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Figure 1. Relative content (a) major and (b) minor phospholipids in cv. Xanthy nc. Leaves after TMV infection. Solid lanes in dicate control plants; dotted lines indicate infected plants. (1, 1`) phosphatidylcholine; (2, 2') phosphatidylglycerol; (3, 3') phosphatidylethanolamine; (4, 4') phosphatidylinositol; (5, 5') phosphatidic acid; (6, 6') phosphatidylserine; (7, 7') diphosphatidylglycerol.
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The content of each phospholipid class is expressed as the percentage of this phospholipid phosphorus in relation to the total phospholipid phosphorus. Means of three independent experiments and their standard errors are presented.
A decline in the content of phosphatidylglycerol during the hypersensitive response development can reflect the degradation of chloroplasts, because this phospholipid class is an essential component of these organelles. At the same time, plant diphosphatidylglycerol was found only in mitochondria, and therefore the rise in the content of diphosphatidylglycerol within three or four days after infection can demonstrate an increase in the proportion of mitochondria in the infected leaf cells. An enhance in the phosphatidic acid content during hypersensitive response development is of special interest. An increase in the proportion of this phospholipid coincided with the development of necrosis (Fig. 1). This increase can reflect an enhanced degradation of other phospholipids, in particular phosphatidylglycerol, under the action of phospholipase D.
Enzyme activity, nmol/(mg15 min)
During the development of the hypersensitive response, phospholipase D activity in cv. Xanthy nc. leaves differed from that in control plants. After 2--2.5 h after infection (0.1 day), water-rubbed control leaves always exceeded the TMV -infected leaves in the phospholipase D activity. After 12 h and, particularly, within 1 day after infection, infected leaves differed little from the control ones in the phospholipase D activity (Fig. 2). Three and four days after infection, when a large-scale cell death in the necrotic lesions took place, infected leaves somewhat exceeded the control ones in the phospholipase D activity (Fig. 2). This was accompanied by an increase in the phosphatidic acid 15 14
2'
13
2
12
1
11 10
1'
9 8 7 0,1 0,5 1
2
3
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5
6
7
Time after infection, days
content (Fig. 1b). Figure 2. Phospholipase D activity in (1, 1') cv. Samsun and (2, 2') cv. Xanthy nc. leaves after the TMV infection. Solid lines indicate control plants; dotted lines indicate infected plants. Phospholipase D activity is presented as nmol of PM formed for 15 min per mg of tissue fr wt. Means of three independent experiments and their standard errors are presented.
Changes in the cell ultrastructure during the hypersensitive response development occurred not only in the local necrotic lesions, but also in the adjacent tissues. The phospholipids and the phospholipase D activity were also studied in the tissues remote from the sites of origination of necrosis. These studies were performed using leaves, one half of which was infected with TMV, and the other one was treated with water. Two days after infection, these leaf halves differed from each other and from the control, water-treated leaves in the content of various phospholipids. Leaf halves with necrotic lesions were characterised by relative decrease in the phosphatidylglycerol content and an increase in phosphatidylethanolamine and phosphatidic acid contents (Figs. 3a, 3b). Phosphatidylethanolamine and, particularly, phosphatidic acid contents in the tissues remote from the necrotic lesions also enhanced. The enhance in the phosphatidic acid percentage was observed not only in the necrotic zones (Fig. 3b), but also in the sites of the same leaf remote from these zones; in the latter case, the phosphatidic acid content is increased to the greatest extent. Such increase of phosphatidic acid level shows a possible signalling role one in the tissues adjacent to the necrotic lesions.
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Phospholipase D activity in both halves of infected leaves changed similarly as the phosphatidic acid content; this activity increased as compared to the control plants (Fig. 3c). However, there was a considerable variation in the phospholipase D activity between replications, and therefore these differences were insignificant. a 60,00
Phospholipid content, %
50,00 40,00 30,00 20,00 10,00
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PE
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0,08
Phosphatidic acid content, nmol/mg
4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5
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c 0,09
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Enzyme activity, nmol/(mg 15 min)
0,00
0,0 1 2 3 Phospholipase D
Figure 3. Relative content of (a) major and (b) minor phospholipids, phosphatidic acid content, and (c) phospholipase D activity in various regions of cv. Xanthy nc. leaves two days after the TMV infection. (1) Control plants; (2) water-treated leaf halves devoid of necrotic lesions, (3) TMV-infected leaf halves with necrotic lesions. (a, b) Content of each phospholipid class expressed as the percentage of this phospholipid phosphorus in relation to the total phospholipid phosphorus; (c) phospholipase D activity as nmol PM formed for 15 min per mg fr wt; phosphatidic acid content was expressed as nmol of PM per mg fr wt. Means of two independent experiments and their standard errors are presented.
We suggest that phospholipase D can be responsible for an increase in the phosphatidic acid proportion in tobacco cv. Xanthy nc. leaves manifesting a hypersensitive response. First, in spite of cell death over the extensive regions of leaves, the enzyme activity did not decrease, but, on the contrary, had the tendency for an enhance (Figs. 2, 3c), whereas the enzyme activity in the infected leaves of a susceptible cv. Samsun virtually did not differ from the control one. Second, cv. Xanthy nc. exceeded cv. Samsun in the phospholipase D activity in the control leaves (Fig. 2). Third, by the example of plants manifesting a hypersensitive response after bacterial infection, Young et al (1996) was demonstrated that phospholipase D can be activated; this activation resulted from a redistribution of the enzyme molecules inside a cell in such a way that the substrate became accessible for the
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enzyme [11]. The evidence obtained here is similar to the results on the effect of severe wounding of tissues on the phospholipid metabolism and phospholipase D activity [26]. These results showed that both the damaged sites and the regions remote from these sites were characterised by an increase in phospholipid hydrolysis and phosphatidic acid content [26]. The physiological role of both phospholipase D and the changes in phosphatidic acid content found in this work in the course of hypersensitive response in leaves remains unknown. This enzyme is involved in ageing processes induced by abscisic acid [27]. Therefore, the accumulation of phosphatidic acid as a product of the hydrolase activity of phospholipase D in leaves can accelerate leaf ageing after the manifestation of a hypersensitive response. Another function of phospholipase D and phosphatidic acid consists in their involvement in the generation of a superoxide anion; this function seems to be important for the manifestation of a hypersensitive response [28]. Within the initial hours after infecting cv. Xanthy nc. leaves, we observed some decline in the phospholipase D activity as compared to the control (Fig. 2); this fact does not exclude a possible involvement of this enzyme at the very onset of the development of infection, i.e., in the course of the first minutes and hours of these processes. 3.3. The phospholipid content and phospholipase D activity in infected leaves of susceptible tobacco plants
No substantial differences in the total content of phospholipids of the susceptible cv. Samsun were found within 7 days after TMV infection. Moreover, we failed to find any noticeable differences between the control and infected plants and in the content of various phospholipid classes. Phospholipase D activity in infected leaves also remained at the control level during 7 days (Fig. 2). The infected cv. Samsun leaves also did not substantially differ from the control ones in the content of phosphatidic acid, a product of hydrolase activity of phospholipase D in vivo. This pattern was observed throughout the entire time period from the viral infection to the occurrence of disease symptoms. Nevertheless, the development of the systemic infection in plants is accompanied by various changes in cell organelles, as well as the occurrence of unusual cell structures and virusinduced inclusions. Ruzicska et al. [18] also stated that there was no changes in the contents of phospholipids and their fatty acids in the susceptible tobacco cv. Xanthy plants within 6 days after TMV inoculation. Changes in the in the polar lipid content were observed after infection with virus of peanut [29] and of barley [30] at later stages of the disease development. The presented results demonstrate that phospholipid content in tobacco plants infected with the virus depend on a reaction type of plant-host and on period of the pathogen multiplication. Changes of phospholipid composition reflect as cell reorganisations under the pathogen influence as the plant defence reaction. 4. The phospholipid content, phospholipase D activity and generation of reactive oxygen species in infected leaves of susceptible tobacco plants on the late stage of viral infection Two groups tobacco plants cv. Samsun of different ages were inoculated with TMV. One group of plants were infected in the age of 14 weeks (vegetation phase), another - in the age of 16 weeks (before a phase of flowering). To inoculate used leaves of different age: unfolded and fully expanded leaves. Samples for the analysis were collected simultaneously when plants were in the age of 18 18,5 weeks. Multiplication of virus occurred in plants from 2 to 4 weeks. Leaf samples was collected separately from upper, middle and lower positions. All inoculated leaves were on the middle position of plants. The symptoms of systemically infection were appeared on young leaves above the inoculated ones. Control leaves of different ages were collected from healthy plants. We observed phospholipid content, phospholipase D activity, generation of reactive oxygen species and viability in tobacco leaves infected with TMV. Leaves of tobacco plants infected with TMV indicated lower viability than leaves of control plants. The loss of viability is evidence of a development of an ageing process in the infected leaves. The total lipid content is decreased in infected plants especially in leaves at upper position. However, in 110
the total content of phospholipids no substantial differences between infected and control plants was observed. Therefore the phospholipid proportion in total lipid content is enlarged. There were observed some changes in the content of major phospholipids in leaves of infected plants at upper and middle position. The changes were rather expressed in leaves at the upper position, which development occurred under viral infection, than in expanded and mature leaves at middle position. The contents of the phosphatidylglycerol and phosphatidylinositol were a little reduced and the phosphatidylethanolamine content was increased in these leaves. The phosphatidylethanolamine content was declined in the infected plants in the leaves at lower position. However, an age influence on the contents of total lipids and phospholip ids in leaves of control plants is more significant in comparison with the influence of a virus infection. The contents of total lipids and phospholipids were much higher in younger leaves at upper position, than in leaves at middle and lower position. Compared to leaves at the upper position, in leaves at middle and lower positions the percentage contents of phosphatidylglycerol and phosphatidylinositol were reduced and of phosphatidylethanolamine and phosphatidic acid were increased. Besides, the changes in the phospholipids content in the leaves at middle and lower positions correlate with faster loss of viability by these leaves. Phospholipase D activity and generation of reactive oxygen species considerably enhanced in leaves at upper and middle positions of infected plants in comparison with the same leaves of the control plants (Figs. 4a, 4b). An activity to reduce nitroblue tetrazolium was not similar in the discs from leaves of different positions of infected tobacco plants (Fig. 4b). The activity to reduce nitroblue tetrazolium was in 2-4 times higher in the leaves at the upper and lower positions of infected tobacco plants, and in 7-10 time higher in the leaves at the middle position (Fig. 4 b), than in the leaves of a control plants. 7,0
Reduction of nitroblue tetrazolium, optical density at 580 nm
Enzyme activity, nmol/ (mg 15 min)
b 6,0 5,0 4,0 3,0 2,0 1,0 0,0 Upper leaf samples
Middle leaf samples
Lower leaf samples
0,120 0,100 0,080 0,060 0,040 0,020 0,000 Upper leaf samples
Middle leaf Lower leaf samples samples
Figure 4. Phospholipase D activity (a) and generation of superoxide anion (b) in leaf samples cv. Samsun on the late stage of viral infection. Each value represents the average of two experiments.
The activity of phospholipase D is increased in the infected leaves of the tobacco plants (4 a). Despite of this fact, the phospholipid content was not reduced. At the same time, the increase of enzyme activity in infected leaves coincided with generation of reactive oxygen species. Probably, phospholipase D plays mainly a signal role in the infected leaves, than a metabolic. Thus, the late stage of viral infection affects not only the metabolism in susceptible plants but also induces a delaying in defence reactions, than what happens at the active response.
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4. Refe rences [1] Munnik, T., Irvine, R.F. and Musgrave A. (1998) Phospholipid Signalling in Plants. Biochim. Biophys. Acta. 389, 222-272. [2] Wang X. (2000) Multiple Forms of Phospholipase D in Plants: the Gene Family, Cat alytic and Regulatory Properties, and Cellular Functions. Prog. Lip. Res. 39, 109-149. [3] Wang X., Wang C., Sang Y., Qin C. and Welti R. (2002) Networking of phospholipases in plant signal transduction. Physiol. Plant. 115, 331-335. [4] Wang, X. M. (1999) The role of phospholipase D in signalling cascades. Plant Physiol. 120, 645 – 651. [5] Pappan, K. and Wang, X.M. (1999). Molecular and biochemical properties and physiological roles of plant phospholipase D. Biochim. Biophys. Acta. 1439, 151-166. [6] Heller, M. (1978) Phospholipase D. Adv. Lipid Res. 16, 267-326. [7] Munnik T. (2001) Phosphatidic Acid: an Emerging Plant Lipid Second Messenger. Trends Plant Sci. 6, 227-233. [8] Huang, J.-s., Goodman, R.N. (1970) The relationship of phosphatidase activity to the hypersensitive reaction in tobacco induced by bacteria. Phytopathology. 60, 1020-1021. [9] Saini, R.S., Chawla, H.K.L. and Wagle, D.S. (1990) Catabolic activity of two phosphoric diester hydrolases in wheat leaves inoculated with brown rust, Puccinia recondita . Biol. Plant. 32, 313-318. [10] Sharma, S., Sanwal, G.G. and Khanna, R. (1985) Lipids of Cuscuta reflexa and changes in lipids of its host plants after infection. Physiol. Plant. 63, 315-321. [11] Young, S.A., Wang, X., Leach, J.E. (1996) Changes in the plasma membrane distribution of rice phospholipase D during resistant interaction with Xanthomonas oryzae pv oryzae. Plant Cell. 8, 1079-1090. [12] Taylor, A. T. S. and Low, P. S. (1997) Phospholipase D involvement in the plant oxidative burst. Biochem. Biophys. Res. Com. 237, 10 - 15. [13] Heath, M.C. (2000) Hypersensitive response-related death. Plant Mol. Biol. 44, 321–334. [14] Murphy, A.M., Gilliland, A., Wong, C. E., West, J., Singh, D.P. and Carr, J. P. (2001) Signal transduction in resistance to plant viruses. European J. Plant Pathol. 107, 121 – 128. [15] Kato, S. and Misawa, T. (1976) Lipid peroxidation during the appearance of hypersensitive reaction in cowpea leaves infected with cucumber mosaic virus. Ann. Phytopath. Soc. Japan. 42, 472 – 480. [16] Doke, N. and Ohashi, Y. (1988) Involvement of an O2 - generation system in the induction of necrotic lesions on leaves infected with tobacco mosaic virus. Physiol. Mol. Plant Pathol. 32, 163-175. [17] Beleid El – moshaty, F. I., Pike, S. M., Novacky, A. J. and Sehgal, O. P. (1993) Lipid peroxidation and superoxide production in cowpea (Vigna unguiculata) leaves infected with tobacco ringspot virus or southern bean mosaic virus. Physiol. Mol. Plant Pathol. 43, 109-119. [18] Ruzicska, P., Gombos, Z. and Farkas, G. (1983) Modification of the Fatty Acid Composition of Phospholipids during the Hypersensitive Reaction in Tobacco. Virology. 128, 60 - 64. [19] Avdiushko, S. A., Ye, X. S., Hildebrand, D. F. and Kug, J. (1993) Induction of lipoxygenase activity in immunized cucumber plants. Physiol. Mol. Plant Pathol. 42, 83 - 95. [20] Bligh, E.G. and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Physiol. 37, 911-917. [21] Vaskovsky, V.E. and Terekhova, T.A. (1979) HPTLC of Phospholipid Mixtures Containing Phosphatidylglycerol. J. HRC/CC. 2, 671672. [22] Kates, M. (1986) Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. Elsevier, Amsterdam. [23] Vaskovsky, V.E., Kostetsky, E.Y. and Vasendin, I.M. (1975) A Universal Reagent for Phospholipid Analysis. J. Chromatogr. 114, 129141. [24] Nekrasov, E.V. and Kotel’nikova, I.M. (2000) Phospholipase D activity in shoots of arboreal plants during the growing season. Russ. J. Plant Physiol. 47, 456-462. [25] Doke, N. (1983) Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol. Plant Pathol. 23, 345 – 357. [26] Ryu, S.B. and Wang, X (1998) Increases in Free Linolenic and Linoleic Acids Associated with Phospholipase D-Mediated Hydrolysis of Phospholipids in Wounded Castor Bean Leaves. Biochim. Biophys. Acta. 1393, 193–202. [27] Fan, L., Zheng, S. and Wang, X. (1997) Antisense Suppression of Phospholipase D α Retards Abscisic Acid – and Ethylene-Promoted Senescence of Postharvest Arabidopsis Leaves. Plant Cell. 9, 2183 – 2196. [28] Sang, Y., Cui, D. and Wang, X. (2001) Phospholipase D and Phosphatidic Acid-Mediated Generation of Superoxide in Arabidopsis. Plant Physiol. 126, 1449–1458. [29] Sai Gopal, D.V.R., Sreenivasulu, P. and Nayudu, M.V. (1990) Effect of Bavistin on Lipid Metabolism in Groundnut (Arachis hypogaea L.) Leaves Infected with Peanut Green Mosaic Virus (PGMV). Physiol. Mol. Plant Pathol. 37, 1-8. [30] Adam, A. and Nagy, P.D. (1989). Variations in Membrane Polar Lipids of Barley Leaves Infected with Three Strains of Barley Stripe Mosaic Virus and with Poa Semilatent Virus. Plant Sci. 61, 53-59.
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ACYL-CoA ELONGASE : GENOMIC STUDIES
R. LESSIRE1 , P. COSTAGLIOLI 1,2 , C. GARCIA 1 , J. JOUBES 1 ., W. DIERYCK1,2 , J. LAROCHETRAINEAU1 , S. CHEVALIER1 , B. GARBAY1,2 1
Laboratoire de Biogenèse Membranaire CNRS FRE 2694,
2
ESTBB Université V. Segalen Bordeaux 2,
146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Introduction The aerial parts of plants are covered by a cuticular wax layer which plays a major role in protecting the plant from uncontrolled water loss [1,2], against ultraviolet radiations and helps minimizing deposition of dust, pollen and air pollutants [3]. In addition, surface wax is believed to play important roles in plant defence against bacterial and fungal pathogens [4]. These properties result of the typical physical and chemical properties of the wax layer, which is mostly constituted of a complex mixture of homologue series of very long chain aliphatics lipids (alcohols, ketones, alkanes, esters). In most plants, including Arabidopsis thaliana, there are two main wax biosynthetic pathways: (Fig 1) an acyl reduction pathway which gives rise to primary alcohols and wax esters, and a decarbonylation pathway leading to the formation of aldehydes, alkanes, secondary alcohols and ketones (for review, see [5]).
Cutin
Transport Ketones Oxidase
Acyl reduction reduction pathway (20%)
Wax esters
Secondary alcohols
Decarbonylation Decarbonylation pathway (80%)
WS
VLCFAs (C20-C32)
Primary alcohol FAR*
KCS
ELONGASE ß-keto acyl-CoA
KCR
Alkanes Decarbonylase
FAR
Long chain acyl-CoA n+2 Long chain acyl-CoA
Hydroxylase
Aldehydes
ECR
trans-2,3-enoyl-CoA
dehydrase 3-hydroxyacyl-CoA
Figure 1: Wax biosynthesis pathway in A.thaliana
In these two pathways, very long chain fatty acids (VLCFA) are the main precursors for the various wax components. They are synthesized by membrane-bound enzyme complexes, the acylCoA elongases, which catalyse the elongation of an acyl-CoA by successive additions of C2-units from malonyl-CoA. Each cycle involves the successive intervention of four different enzymes: a 3-
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keto acyl-CoA synthase (KCS), a 3-ketoacyl-CoA reductase (KCR), a 3-hydroxy acyl-CoA deshydratase (HCD) and a 2,3 enoyl-CoA reductase (ECR) (Figure 2). O
CoA
S Oleoyl-CoA O
CoA
CoA CoA
OH
S
Malonyl-CoA
3-ketoacyl-CoA Synthase (condensing enzyme
O
O
SH
+ CO
2
O
S 3-ketoacyl-CoA NAD(P)H
3-ketoacyl-CoA Réductase NAD(P) O
CoA
OH
S 3-hydroxyacyl-CoA
3-hydroxyacyl-CoA Dehydratase H O 2
O
CoA
S trans-2,3-énoyl-CoA NAD(P)H
Trans-2,3-énoyl-CoA Reductase NAD(P)
O
CoA
S Acyl-CoA (n+2 carbon atoms)
Figure 2: Reactions catalyzed by the acyl-CoA elongases
The first KCS gene characterized in Arabidopsis thaliana was FAE1 [6]. So far, several related-genes have been identified in the A. thaliana genome. The gene encoding the second enzyme, the 3-ketoacyl-CoA reductase, has been cloned and characterized in many species. Indeed, the GLOSSY8 gene has been isolated using a maize mutant which was deficient in the synthesis of the very-long chain compounds of cuticular waxes [7]. The
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ortholog sequences from Arabidosis thaliana, barley and leek were cloned at the same time [7]. Xu et al. [8] showed that the GLOSSY8 protein possesses a 3-ketoacyl-CoA reductase activity. In the same time, the YBR159w gene of Saccharomyces cerevisiae was identifie d as a member of the short chain alcohol dehydrogenase reductase superfamily. Both Ybr159p and its Arabidopsis ortholog (GenbankTM accession no. NP_564905) were able to restore an elongation activity when expressed in ybr159∆ mutant [9]. The final step of the elongation process is catalysed by a trans-2,3-enoyl-CoA reductase. In yeast, the TSC13 gene encodes a protein which is localized in the endoplasmic reticulum and which co-immunoprecipitates with the condensing enzymes Elo2p and Elo3p [10]. Both are responsible for the synthesis of VLCFAs enriched in the sphingolipids of the yeast plasma membrane. Regarding to these properties, a trans-2,3-enoyl-CoA reductase activity has been assigned to TSC13. The gene encoding the HCD has not been characterized so far, although several genes encoding deshydratase/hydratase enzymes have been identified raising questions about their role and their involvement in different biosynthetic pathways. The goal of our study was to complete the knowledge of the acyl-CoA elongase by searching gene candidates for the HCD (See Garcia et al, this book), and to identify among the different KCS genes present in the Arabidopsis thaliana genome, which ones are involved in the synthesis of wax precursors. Results: As stated above, several genes have been identified for the KCS activities. To identify which ones are involved in the wax biosynthesis pathway, we performed microarray experiments to measure the steady-state levels of candidate mRNAs in 15-days-old Arabidopsis shoots, a developmental stage for which wax synthesis is maximal. Nine Agilent Arabidopsis 1 microarrays containing 60-mers oligonucleotide probes representing 13704 genes were used in this study. The average intensity signal was around 1600 arbitrary units (AU) for the 13704 genes studied, and the average intensity of the negative controls (180 blank spots on each array) was 246. Therefore, genes were separated in three classes according to their average expression levels in the nine arrays. Genes which were not expressed (average signal intensity below 500 AU), genes moderately expressed (between 500 and 1600 AU) and genes strongly expressed (AU above 1600 AU). A search in the Arabidopsis Lipid Gene Database [11] revealed the existence of 21 KCSrelated genes. When the corresponding sequences were aligned with Clustal W (1.83), we were able to class them into four main families: KCS1-, FAE 1-, FDH- and CER 6-like (Fig. 3). The smallest family comprised only two members, CER6 and CER60. As expected from previous studies, CER 6 was highly expressed in 15-days-old shoots. However, the level of expression of CER60 was higher than expected from literature data, which may indicate that CER60 plays a particular role in wax deposition at certain stages of development that require high levels of wax production [12]. From the seven genes belonging to the FDH family, three were not expressed in our samples. Both At5g49070 and At1g71160 could be pseudogenes as no EST were present in the databases. The last one At3g52160, could be specifically expressed in flower tissues and have probably no role in stems or leaves wax biosynthesis. As expected, FDH was expressed at high level. Three other genes, closely related to each other, At5g04530, At1g07720 and At2g28630 were expressed at moderate levels. Their possible implication in wax biosynthesis has to be evaluated more precisely. Six genes closely related to the seed-specific condensing enzyme FAE1 were identified in the Arabidopsis genome. One of them, At4g34520 was poorly expressed which is in good agreement with its specific role in seed oil production. Three genes belonging to this family appeared to be expressed to moderate levels in our samples: At1g19440, At2g16280 and At2g15090.
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The last family, composed of six members, corresponds to the KCS1 gene characterized by Todd et al. [13]. From the four sequences present on our array, two genes were expressed at moderate level, At1g01120 (KCS1) and At2g26640. Surprisingly, we found that At5g43760, a gene which has never been studied before, was expressed to levels comparable to those measured for CER6 and FDH. The abundance of At5g43760 transcripts in shoots suggested that this KCS may play an important role in wax deposition in shoots at this stage of development. Figure 3: Phylogenetic analysis of the Arabidopsis thaliana KCS and expression levels in 15-days-old shoots
Families KCS1
FAE 1
FDH CER6
Gene Level of locus Expression At1g01120 At2g46720 At3g10280 At2g26640 At1g04220 At5g43760 At1g19440 At2g16280 At4g34510 At4g34520 At2g15090 At4g34250 At5g04530 At1g07720 At2g28630 At5g49070 At1g71160 At2g26250 At3g52160 At1g25450 At1g68530
Moderate (KCS1) ND ND Moderate Not Expressed Elevated Moderate Elevated Not Expressed Not Expressed (FAE1) Moderate ND Moderate Moderate Moderate Not expressed Not Expressed Elevated (FDH) Not Expressed Elevated Elevated (CER6)
BIO
In conclusion, using microarray approach we have studied the expression of candidate genes probably involved in the acyl-CoA elongase responsible of the VLCFA precursors of the wax components. We showed that twelve KCS out of the 18 studied are expressed in 15-days-old shoots, and three presented quite high levels of mRNA expression: CER6, FDH and At5g43760. We are currently investigating the putative role of the latter gene by studying the corresponding tagged mutant. References [1] Kerstiens G (1996) Cuticular water permeability and its physiological significance. J Exp Bot 47: 1813-1832 [2] Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52: 20232032 [3] Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42: 51-80 [4] Jenks MA, Joly RJ, Peters PJ, Rich PJ, Axtell JD, Ashworth EN (1994) Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench. Plant Physiol 105: 1239-1245
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[5] Von Wettstein-Knowles P (1995) Biosynthesis and genetics of waxes. In RJ Hamilton, ed, Waxes: Chemistry, Molecular Biology and Functions. Oily Press, Dundee, pp 91-129 [6] James DW, Lim E, Keller J, Plooy I, Ralston E, Dooner HK (1995) Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator. Plant Cell 7: 309-319 [7] Xu X, Dietrich CR, Delledonne M, Xia Y, Wen TJ, Robertson DS, Nikolau BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a beta-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physiol 115: 501-510 [8] Xu X, Dietrich CR, Lessire R, Nikolau BJ, Schnable PS (2002) The endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase involved in the production of cuticular waxes. Plant Physiol 128: 924-934 [9] Beaudoin F, Gable K, Sayonova O, Dunn T, Napier JA (2002) A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-keto-reductase.,J Biol Chem.;277:1 1481-8 [10] Kohlwein SP, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T. (2001) Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol. Cel. Biol.,21: 109-125. [11] Beisson F, Koo AJ, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, Savage L, Milcamps A, Mhaske VB, Cho Y, Ohlrogge JB. (2003) Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol 132: 681-97 [12] Hooker TS, Millar AA, Kunst L (2002) Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiol 129: 1568-1580 [13] Todd J, Post -Beittenmiller D, Jaworski JG (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J. 17: 119-130
Acknowledgements: The help of Conseil regional d’Aquitaine is gratefully acknowledge. One of us (C.G). was a recipient of Conseil Régional d’Aquitaine funding.
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CHARACTERIZATION OF A LYSO-PC ACYLTRANSFERASE FROM S. CEREVISIAE ERIC TESTET*, JEANNY LAROCHE-TRAINEAU§, ABDELMAJID NOUBHANI*, DENIS COULON*, ODILE BUNOUST‡ RENÉ LESSIRE§ AND JEAN-JACQUES BESSOULE§
*Laboratoire de Biogenese Membranaire, FRE2694 CNRS-Université Victor Segalen, Bordeaux II/ESTBB, case 92, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. § Laboratoire de Biogenese Membranaire FRE 26 94, CNRS-Université Victor Segalen, Bordeaux II, case 92, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. ‡IBGC UMR 5095, CNRS-Université Victor Segalen, Bordeaux II, 1 rue Camille Saint Saens 33077 Bordeaux cedex France
1. Abstract We cloned a yeast gene encoding for a protein described as a putative acyltransferase. When this protein (that we called LPCAT1) was expressed in E. coli, a lyso-PC acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein able to catalyze the acylation of lyso-PC molecules to form PC. We first compared the growth of wild type cells and the mutant (deleted for LPCAT1). Whatever the culture media used (supplemented with 2% glucose or containing 2% lactate) no significant difference in growth rate was observed.We further determined the lipid composition of the yeast mutant deleted for the corresponding gene and the lipid composition of the wild type cells. When cells were grown in the presence of 2% glucose, no significant differences in lipid amounts were observed, irrespective of the phase chosen to harvest cells (exponential or stationary phase). In contrast, when yeast cells were grown in the presence of lactate, the mutant synthesized two-fold more triglycerides and steryl esters than the wild type. This increase in the amount of triglycerides and steryl esters was not accompanied by significant changes in the fatty acid composition of the various lipids. Moreover the mitochondrial membranes from the mutant contained a reduced amount of phosphatidylcholine and cardiolipin, and the fatty acid composition of the latter was greatly changed. 2.Introduction Lyso-phosphatidylcholine (Lyso-PC) acyltransferases (EC 2.3.1.23) which catalyze the acylation of lyso-PC molecules to form phosphatidylcholine (PC) are involved in several important physiological processes. In plant cells, we previously showed that a lyso-PC acyltransferase located in the plastid envelope [1] could be a key enzyme for the import of lipids from the ER membranes into chloroplasts, and therefore for plastid membrane biogenesis. Briefly, plastids require the import of extraplastidial lipid precursors for the synthesis of the envelope and of thylakoid membranes ([2] for review). Based on in vitro [1, 3] and in vivo [4, 5] studies we have proposed that this import involves a release of lyso-PC from extraplastidial membranes, a transfer of these molecules to chloroplasts, and their acylation in the envelope to synthesize plastidial PC. Hence, since this plastidial PC is further used as substrate for the biosynthesis of 50 to 90% of other plastid lipids -depending on plant species [6], the plastidial lyso-PC acyltransferase located in the envelope could play an essential role in plastid membrane biogenesis. In seeds, any enzyme able to catalyze the transfer of an acyl chain to the sn-2 position of lyso-PC could be used to engineer oil of interest with specific fatty acids. This could be useful for producing
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oil seed with a fatty acid composition of industrial value, such as oil with a high erucic acid content in Brassica napus seeds ([7] for review). In addition, it has been shown that the oil synthesis in various seed crops can occur via a transfer of the acyl chain esterified to the sn-2 position of phosphatidylcholine to diacylglycerol molecules [8]. Moreover, recent data [9] strongly suggest that the reverse reaction catalyzed by lyso-PC acyltransferase [10] might be the rate-limiting step for the accumulation of very long chain polyunsaturated fatty acids (PUFAs) both in transgenic yeast and in transgenic seed crops. Since numerous health benefits are attributed to these very long chain PUFAs, the production of these molecules by fungi and/or by plants –and therefore the identification of a lyso-PC acyltransferase gene- appear of great interest.
3•Results 3.1 Looking for a gene encoding for a Lyso-PC acyltransferase We used the sequence of a protein thought to be a lysolipid acyltransferase to identify some ORFs of interest in S. cerevisiae. From our analysis, it appeared that only one yeast gene (LPCAT1) encodes for a protein having a significant homology with this putative lysolipid acyltransferase. The LPCAT1 Saccharomyces cerevisiae gene was amplified by PCR. The PCR product was first subcloned in pGEM T Easy vector (Promega, Charbonnieres les Bains, France). Then, we used a set of sense and antisense primers containing the appropriate restriction sites for cloning sequences of interest in the pET-15b vector (Novagen, Merck Biosciences, Badsoden, Germany). To obtain a sequence encoding for the LPCAT1::HA fusion protein, the HA tag encoding the following peptide: MYPYDVPDYASL) was included in the primer. All constructs were verified by sequencing. C41(DE3) E. coli bacteria (Avidis, Saint-beauzire, France) were further transformed with the pET15b containing putative lyso-PC acyltransferase gene with or without the HA tag. 3.1 Lyso-PC acyltransferase associated with bacterial membranes expressing LPCAT1p The first sought to determine the activity associated with membranes of C41 (DE3) E. coli bacteria transformed with a plasmid containing the sequence encoding for LPCAT1p. After IPTG induction, the amount of LPCAT1p synthesized by such bacteria was too low to be evidenced by a mere electrophoresis staining. However, when the cells were transformed with the plasmid containing the sequence encoding for the LPCAT1::HA fusion protein, induction of protein synthesis could be evidenced by using anti-HA antibodies (data not shown). Unfortunately, as shown in the figure 1, no significant lyso-PC acyltransferase activity (0.16 nmol and 0.19 nmol PC / 30min / mg) was associated with the membrane of bacteria expressing the LPCAT1::HA fusion protein. By contrast, when C41 (DE3) E. coli bacteria expressed LPCAT1p, their membranes were able to catalyze the acylation of lyso-PC to form PC. The rate of PC synthesis with such membranes was of 1.92 ± 0.16 nmol / 30 min/ mg of membrane-bound proteins (n = 3; 2 different clones). This activity was (almost) undetectable in the absence of IPTG (0.17 ± 0.01 nmol/ 30 min / mg, n = 3; 2 different clones) or when bacteria were transformed with the plasmid devoid of the coding sequence (0.15 and 0.20 nmol of PC synthesis/ 30 min/ mg).
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nmol PC / 30 min / mg
Figure 1 : Lyso-PC acyltransferase activity associated with bacterial membranes 2,00 1,80 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0,00 C41 + pET15b
clone 5.3 (Tag HA)
clone C41 5.2
clone C41 5.3
Lyso -PC acyltransferase reactions were conducted in 100 µl assay mixtures containing 2 nmol [ 14 C] lyso -PC, 5 nmol oleoyl-CoA, 25 mM Tris-HCl (pH 7.0) and proteins from the various fractions. Incubations were carried out at 30°C for 10 min (30 min when bacterial membranes were used) and reactions were stopped by the addition of 2 ml of chloroform/methanol (2/1; v/v) and 500 µl of H2 O. The organic phase was isolated and the aqueous phase was reextracted with CHCl3 (2 ml). These combined lipid extracts were dried, redissolved in 50 µl CHCl3 /CH3 OH (2/1 v/v) and the lipids were separated by HPTLC as described above. The radioactivity incorporated in PC was quantified by using a PhosphorImager (Amersham Pharmacia Biotech-Molecular Dynamics). C41 (DE3) + pET15b E. coli bacteria transformed with the plasmid pET15b. Clone 5.3 (tag HA): E. coli bacteria transformed with the same plasmid containing the sequence encoding for the LPCAT1::HA fusion protein. Clones C41 5.3 and C41 5.2 : E. coli bacteria transformed with the same plasmid containing the sequence encoding for LPCAT1p. Open histograms : absence of IPTG, Closed histogramms : + 1mM IPTG
3.2 Growth rate of wild type and mutant cells
We first compared the growth of wild type cells and the mutant (deleted for LPCAT1). Whatever the culture media used (supplemented with 2% glucose or 2% lactate) no significant difference in growth rate was observed (figure 2).
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OD (600 nm)
Figure 2: Growth rate of wild type and mutant cells media in the presence of 2% glucose or 2% lactate 30
A
25 20 15 10 5 0
0
10
20
30
40 Time (h)
OD (600 nm)
25
B
20 15 10 5 0
0
10
20
30
40 Time (h)
The cells were grown in a shaking incubator at 30°C overnight in 500 ml Erlenmeyer flasks containing 100 ml of liquid medium YP (yeast extract 10g, peptone 10g, potassium phosphate 1g, ammonium sulfate 1.2 g) supplemented with 2% glucose (A) or 2% lactate (B) as carbon substrate. The pH was set at 5.5. Circles : Wild type cells, triangles: lpcat1? mutant.
3.3 Increase in the oil content in mutant cells We further determined the lipid composition of the wild type and the mutant (deleted for LPCAT1) cells grown in media supplemented with 2% glucose or 2% lactate. When cells were grown in the presence of 2% glucose, no significant differences in neutral lipid amounts were observed, irrespective of the phase chosen to harvest cells (exponential or stationary phase, data not shown). By contrast, following a culture in a medium supplemented with 2% la ctate, the mutant cells harvested during the stationary phase contained approximately two-fold more triglycerides than the wild type cells. (Table 1). The amount of steryl esters was also slightly higher in the mutant, whereas the amount of all other neutral lipids (DAG, FFA) did not vary (not shown). This repeatedly observed increase in the amount of triglycerides and steryl esters in the mutant cells grown in the presence of lactate and harvested during the stationary phase was not accompanied by significant changes in the fatty acid composition of the various lipids, except for a slight increase in the percentage of 16:1 fatty acid (Table 1). Nevertheless, by itself, this increase could not account for the increase in the amount of triglycerides. In other words, the deletion of LPCAT1 induced an increase in the oil synthesis in yeast but did not seem to greatly affect the “specificity” of the overall process towards the various acyl chains. Moreover, no variation in the fatty acid composition of the various lipids was observed when cells were harvested during the exponential phase. Similar results (i.e. no change in the fatty acid composition) were obtained when cells were harvested during the stationary or the exponential phase from a medium supplemented with 2% glucose (data not shown).
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TABLE 1: Amount and fatty acid composition of TAG and steryl esters from mutant and wild type cells grown in the presence of 2% lactate and harvested during stationary phase Amount of total Lipids
lipids (a.u)
16:0 (%)
16:1 (%)
18:0 (%)
18:1 (%)
Triglycerides Wild type
26 ± 3
14.9 ± 0.24
42.3 ±0.32
7.3 ± 0.7
35.5 ± 0.1
Mutant
46 ± 8
12.7 ± 0.2
50.2 ± 0.35
5.4 ± 0,06
31.8 ± 0.1
Wild type
3 ± 0.5
16.8 ± 0.63
41.0 ± 1.1
7.0 ± 0.45
35.1 ± 0.6
Mutant
4.1 ± 1
17.2 ± 0.32
53 ± 0.44
5.1 ± 0.2
24. 6 ± 0.6
Steryl Esters
Neutral lipids were purified from the extracts by monodimensional thin layer chromatography using Hexane/Diethylether/Acetic acid (90/15/2; v/v/v) as solvent [11]. The lipids were then located by spraying the plates with a solution of 0.001% (w/v) primuline in 80% acetone, followed by visualization under ultraviolet light. The silica gel zones corresponding to the various lipids were then scraped from the plates and added to 1 ml of methanol/2,5% H2 SO4 containing 5µg of heptadecanoic acid methyl ester. After 1 hour at 80°C, 1.5 ml of 1M NaCl was added and fatty acid methyl esters (FAMES) were extracted by 0.75ml of hexane. Separations of FAMES were performed by gas chromatography (Hewlett Packard 5890 series II). The retention times of FAMES were determined by comparison with standards, and they were quantified using heptadecanoic acid methyl ester as standard. Results represent the mean values ± SD of four analyses carried out in two separate cultures
3.4 Changes in the polar lipid composition in mutant cells A further set of experiments was carried out to determine the distribution of the various phospholipids in the mutant and wild type cells. grown on a medium supplemented with 2% glucose or 2% lactate. In the presence of glucose, no significant difference was observed in the phospholipid composition of wild type and lpcat1 ? cells. TABLE 2: Fatty acid composition of PC and cardioloipin extracted from microsomes and from purified mitochondria of mutant and wild type cells grown on a medium supplemented with 2% lactate % of total 16:0 (%) 16:1 (%) 18:0 (%) 18:1 (%) Phospholipids Microsomes PC
Wild type
44.1 ± 2
13.5 ± 0.7
45.9± 0.5
7.5 ± 0.3
33.1 ± 0.5
Mutant
46.4 ± 1.4
12.5 ± 0.5
45 .4 ± 0.2
7.7 ± 0.05
34.3 ± 0.3
Wild type
37.1 ± 0.6
9.2 ± 0
57 ± 0.2
4.1 ± 0
29.8 ± 0.1
Mutant
33.7 ± 1.1
8.4 ± 0.3
55.3 ± 0.7
4.0 ± 0
32.9 ± 0.4
Wild type
16.8 ± 0.2
5.8 ± 0.6
40.9± 3.2
2 ± 0.9
51.3 ± 1.6
Mutant
9.8 ± 3
16.9 ± 1.1
42.6 ± 2.1
4 ± 1.8
36.4 ± 1.5
Mitochondria PC
Cardiolipin
Polar lipids were purified from the extracts by monodimensional thin layer chromatography using chloroform/methanol / 1-propanol / methyl acetate / KCl 0.25% (10/10/10/10/3.6; v/v/v/v/v) as solvent [12]. The lipids were then located by spraying the plates with a solution of 0.001% (w/v) primuline in 80% acetone, followed by visualization under ultraviolet light. The silica gel zones corresponding to the various lipids were then scraped from the plates and added to 1 ml of methanol/2,5% H2 SO4 containing 5µg of heptadecanoic acid methyl ester. After 1 hour at 80°C, 1.5 ml of 1M NaCl was added and fatty acid methyl esters (FAMES) were extracted by 0.75ml of hexane. Separations of FAMES were performed by gas chromatography (Hewlett Packard 5890 series II). The retention times of FAMES were determined by comparison with standards, and they were quantified using heptadecanoic acid methyl ester as standardResults represent the mean values ± SD of three analyses. Note that cardiolipin was not detected in the microsomal fraction.
By contrast, when cells were grown in the presence of lactate, a significant decrease in the percentage of cardiolipin + phosphatidylcholine was observed in mutant cells harvested during the exponential
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and stationary phases. This difference in phospholipid distribution was accompanied by a change in the composition of fatty acids esterified to cardiolipin since cardiolipins from mutant cells contained much less oleic acid (18:1) than wild type cells, this decrease being compensated by an increase in the percentage of the palmitic acid (16:0). This result was observed with cells harvested both during the exponential and stationary phases. The fatty acid composition of other phospholipids purified from mutant and wild type cells did not significantly vary whatever the culture media used (glucose or lactate), and whatever the phase chosen to harvest cells (data not shown). Since cardiolipins are exclusively associated with mitochondria, and in order to confirm the decrease in cardiolipins content and the changes observed in their fatty acid composition in mutant cells, we further analyzed the phospholipid composition of mitochondria purified from mutant and wild type cells. The data in Table 2 confirm those obtained following the purification of lipids from the whole cells, i.e.: there was a decrease in the PC and cardiolipin contents in purified mitochondria from the mutant, and the main effect on the fatty acid composition of individual lipids was a decrease in the percentage of 18:1 associated with cardiolipins. Moreover, as observed with whole cells, this decrease was compensated mainly by an increase in the percentage of 16:0 fatty acid. By contrast, when lipids from the microsomal membranes of mutant and wild type cells were analyzed, no clear-cut differences appeared, either in the phospholipid distribution or in their fatty acid composition. 4.Conclusion To our knowledge, this is the first report of a coding sequence of a protein that catalyzes the acylation of lyso-PC molecules to form PC. Deletion of the gene induced an increase in the oil content of the cell. As expected and as generally observed (for example [13]), by staining cells with oil red, we found that these triacylglycerols were located in lipid droplets present in the cytosol (data not shown). In good agreement, no or few neutral lipids were found associated with purified mitochondria or microsomal membranes. The relatively high yield of triglycerides in mutant cells grown in the presence of lactate is of interest for increasing the oil content of oleaginous seeds, but the reasons for such a phenotype remain to be elucidated.
5. References [1] Bessoule, JJ, Testet, E., and Cassagne, C. (1995). Synthesis of phosphatidylcholine in the chloroplast enveloppe after import of lysophosphatidylcholine from endoplasmique reticulum membranes. Eur J Biochem 228, 490-497. [2] Browse, J., and Somerville, C.R. (1991). Glycerolipid synthesis: biochemistry and Regulation. Ann. Rev Plant Physiol. Plant Mol Biol 42,467-506. [3] Testet, E., Verdoni, N., Cassagne, C., and Bessoule, J.-J. (1999). Transfer and subsequent metabolism of lysolipids studied by immobilizing subcellular compartments in alginate beads. Biochim. Biophys. Acta 1440, 73-80. [4] Mongrand, S, Bessoule, J.-J., and Cassagne, C. (1997). A re-examination in vivo of the phosphatidylcholine-galactolipid metabolic relationship during plant lipid biosynthesis. Biochem J 327, 853-858. [5] Mongrand, S., Cassagne, C., and Bessoule, J. -J. (2000). Import of lyso -PC into chloroplasts likely at the origin of eukaryotic plastidial lipids. Plant Physiol. 122, 845-852. [6] Mongrand, S, Bessoule, J.-J., Cabantous F., and Cassagne, C. (1998). Phytochem. The C16:3/C18:3 fatty acid balance in photosynthetic tissues from 468 species. 49, 1049-1064. [7] Weselake, R.J., and Taylor, D.C. (1999). The study of storage lipid biosynthesis using microspore-derived cultures of oil seed rape. Prog. Lipid Res. 38, 401-460. [8] Dahlqvist, A, Stahl, U., Lenman, M., Banas, A., Lee, M., Sandager, L., Ronne, H., and Stymne, S. (2000). Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad. Sci. 97, 6487-6492. [9] Domergue, F., Abbadi, A., Ott, C., Zank, T.K., Zahringer, U., and Heinz, E. (2003). Acyl carriers used as substrates by the desaturases and elongases involved in very long chain polyusaturated fatty acid biosynthesis reconstituted in yeast. J. Biol. Chem. 278, 3511535126. [10] Stymne, S., and Stobart, A.K.. (1984). Evidence for the reversibility of the acyl- CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver. Biochem J., 223, 305-14.
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[11] Vitiello, F., and Zanetta, J.P. (1978). Thin layer chromatography of phospholipids. J. Chromatogr. 166, 637-640. [12] Juguelin, H., Heape, M.A., Boiron, F., and Cassagne, C. (1986). A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and Trembler mice. Dev. Brain. Res. 25, 249-252. [13] Sandager, L., Gustavsson, M.H., Stahl, U., Dahlqvist, A, Banas, A., Lenman, M., Ronne, H., and Stymne, S. (2002). Storage lipid synthesis is non-essential in yeast. J. Biol. Chem. 277, 6478-6482. ACKNOWLEDGMENTS: Special thanks to Dr S. Mongrand (FRE 26 94, CNRS Bordeaux) for his helpful comments throughout this project. Many thanks to Pr Marc Bonneu (ESTBB/ Université Victor Segalen, Bordeaux II), and Pr M. Rigoulet (IBGC UMR 5095, CNRSUniversité Victor Segalen, Bordeaux II) for their help and comments. We are grateful to Francesca Ichas (INSERM, CNRS IECB Bordeaux) for her contribution to the visualization of the lipid droplets by microscopy, and to Giselle Velours (IBGC UMR 5095, CNRS, Bordeaux) for the gift of the yeast DNA. The helpful reading of the manuscript by Ray Cooke is acknowledged. This work was in part supported by the Conseil Régional d'Aquitaine (France).
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Posters
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ENZYMATIC PROPERTIES OF AN ARABIDOPSIS THALIANA PHOSPHOLIPID: STEROL ACYLTRANSFERASE A. BANA S1 , A.S. CARLSSON1 , W. BANAS2 , B. HUANG3 , A. NOIRIEL4 , P. BENVENISTE4 , H. SCHALLER4 , P. BOUVIER-NAVE4 , S. STYMNE1 1
Department of Crop Science, Swedish University of Agricultural Sciences, Alnarp, Sweden Institute of Biology, University of Podlasie, Siedlce, Poland 3 College of Life Science, Hubei University, Wuhan, P.R.China 4 Institut de Biologie Moleculaire des Plantes du CNRS, Departement Isoprenoides, Institut de Botanique, 28 rue Goethe, 67083 Strasbourg Cedex, France 2
1.
Introduction
A gene encoding an enzyme catalysing transacylation of acyl groups from position sn-2 of phospholipids to diacylglycerols, forming triacylglycerols in an acyl-CoA-independent reaction was first cloned from yeast (Dahlqvist et al. 2000). The enzyme was named phospholipid: diacylglycerol acyltransferase (PDAT). Based on sequence homologies to the yeast PDAT we identified six PDAT-like putative proteins in Arabidopsis thaliana by searching the genome database. The genes encoding five of these proteins were overexpressed in Arabidopsis plants. In studies of the overexpressers, we could show that one of these proteins (Atg13640) catalysed a PDAT reaction (Banas et al. 2003). In the present study we have characterised another of the Arabidopsis PDAT-like proteins (Atg04010) and we could show that it catalysed a phospholipid: sterol acyltransferase reaction. 2.
Material and methods
A cDNA encoding Atg04010 was inserted downstream the CaMV35S promoter into the binary vector pGVTVKAM (Becker et al. 1992). The resulting construct pG35s3027-178 was transformed into Arabidopsis cv Columbia using Agrobacterium and floral-dip method (Clogh and Bent, 1998). T3 seeds were germinated on agar containing kanamycin. Three different homozygous lines expressing the gene were selected for further studies. The seeds of the selected lines and control plants were germinated on agar plates (0,8% agar, 1/3 MS, 1 % sucrose) for two weeks after which the seedlings were transferred into liquid 1/2 MS-medium supplemented with 1% sucrose. The plants were grown for further 14 days in growth chamber (16 hrs light/ 23o C, 8 hrs dark/ 18o C) before used in experiments. Total RNA was extracted from leaves (according to modified “phenol extraction method”; Sambrook et al., 1989), and aliquots of extracted RNA (10 and 20 µg) was then run on 1,5% agarose gels with 5% formaldehyde and transferred to nylon (Hybond-N+) membranes. Blots were hybridised with the Atg04010 cDNA in 0,5 M NaHPO4 (pH 7,2), 7% SDS and 1 mM EDTA at 64o C over night. After washing, the hybridisation was visualised by electronic autoradiography. Microsomes were prepared according to procedure of Stobart and Stymne (1985) from leaves of transformed and control plants. Microsomal fractions (corresponding to 12, 24 or 48 nmol of microsomal PC) were lyophilised over night and used for enzyme assays. Substrates (in which either the acyl donor or acyl acceptor was labelled with 14 C) dissolved in a small volume of benzene were added to dried microsomes. The solvent was immediately evaporated under a stream of nitrogen and 0,1 ml of 50 mM potassium phosphate (pH 7,2) was added. The suspension was thoroughly mixed and incubated at 30o C for 60 min. Lipids were extracted from the reaction mixture into chloroform (Blight & Dyer, 1959) and separated by thin layer chromatography (TLC). The radioactive lipids were visualised and quantified on the plates by electronic autoradiography. 3.
Results and discussion
The expression of Atg04010 gene was clearly detected in the three transformants. The highest expression was in “D28-1-5-8 line and the lowest in the “D28-6-2-2” line. The expression was hardly detectable in control plants.
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Typical PDAT assays were performed with sn-2-[14 C]acyl-PE as a fatty acid donor and non-radioactive DAG as an acyl acceptor and did not reveal any increase in TAG synthesis in microsomal preparations from the transformed lines compared to the control. However, there was an increase in the radioactivity in the area of sterol esters and wax esters on the TLC after separation of the lipids from assays with membranes from transformants compared to the controls. Replacement of the diacylglycerols with different fatty alcohols did not increase the amount of these radioactive compounds. However, additions of cholesterol increased its synthesis significantly (Fig.1A). To verify that cholesterol was the acyl acceptor, we performed the assays with radioactive cholesterol as the only added substrate. The amount of 14 C-cholesterol esters synthesised reflected the level of expression of the Atg04010. The synthesis of 14 C-cholesterol esters, as a function of cholesterol concentration in microsomal preparation from the different plants, is given in Fig.1B. These results show unequivocally that the Atg04010 gene encodes an enzyme, which is using phospholipids as the acyl donor and sterols as the acyl acceptor in acyl-CoA-independent formation of sterol esters. We prefer the name phospholipid: sterol acyltransferase (PSAT) for this type of enzyme instead of the commonly used lecithin: cholesterol acyltransferase (LCAT) that is used for the animal enzyme catalysing the same reaction.
[14C]sterol esters formed (pmol)
A 200 150 100 50 0 WT
D28-6-2-2
D28-3-4-8
D28-1-5-8
D28-6-2-2
D28-3-4-8
D28-1-5-8
[14C]sterol esters formed (pmol)
B 250 200 150 100 50 0 WT
Fig.1. Sterol ester acyltransferase activity in microsomal preparations (equivalent to 12 nmol microsomal PC) of leaves from Arabidopsis Wt plants (null segregant of line D28-1-5) and three transgenic lines (D28-6-2-2, D28-3-4-8 and D28-1-5-8) expressing AtPSAT at different levels. A - assays were performed with sn -2-[ 14 C]18:2-sn-1-16:0 -PE (5 nmol) without added cholesterol (grey bars) or with 5 nmol of added cholesterol (black bars). B - assays were performed with [14 C]cholesterol added in following amounts: 1.5nmol, white bars; 3 nmol, light grey bars; 6 nmol, dark grey bars; 12 nmol, black bars.
Sterols and lipids specificity of the AtPSAT was tested with the microsomal preparation of leaves fro m “D28-15-8” line (the highest expresser). The enzyme activity was strongly dependent on the polar head group of the
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120
60
-60
Si to ste ro l La no ste ro Ca l m 24 pe -M ste eth ro l yle Er ne go -cy st er clo ol ar ten ol Cy clo ar te no St l ig Ch m ol a st es er ta ol -5 ,7di en ol
0 Ch ole ste ro O l Di bt hy us dr ifo 24 oc lio -E ho l th l e yli ste de ro ne l lo p St he igm no l as Z taym 8,2 os 4(2 ter 4`) ol -d ien ol
Stimulation of formation of [14C]sterol esters
phospholipid. Phosphatidylethanolamine was utilised at least five times better than phosphatidylcholine and phosphatidic acid. The enzyme had strong preference for the sn-2 position. Palmitate, stearate and oleate were utilised at similar rate whereas linoleate and linolenate was utilised at a rate of two and three times that of the saturated and monounsaturated acyl groups, respectively. Enzyme also showed different preferences towards the acyl acceptors. The specificity towards 14 different sterols was tested of which 11 increased the incorporation of radioactivity from [14 C]PE into the sterol esters (Fig.2).
Fig.2. The effect of addition of different sterols on sterol ester acyltransferase activity in microsomal preparations (equivalent to 24 nmol microsomal PC) of leaves from Arabidopsis AtPSAT overexpressor (line D28-1-5-8). Assays were performed with 5 nmol of sn -1-18:1-sn2-[ 14 C]18:1-PE and 5 nmol of added sterols. The stimulation of activity is given as percentage of activity of incubations in absence of added sterols. Corresponding assays with microsomal preparations from Wt (null segregants from D28-1-5 line) yielded one tenth of the activity of the transformant in the absence of sterols and no difference in activity was observed with any of the sterols added (data not shown).
Acknowledgement: This work was supported by the Swedish University of Agricultural Science’s strategic research grants ‘The Biological Factory’ , ‘AgriFunGen’, ScanBi AB, Stiftelsen Svensk Ojeväxtforskning, the Swedish Farmers Foundation for Agricultural Research, Stiftelsen Västsvenska Lantmännen Odal and by the European Commissions research grant (QLRT1999-00213) 4. References
Banas, A., Lenman, M., Dahlqvist, A., Stymne, S. (2003) Cloning and characterisation of phospholipid: diacylglycerol acyltransferase from Arabidopsis thaliana. In „Advanced Research on Plant Lipids” (ed. N. Murata, M. Yamada; I. Nishida, H. Okuyama, J.Sekiya, H. Wada), 179-182. Kluwer Academic Publishers. Dordrecht, Netherlands. Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left t-dna border. Plant Mol Biol 20: 1195-1197 Blight, E.G. and Dyer, A. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911-917. Clough, S.J. and Bent, A.F. (1998) Floral dip: simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. The Plant Journal. 16 (6), 735-743. Dahlqvist, A., Ståhl, U., Lenman, M. Banas, A., Lee, M., Sandager, L., Ronne, H., Stymne, S. (2000) Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS, 97 (12), 6487-6492. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press. Stobart, A.K. and Stymne, S. (1985) The regulation of fatty acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower. Planta, 163, 119-125.
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THE ACYL-CoA ELONGASE IN Arabidopsis thaliana : CHARACTERIZATION OF A CANDIDATE GENE PRESUMABLY ENCODING THE 3-HYDROXYACYL-CoA DEHYDRATASE C. GARCIA 1 , S. CHEVALIER1 , A BRETON3 , R. LESSIRE1 AND W. DIERYCK1,2 . 1 Laboratoire de Biogenèse Membranaire CNRS FRE 2694 Université V. Segalen Bordeaux 2, 146, Rue Léo Saignat, 33076 Bordeaux Cedex France. 2 ESTBB Université V. Segalen Bordeaux 2, 146, Rue Léo Saignat, 33076 Bordeaux Cedex France. 3 Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, 1 Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France.
1. Introduction To assume their protection against environnemental stresses including drought [1], fungal pathogens [2] and phytophagous insects [3], plants synthesize epicuticular waxes which form the outermost layer of aerial organs. The predominant epicuticular waxes constituents are derivated from saturated very long chain fatty acids (VLCFA). These VLCFA result from the elongation of C18:0-CoA by malonyl -CoA [4,5]. This elongation takes place in microsomes and involves four successive reactions: condensation, ketoacyl-CoA reduction, hydroxyacyl-CoA dehydration and enoyl-CoA reduction. Each reaction is catalysed by a specific enzyme and theses proteins are organized in a membrane-bound elongation complex called acyl-CoA elongase. Most of the genes encodi ng the acyl-CoA elongase proteins have been characterized in different organisms. In A. thaliana, many genes differentially expressed encode 3-ketoacyl-CoA synthases (condensing enzymes): CUT1, FAE1, KCS1...[6-8]. The 3-ketoacyl-CoA reductase and the enoyl-CoA reductase are respectively encoded by AtYBR159 and AtTSC13 [9,10], but the gene encoding the third enzyme, 3-hydroxyacyl-CoA dehydratase (HCD), still remains unknown. We have developed a candidate gene strategy which allowed us to identify a protein which could correspond to HCD. Characterization of this protein using yeast and A. thaliana mutants has been undertaken. 2. Material and Methods 2.1. Bioinformatic study Researches in databases were carried out with the BLAST algorithm at the following web sites : www.yeastgenome.org, www.ncbi.nlm.nih.gov, www.tigr.org, www.arabidopsis.org and mips.gsf.de. Alignments were made with ClustalW software and dendrogram with TreeView software. 2.2. Yeast strains wt : Mat α, his 3∆1, leu2∆0, lys2∆0, ura3∆0 ydr036? : Mat α, his 3∆1, leu2∆0, lys2∆0, ura3∆0, YDR036c::KanMX4 2.3. Yeast microsomes preparation and elongation /dehydration assays Microsomes were prepared from the wild-type or mutant cells as previously described [11]. Microsomal protein contents were determined using Bradford’s method [12]. Total elongase activity was measured in a total volume of 100 µl containing 60 µg of microsomal protein, 0.5 mM NADH, 0.5 mM NADPH, 1 mM MgCl2 , 2 mM DTT, 9 µM [2-14 C]malonyl-CoA and 50 µM C16:0-CoA or 10 µM C18:0-CoA. After saponification and acidification, fatty acids were extracted with
129
chloroform and radioactivity was measured. The nature of elongation products was identified by silica gel TLC using hexane/diethyl ether/acetic acid (30:70:1) as developing solvent. The fatty acid chain length was analyzed after conversion of extracted fatty acids into methyl esters by reverse phase TLC using RP-18 HPTLC plates (Merck, Darmstadt) with acetonitrile/tetrahydrofurane (80:20) as developing solvent. The radiolabeled fatty acids were detected using a PhosphorImager and quantified with ImageQuaNT software (Molecular Dynamics Inc, Sunnyvale, CA). 3-hydroxy -C20:0-CoA dehydration activity was measured during 5 min in a total volume of 100 µl with 60 µg of microsomal protein, 1 mM MgCl2 , 1 mM DTT, 75 µM triton X-100, and 11.5 µM [1-14 C]-3hydroxy -C20:0-CoA. Fatty acids were extracted with chloroform and resolved by TLC as described above. 3. Results and Discussion 3.1. Bioinformatic study A protein family which catalyses the reverse reaction of the 3-hydroxyacyl-CoA dehydratase was used to screen Arabidopsis proteins databases in order to list the homologous sequences without assigned function. A total of 12 putatives and uncharacterized proteins were identified as possible candidates. These proteins are divided into 5 classes according to their amino acid sequences (Figure 1) and analysis of each protein with different prediction algorithms shows that each class has proper features for subcellular location and hydropathy. Classes A and E contain predicted membrane proteins with various putative subcellular locations. In class B, proteins are predicted to be mitochondrial or peroxisomal soluble proteins. Members of class C are putative soluble mitochondrial proteins and members of class D putative soluble proteins of the secretory pathway. AtHCD1 AtHCD2 AtHCD3 AtHCD4 AtHCD5 AtHCD6 AtHCD7 AtHCD8 AtHCD9 AtHCD10 AtHCD11 AtHCD12
A B C D E
Figure 1. Dendrogram of Arabidopsis candidate proteins for 3-hydroxyacyl-CoA dehydratase function in acyl-C oA elongation. Sequence were named arbitrarily.
In these different classes, one protein that we named AtHCD1 has the characteristics thought to be required to take part in the acyl-CoA elongase complex. Indeed, this protein has a putative transmembrane domain and, interestingly, a dilysine motif near the C-terminus for endoplasmic reticulum targeting. Expression data from semi-quantitative PCR show that the gene encoding this protein is expressed during wax biosynthesis (data non shown). These properties make this protein the best candidate to ensure the function of 3-hydroxyacyl-CoA dehydratase of the endoplasmic reticulum membrane-bound acyl-CoA elongase. The AtHCD2 protein shares 49% of homology with AtHCD1 but it is predicted as mitochondrial or chloroplastic protein. To demonstrate that AtHCD1 catalyses the dehydration activity, two strategies implicating tagged mutants were developped in parallel: the study of an A. thaliana T-DNA insertion mutant interrupted in the coding sequence of AtHCD1 gene and the analysis of a Saccharomyces cerevisiae strain mutated in an orthologous sequence of AtHCD1. 3.2. Characterization of the ydr036c? yeast strain Orthologous sequences of AtHCD1 were researched in S. cerevisiae proteins database. Only one sequence, Ydr036c, was found to contain the same pattern shared by all our candidate dehydratases. This protein shared about 30 % of amino acid homology with AtHCD1 in the overlapping regions but its subcellular location and its molecular function were unclear.
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In a first step, the knock-out yeast strain for this gene was checked for fatty acid elongation activity and 3-hydroxyacyl-CoA dehydration reaction. Our results showed a reduction of the C18:0-CoA elongation activity in the mutant strain (Table 1) and a decrease by about 33 % of the 3-hydroxy -C20:0-CoA dehydration activity (Table 2). On the other hand, C16:0-CoA elongation activity was not reduced. These results suggest that the deletion of the YDR036c gene affects the 3-hydroxyacyl-CoA dehydratase activity of the C18:0-CoA elongase and probably that this gene is encoding this enzyme. Table 1. Elongation specific activity in ydr036c? yeast cells % of the wt maximal value for elongation specific activity after 1h C16:0 -CoA elongation
95
C18:0 -CoA elongation
63,4
Table 2. Comparison of 3-hydroxy-C20:0-CoA dehydration activity in wt and ydr036c? yeast cells Strain
3-hydroxy-C20:0 -CoA dehydration activity (nmol/mg/h)
wt
62,6
ydr036c?
20,9
Moreover, the presence of a residual activity in the mutant and the detection of the different reaction intermediates suggest that a second enzyme is able to ensure the same function as Ydr036c. To verify this hypothesis, we searched in S. cerevisiae database homologs of Ydr036c. The two proteins identified have a quite low homology with Ydr036c. These proteins are Yor180c, which has an assigned function of delta(3,5)-delta(2,4)-dienoyl-CoA isomerase and Ybr281c, which function is unclear. Complementation experiments in ydr036c? using AtHCD1 are in progress. 3.3. Characterization of an A. thaliana insertion mutant line for AtHCD1 In parallel, characterization of an A. thaliana T-DNA insertion mutant interrupted in the open reading frame of AtHCD1 from the Arabidopsis mutants library of the SALK Institute [13] has been undertaken. Preliminary studies to isolate an homozygous mutant line have been realized and the absence of cDNA corresponding to the AtHCD1 gene has been verified. Wax composition analysis of this mutant line and fatty acid elongation assays are carried out. Acknowledgement: This work is supported by a Conseil Régional d’Aquitaine fellowship to C.G. 4. References [1]
W.R. Jordan, et al. (1984). Environnemental physiology of sorghum. II. Epicuticular wax load and cuticular transpiration. Crop Sci 24 1168-1173. [2] M.A. Jenks, et al. (1994). Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench. Plant Physiol 105 1239-1245. [3] S. Eigenbrode, K. Espelie (1995). Effects of plant epicuticular lipids on insect herbivores. Annu ReV Entomol 40 171-194. [4] P.M. Von Wettstein -Knowles (1995). Genetics ans biosynthesis of plant epicuticular waxes. In Waxes : Chemistry, Molecular Biology and Functions, ed. R.J. Hamilton. Alloury, Ayr, Scotland : Oily Press. 91-130. [5] D. Post -Beittenmiller (1996). Biochemistry and Molecular Biology of Wax Production in Plants. Annu Rev Plant Physiol Plant Mol Biol 47 405-430. [6] A.A. Millar, et al. (1999). CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11 825-38. [7] D.W. James, Jr., et al. (1995). Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator. Plant Cell 7 309-19. [8] J. Todd, et al. (1999). KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J 17 119-30. [9] F. Beaudoin, et al. (2002). A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-keto-reductase. J Biol Chem 277 11481-8. [10] K. Gable, et al. (2004). Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system. J Exp Bot 55 543-5.
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[11] K. Gable, et al. (2000). Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity. J Biol Chem 275 7597-603. [12] M.M. Bradford (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding. Anal. Biochem 72 248-254. [13] J.M. Alonso, et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301 653-7.
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ALMOND LIPOXYGENASES JUDIT KOSÁRY1 , MARGIT KORBÁSZ2 , NIKOLETTA KISS2 , TERÉZ BALOGH3 1 Department of Applied Chemistry, Faculty of Food Science, Budapest University of Economic Sciences and Public Administration, Budapest, Hungary 2 Undergraduate 3 PhD student Lipoxygenases (LOX) (EC 1.13.11.12) are dioxygenase enzymes containing nonheme iron protein [1]. They catalyze reactions of polyunsaturated fatty acids containing 1(Z),4(Z)-pentadiene units with oxygen [2]. In these reactions optically pure (S)-hydroperoxy fatty acids are formed via hydroperoxides [3]. The formation of hydr operoxides, an important step in lipid peroxidation can be catalyzed by lipoxygenases but an alternative way, a simple autoxidation can play a significant role in the process, as well. It is known that different plants contain so many kinds of lipoxygenase that they are often considered as different lipoxygenase groups instead of lipoxygenase isoenzymes [4]. A lot of lipoxygenases were detected in plants e.g. soybean, bean, potato, cucumber, rice and barley. Lipoxygenases 9-LOX or 13-LOX oxygenate unsaturated fatty acids C18 linolic acid and linoleic acid to 9- or 13-hydroperoxides. Substrate of lipoxygenase 15-LOX is arachidonic acid (C20 ) that is starting material of prostaglandines [3]. The structure, activity and types of lipoxygenases were studied above all in soybean because its lipoxygenases are far more active than those of other plants [5]. It was found that substrates of soybean lipoxygenases are unsaturated fatty acids C18 and their esters and the isoenzymes can be distinguished on the basis of their pH optima, substrate specificity, molecular weight, stability and propensity for increasing in activity [6]. Deactivation of soybean lipoxygenases has not only theoretic but also practical importance because these lipoxygenases are significant quality deteriorating agents in processing of this popular plant. On the basis of recent studies it was found that not only the activity but the formation of lipoxygenases too can be increased by different stresses in plants e.g. injuries, physical effects, drought, infections, effects of extreme cold or hot temperatures, effect of ozone and ultraviolet light [7,8,9]. It means that an increase in lipoxygenase activity could be an indicator in the case of different types of stress in a plant (like peroxidase and polyphenoloxydase). There were no results about the participation of different isoenzymes in this increase of lipoxygenase activity in plants or plant organs exposed to different effects or during their germination. In the last few years we studied the behaviour of plant lipoxygenase isoenzymes deduced from the pH dependence of lipoxygenase activity for different practical reasons. We found that the examination of the kinetics of lipoxygenase activity could be one of the characteristic parameters to estimate the quality deterioration caused by lipid peroxidation in stored ground poppy-seed samples [10,11]. Later we tried to characterize the germinating status of stored onion by the change in isoenzyme content of onion lipoxygenases. We found a significant high activity of lipoxygenase isoenzymes in acidic range only for germinating onion and we supposed that this difference in activity of lipoxygenase isoenzymes could be a characteristic biochemical parameter for the examination of stored onion [12]. Then a detailed study was carried out on the leaves of ginkgo tree plants of characterized sex to establish whether the lipoxygenase profile of ginkgo tree organs can be exploited to identify the sex of the individual ginkgo tree plants. It was found that the lipoxygenase profiles in themselves are inadequate for this purpose but they provide a useful supplementary tool to support the results of other methods e.g. isoelectric focusing [13]. Now we studied the behaviour of different sweet almond varieties, that of marzipan raw materials made from them during rancidification and the activity of lipoxygenase isoenzymes playing a an important role in the rancidification. Some hydrolases were also studied.
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MATERIALS AND METHODS Different commercial sweet almond (Prunus amygdalus) samples produced in Hungary were used. Marzipan samples containing about 50% almond was made in Szamos Marcipán Kft. Pilisvörösvár, Hungary. Chemicals were purchased from SIGMA. All data are mean values obtained from three parallel experiments. The formation of hydroperoxides in lipid peroxidation of the samples was characterized by the peroxide value measured iodometrically following a modified method described in the literature [14] combined with our intensive test for lipid peroxidation [15]. After treatment the different samples (100 mg) were extracted with an acetic acid – dichloroethane mixture (1:1) (2 ml) by shaking in the closed vessel for 5 min at room temperature then a saturated aqueous potassium iodide solution (0.10 ml) was added and the shaking was continued. After 15 min the mixture was diluted with water (7.5 ml) and titrated with sodium thiosulfate (0.005 M) in the presence of a starch indicator (1%). One unit of peroxide value is defined as the amount (g) of liberated iodine from potassium iodide by the sample (100 g). The LOX activity of the samples was determined using linoleic acid as substrate and measuring changes in concentration of conjugated dienes at 234 nm in buffers of different pH values following a modified method described in the literature [5]. The extracts of the samples (100 mg ml-1 ) were made with 0.05 M TRIS acetate buffer pH 8.2 containing sucrose (0.38 M) and calcium chloride (0.02 M). For the linoleic acid substrate solution (8x10-3 M), the mixture of 0.050 M TRIS acetate buffer (pH 9.2) (5.5 ml), TWEEN 20 (0.025 ml), linoleic acid (0.025 ml) and 1M NaOH (0.10 ml) was diluted to 10 ml with water. Reaction mixtures contained linoleic acid substrate solution (1.5x10 -4 M), the almond extract (1,8 mg ml-1 ) in buffers of different pH values. Controls with only linoleic acid substrate solution and buffers were used to detect autoxidation. One unit of lipoxygenase activity was defined as the amount of enzyme that caused one unit of change in absorbance of the reaction mixture (1.0 ml) in 1 min at 234 nm. The β-glucosidase activity of the extractions was measured according to the literature [16] with substrate 0.25 M cellobiose in 0.1 M sodium acetate buffer pH 5.1. One unit of β-glucosidase activity was defined as the amount of enzyme required to release 1 µmol glucose per minute. The pectinmethylesterase activity of the extractions (1 g ml-1 ) made by a mixture of Triton-X100 (1%), glycerol (20%), polyvinylpyrrolidone (5%), sodium chloride (2 M) and dithiothreitol (0.02 M) in 0.5M TRIS chloride buffer pH 8.5 was measured following a modified method described in the literature [17] by titrating with 0.01 M sodium hydroxide the free carboxyl groups released from a 0.5% pectin solution (30 ml) containing sodium chloride (0.5 M) (pH 7.0) at 35 °C. One unit of pectinmethylesterase activity was defined as the amount of enzyme required to release 1 µmol carboxyl group per minute, under the mentioned assay conditions. RESULTS AND DISCUSSION Lipid peroxidation in almond and marzipan samples The tendency for rancidification was tested by our intensive test for lipid peroxidation [15] at elevated temperature (50 °C) generated by infrared light. This test was originally used for the examination of lipid peroxidation of oils and for screening chemical agents, especially that of new antioxidants. In our previous studies on lipid peroxidation [11, 15] a special ratio called activation coefficient (AC) of the parameters (e.g. peroxide value) was introduced. AC is the ratio of the value of the given parameter at the time of the measurement and the value at the beginning of the investigation. Therefore the higher the AC value of a particular parameter, the higher is the degree of lipid peroxidation. It was found that AC values could illustrate changes in lipid peroxidation better than the measured data. We found that the tendency for rancidification of almonds of all varieties examined was low. This tendency in marzipan raw materials was higher because during their preparation lipid peroxidation got started. Due to the intensive interaction between almond lipids and oxygen on the expanded surface in ground almonds the tendency for rancidification was quite high (Fig. 1).
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14
AC value
12 10
1 2 3
8 6 4 2 0
0
1
2
3
4
Storage time (d) Fig. 1: Change in AC values of peroxide values with respect to storage time in the samples at 55 °C generated by infrared light; (1) almond, (2) ground almond, (3) marzipan.
Isoenzyme content of lipoxygenases in almond and marzipan samples Isoenzyme content of almond lipoxygenases was deduced from the pH dependence of LOX activity. This was determined in different buffers. It is known that for different types of lipoxygenases different substrates are the best: e.g. linoleic acid for lipoxygenase-1 types (pH optima>pH 8.0), arachidonic acid and methyl linoleate for lipoxygenase-2 types (pH optima between pH 6.1-7.9). Linoleic acid was found to be a satisfactory substrate for the different types of lipoxygenases; therefore for preliminary tests only this substrate was used. We found that the activity of LOX isoenzymes could be registered not only in potassium phosphate and sodium borate buffers, as described in the literature, but also in TRIS acetate buffer. The best results were detected when we used a combination of these buffers: TRIS acetate (0.050 M) (pH 4.5-6.1) potassium phosphate (0.050 M) (pH 6.1-7.5) and sodium borate (0.05 M) (pH 7.5-9.3).
Activity (U g-1)
100 80 60
1
40
2
20 0 4,3
6,3
8,3
pH Fig. 2: The characteristic differences in pH dependence of LOX activity of two different sweet almond varieties
In the LOX isoenzyme content measured by pH dependence of LOX activity of different sweet almond varieties smaller or bigger differences were detected (Fig. 2). To illustrate these divergences five different groups were distinguished: (pH 4.5-5.3), (pH 5.5-6.7), (pH 6.9-8.1), (pH 8.3), (pH 8.5-9.3). A strong deviation from mean values of LOX activity was found in two of these groups: (pH 5.5-6.7) and (pH 8.5-9.3). We presume that these deviations could be characteristic parameters in distinguishing the different sweet almond varieties. Marzipan is made from scalded and peeled almond therefore sweet almond varieties after this treatment were also examined. A slight
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decrease in LOX activity and almost the same deviation values were found. In marzipan samples the deviation values were significantly higher than in other samples but LOX activities were almost the same as in almonds after treatment, even in group (pH 5.5-6.7) LOX activity was almost as high as it was in the original almond samples. It is supposed that the reason for this high deviation is the fact that marzipan samples were made from a mixture of different almond varieties (Table 1). Table 1 The mean activity values of almond LOX isoenzymes characterized by pH dependence (given in pH ranges) of LOX activity (U g -1 ) with deviation values in parentheses
Characteristic pH ranges (data) 4.5-5.3 (5) 5.5-6.7 (7) 6.9-8.1 (7) 8.3 (1) 8.5-9.3 (5)
In almonds 65.99 (±2) 62.16 (±8) 28.50 (±1) 41.60 (±1) 19.18 (±9)
In scalded and peeled off almonds 40.42 (±3) 20.62 (±6) 15.14 (±1) 34.30 (±3) 14.56 (±4)
In marzipans 42.78 (±9) 59.47 (±12) 25.71 (±8) 35.53 (±8) 15.56 (±5)
A correlation between LOX activity and lipid peroxidation in almond and marzipan samples In order to study the possibility of a correlation between almond LOX activity and lipid peroxidation the change of the mean LOX activity values in ground almond samples was examined at the characteristic parameters of intensive test for lipid peroxidation at elevated temperature (50 °C) generated by infrared light. We found that the activity of LOX isoenzymes decreased only the acidic region (pH 4.5-5.3) and (pH 5.5-6.7), and in other regions the LOX activity slightly increased. On the basis of the dramatic increase in the concentration of hydroperoxides in the fourth day of the test (Fig. 1) as compared to the change in LOX activity in different pH regions (Fig. 3) we suppose that after a few days the autoxidation became the determinant mechanism during storage of ground almond at elevated temperature (50 °C) generated by infrared light. Activity of some hydrolases in almond and marzipan samples In the decay of raw materials of plant origin not only oxidative but also hydrolytic processes can be accomplished. Therefore the change in activity of two of different hydrolases during storage at elevated temperature (50 °C) generated by infrared light was tested, as well. The mean β-glucosidase activity of different sweet almond varieties was 59.74 U g-1 (±5). The activity of β-glucosidase disappeared during a four-day storage at elevated temperature (50 °C) generated by infrared light. The mean pectinmethylesterase activity of different sweet almond varieties was 1.30 U g-1 (±3). The activity of pectinmethylesterase decreased to 22% of its original activity during a four-day storage at elevated temperature (50 °C) generated by infrared light. It is supposed that neither β-glucosidase nor pectinmethylesterase affect the behaviour of stored almond.
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LOX activity (U g-1)
80 70 60 50 40 30 20 10 0
1 2 3 4
pH 4.5-5.3 pH 5.5-6.7 pH 6.9-8.1
pH 8.3
pH 8.5-9.3
Fig. 3: The change in isoenzyme content of almond LOX characterized by pH dependence (given in pH ranges) of LOX activity (U g -1 ) during storage of ground almond at elevated temperature (50 °C) generated by infrared light Storage time: 0 day (1), 1 day (2), 2 days (3), 3 days (4), 4 days (5).
ACKNOWLEDGEMENT This research was supported by Szamos Marcipán Kft. Pilisvörösvár, Hungary.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bergström, S., Holman, R.T.: Lipoxidase and the autoxidation of unsaturated fatty acids. Advances in Enzymology (Ed.: Nord, F.F.) 8, 425-457 1948. Boyer, P.D. (Ed.): The Enzymes 12. Oxidation-Reduction Part B. Electron Transfer, Oxygenases, Oxidases. Academic Press, New York-San Francisco-London, 1975 p. 150. Feussner, I., Wasternack, C.: Lipoxygenase catalyzed oxygenation of lipids. Fett/Lipid 100, 146-152 1998. Gasztonyi K., Lásztity R. (Szerk.): Élelmiszer-kémia 1. Mezôgazda Kiadó, Budapest, 515-517 1993. Song, Y., Love, M.H., Murphy, P.: Subcellular localization of lipoxygenase-1 and -2 in germinating soybean seeds and seedlings. J. Am. Oil Chem. Soc., 67, 961-965 1990. Boyington, J.C., Gaffney, B.J., Amzel, L.M.: Structure of soybean lipoxigenase-1. Biochem. Soc. Trans. 21, 744-748 1993. Wismer, W.V., Wirthing, W.M., Yada, R.Y., Marangoni, A.G.: Membrane lipid dynamics and lipid peroxidation in the early stages of low temperature sweetening in tubers of Solanum tuberosum. Physiol. Plantanarum 102, 396-410 1998.
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Maccarone, M., Veldink, G.A., Vliegenthart, J.F.G., Agro, A.F.: Ozone stress modulates amine oxidase and lipoxygenase expression in lentil (Lens culinaris) seedlings. FEBS Letters 408, 241-244 1997.Gamborg, O.L., Zalik, S.: A lipoxidase system from sunflower seed. Can. J. Biochem. Physiol., 36, 1149-1157 1958. 9. Franck, T., Kevers, C., Penel, C., Greppin, H., Hausman, J.F., Gaspar, T.: Reducing properties, and markers of lipid peroxidation in normal and hyperhydrating shoots of Prunus avium (L). J. Plant Physiol. 153, 339-346 1998. 10. Kosáry J., Csalári J., Perédi J., Takács M., Polgár A., Síró I.: A mák lipoxigenázok. Olaj, szappan, kozmetika 48, 1-3 1999. Kosáry, J., Csalári, J., Siró, I.: Studies on rancidification of ground poppy seed. Acta Aliment. 31, 161-168 2002. 11. Kosáry J., Szollosi D., Füstös Zs.: A lipoxigenázok izoenzim összetételének változásai a növényekben 1. A tárolt hagyma csírázása. Olaj, szappan, kozmetika 50, 169-172 2001. 12. Kosáry J., Koczka N., Stefanovitsné Bányai É.: A lipoxigenázok izoenzim összetételének változásai a növényekben 2. A ginkgofa (Gingko biloba L.) lipoxigenázainak vizsgálata Olaj, szappan, kozmetika 51, 11-13 2002. 13. Dahle, L. K., Hill, E. G., Holman, R. T.: The thiobarbituric acid reaction and autoxidations of polyunsaturated fatty acid methyl esters. Arch. Biochem. Biophys., 98, 253-261 1962. 14. Kosáry, J., Takács M., Siró I.: Intensive peroxidation test for studying lipid peroxidation. Acta Aliment. 31, 57-62 (2002). 15. Larner, J.: Other glucosidases. In: The Enzymes (2nd edn) Vol. 4. Boyer, P.D.; Lardy, H. és Myrbäck K. (szerk) Academic Press, New York, 369-378 1960. 16. Barnavon, L., Doco, T., Terrier, N., Ageorges, A., Romieu, C., Pellerin, P.: Involvement of pectin methyl- esterase during the ripening of grape berries: partial cDNA isolation, transcript expression and changes in the degree of methyl- esterification of cell wall pectins. Phytochemistry 58, 693-701. 2001.
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EFFECT OF ALTERED EXPRESSION OF LYSOPHOSPHATIDIC ACID ACYLTRANSFERASES ON TRIACYLGLYCEROL SYNTHESIS IN ARABIDOPSIS THALIANA SEEDS MAISONNEUVE, Sylvie ; CHIRON, Hélène ; DELSENY, Michel and ROSCOE, Thomas
Laboratoire Génome et Développement des Plantes, CNRS UMR5096, Université de Perpignan, 66860 Perpignan, FRANCE
Abstract
Two cDNAs were isolated from a Brassica napus immature embryo library that were predicted to code for distinct membrane proteins possessing the glycerolipid acyltransferase molecular signature. Comp lementation of an acyltransferase-deficient mutant of E. coli suggested that each of the cDNAs encoded a functional lysophosphatidic acid acyltransferase. The overexpression of the isoform BAT1.13 in Arabidopsis seeds led to substantial increases in seed mass and lipid content, whereas reducing the level of expression of the homologous Arabidopsis gene using antisense suppression by the rapeseed sequence led to a reduced lipid content. In contrast, both overexpression and reduced expression of the homologue of the second isoform, BAT1.3, in seeds of Arabidopsis led to high frequency of aborted seed and a lower triacylglycerol content in surviving seeds. These results indicate that these two enzymes have different functions in planta, and suggest that the BAT1.13 isoform plays an important role in glycerolipid biosynthesis, contributing to triacylglycerol biosynthesis in seeds. In contrast, BAT1.3 does not seem to influence directly structural or storage lipid biosynthesis in seeds and a role for the phosphatidic acid produced by this isoform remains obscure. Introduction Isozymes of lysophosphatidic acid acyltransferase (LPAAT) located in organellar and cytoplasmic compartments play a central role in determining the acyl composition of phosphatidic acid, a key intermediate in the biosynthesis of membrane and storage lipids. Despite this importance, little is known of the function of the diverse isoforms that exist in plants. Two distinct microsomal LPAATs have been characterised in Limnanthes, one of which controls the incorporation of Very Long Chain Fatty Acids into the sn-2 position of triacylglycerol. Crucifer species lack a homologue of the seed specific Limnanthes enzyme and thus it is unclear as to whether a common pool of phosphatidic acid is used for both phospholipid and TAG synthesis or whether a specific LPAAT isoform produces phosphatidic acid destined for TAG synthesis in seeds. Using a genomic approach, we have identified a large gene family encoding LPAAT-like proteins in Arabidopsis (Maisonneuve et al., 2002). In this report, we describe the functional characterisation of two microsomal LPAATs of Brassica napus, BAT1.13 and BAT1.3, and assess the contribution to TAG biosynthesis by overexpression and by repression of the homologous genes in transgenic Arabidopsis seeds. Methods cDNA isolation : A cDNA named BAT1.13 was isolated by screening a Brassica napus immature embryo cDNA library with a cDNA probe derived from microsomal LPAAT of maize (accession number : CAA82638.1). A second cDNA named BAT1.3 was isolated with a probe derived from the Arabidopsis sequence (accession number : AC006085.11). Complementation of JC201 : The two rapeseed cDNAs encoding putative LPAATs were transformed into the temperature sensitive E. coli mutant JC201, deficient in LPAAT activity, (Coleman, 1990). The complementation of the strain JC201 was verified by growth of transformed bacteria at non-permissive temperature of 38°C on plates and confirmed in liquid culture in the presence of ampicillin and IPTG. Plant transformation : The sequences corresponding to the ORFs contained within the BAT1.13 et BAT1.3 cDNAs were cloned under the control of a seed-specific promoter (napin) and this cassette was transfered into the plant transformation vector (pEC2 provided by Dr P. Guerche and transformed, according to the protocol described by Cartea et al. (1998). Transgenic lines and Arabidopsis ecotype Columbia WT were grown under the same constant conditions to respect reproduciblity of oil content and seed weight measurements.
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Lipid Analysis:The quantification of fatty acids in triacylglycerol (TAG) fraction of different Arabidopsis homozygous lines (T3) was performed according to the protocol of Baud et al. (2002). Results and Discussion 1. Characterisation of cDNAs encoding putative LPAATs of Brassica napus The cDNA sequence BAT1.13 revealed the presence of a single ORF encoding a protein of 390 amino acids, whereas the cDNA sequence BAT1.3, contained two ORFs including one of 376 residues. The deduced protein sequences were homologous to known LPAAT sequences, for example, BAT1.13 shared 82% homologies with the functionally characterized LPAAT of maize, MAT1 (Brown et al., 1995). The predicted hydropathy profile revealed the presence of hydrophobic regions which may correspond to transmembrane domains (Figure 1A and B). In the case of BAT1.13 three potential transmembrane domains are located between residues 6 and 22, 304 and 320, and 337 and 353 whereas for BAT1.3, there are four potential transmembrane domains predicted to be located between residues 7 and 23, 25 and 41, 305 and 321, and 338 and 354. The deduced LPAATs BAT1.13 and BAT1.3 shared 55% similarity over 376 residues. Present within the deduced amino acid sequence of each cDNA are the characteristic motifs of glycerolipid acytransferases NH(X4 )D and FPEGTR (Lewin et al., 1999) located between residues 90 and 96, and 169 and 174 for BAT1.13, and 91 and 97, and 170 and 175 for BAT1.3. In contrast, the deduced amino acid sequences of BAT1.13 and BAT1.3 are very divergent from microsomal LPAATs possessing a substrate preference for unusual fatty acids such as the LPAAT (LAT2) of Limnanthes (Brown et al.,1995) and from prokaryotic LPAATs. 300
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Figure 1: Characteristics of two microsomal LPAATs A and B : Hydropathy analysis of BAT1.13 (A) and BAT1.3 (B) protein. Hydropathy profiles were determined using Kyte –Doolittle algorithm. Horizontal number indicate amino acid positions in the sequence and positive numbers on the vertical scale indicate a hydrophobic tendency. C : Complementation of JC201 mutant by each Brassica napus cDNAs. JC201 : strain JC201 ; pBSK (control -): strain JC201 transformed with only the vector pBluescript ; BAT2 (control +) : strain JC201 t ransformed with the vector pBluescript containing the plastidial LPAAT BAT2 (Bourgis et al., 1999) ; BAT1.3 : strain JC201 transformed with the vector pBluescript containing the cDNA BAT1.3 ; BAT1.13 : strain JC201 transformed with the vector pBluescript containing the cDNA BAT1.13. This experiment was carried out independently three times.
2. Functional analysis of proteins encoded by BAT1.13 and BAT1.3 The ability of the proteins encoded by the BAT1.13 and BAT1.3 cDNAs to complement the deficiency of LPAAT activity that is characteristic of the JC201 strain (Coleman, 1990) was assessed by the restoration of growth at non-permissive temperature of 38°C compared with that of the plastidial LPAAT BAT2. The strain JC201 and the strain transformed with the vector pBluescript are unable to grow at a non-permissive temperature of 38°C (Figure 1C). Nevertheless, the plastidial LPAAT and the proteins coded by the cDNAs BAT1.13 and BAT1.3 are able to restore bacterial growth at the non-permissive temperature. Moreover, the growth of JC201 expressing BAT1.3 was stronger than JC201 expressing BAT1.13 but less strong than the complementation by the plastidial LPAAT. In addition, we showed that the proteins BAT1.13 and BAT1.3 are able to synthetise PA in the presence of C18:1-CoA and lysophosphatidic acid (data not shown). Based on the LPAAT sequence homologies and the complementation experiments, we concluded that the cDNAs BAT1.13 and BAT1.3 encode functional microsomal LPAATs.
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3. Effect of microsomal LPAATs on the biosynthesis of triacylglycerols in Arabidopsis seeds With the goal to evaluate the contribution of the two microsomal LPAATs to the biosynthesis of triacylglycerols in developing seeds, we have created Arabidopsis transgenic lines expressing the Brassica napus LPAATs under the control of specific seed promoter in sense and antisense orientations. The effect of overexpressing BAT1.13 and BAT1.3 rapeseed cDNAs and the repression of expression of the homologous genes in Arabidopsis was examined. The overexpression of BAT1.13 in Arabidopsis seeds led to substantial increases in seed mass and triacylglycerol content, whereas reducing the level of expression of the Arabidopsis homologue using antisense suppression by the BAT1.13 coding sequence led to a reduced seed mass, triacylglycerol content and a wrinkled seed phenotype. The overexpression of BAT1.13 resulted in a greater seed mass and approximately a 25% greater TAG content compared to non-transformed plants grown under the same conditions. In the case of antisense lines, approximatly 10% lower TAG content was observed (Figure 2). Zou et al. (1997) showed that the overexpression of a yeast SLC1-1 LPAAT that preferred very long chain fatty acyl CoAs in rapeseed altered acyl composition but also dramatically increased the seed oil content and possibly because expression of the diverged yeast gene was unregulated. Remarkably, our results suggest that the overexpression of an homologous microsomal LPAAT can substantially increase lipid content in seeds. Taken together, these results suggest that BAT1.13 provides phosphatidic acid for both storage lipid biosynthesis and phospholipid synthesis (data not shown) in seeds.
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Figure 2 : Relative content of fatty acids in triacylglycerols in seeds of Arabidopsis transgenic plants For each line, the content of TAG was determined by gas chromatography of fatty acids. In gray : Col0 lines ; in hatched: antisense lines ; in dotted: sense lines. A-1 to A-4: antisens lines BAT1.13 ; A-5 to A-8: sense lines BAT1.13 ; A-9 to A -12: antisense lines BAT1.3 ; A-13 to A-16: sense lines BAT1.3. Col 0 : control. Four representative lines from this experiment are represented.
In contrast, the overexpression of BAT1.3 isoform in seeds of Arabidopsis led to impaired seed development and a lower triacylglycerol content. Similarly, reduced expression of the Arabidopsis homologue of BAT1.3 resulted in a lower seed mass and reduced triacylglycerol content. The observations together with a restricted profile of exp ression (data not shown) suggest a role for BAT1.3 in the provision of phosphatidic acid for the synthesis of specialised lipids in specific tissues. Acknowledgements: We thank Drs J-J Bessoule and R Lessire for assistance with lipid analysis References : Baud, S., J-P. Boutin, M. Miquel, L. Lepiniec, C. Rochat (2002). An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol. Biochem. 40 : 151-160. Bourgis, F., J. C. Kader, P. Barret, M. Renard, D. Robinson, C. Robinson, M. Delseny and T. J. Roscoe (1999). A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol 120(3): 913-22. Brown, A. P., C. L. Brough, J. T. Kroon and A. R. Slabas (1995). Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii. Plant Mol Biol 29(2): 267-78. Cartea, M. E. M., M.;Gall, A.M.; Pelletier, G; Guerche, P. (1998). Comparison of sense and antisense methodologies for modifying the fatty acid composition of Ara bidopsis thaliana oilseed. Plant Science 136: 181-194. Coleman, J. (1990). Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3- phosphate acyltransferase activity. J Biol Chem 265(28): 17215-21. Lewin, T. M., P. Wang and R. A. Coleman (1999). Analysis of amino acid motifs diagnostic for the sn -glycerol-3-phosphate acyltransferase reaction." Biochemistry 38(18): 5764-71. Maisonneuve, S., R. Guyot, M. Delseny and T. J. Roscoe (2002). A multigene family of lysophosphatidic acid acyltransferases of Arabidopsis thaliana. Advanced research on plant lipids. N. Murata, M. Yamada, I. Nishidaet al, Kluwer Academic Publishers. Zou, J., V. Katavic, E. M. Giblin, D. L. Barton, S. L. MacKenzie, W. A. Keller, X. Hu and D. C. Taylor (1997). Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn -2 acyltransferase gene. Plant Cell 9(6): 909-23
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THE PARTICIPATION OF PHOSPHOLIPASE D IN PLANT GROWTH PROCESSES AND RESPONSE TO VIRAL INFECTION E.V. NEKRASOV1 , I.M. KOTEL’NIKOVA2 , V.E. VASKOVSKY3 1 Botanical Garden of Amur Research Center of Far Eastern Branch of Russian Academy of Sciences 1, Relochny Lane, Blagoveshchensk, 675000 Russia; e-mail:
[email protected] 2 Amur Research Integrated Institute of Amur Research Center of Far Eastern Branch of Russian Academy of Sciences 1, Relochny Lane, Blagoveshchensk, 675000 Russia 3 Institute of Marine Biology of Far Eastern Branch of Russian Academy of Sciences 17, Palchevskogo St., Vladivostok , 690041 Russia
1. Introduction Phospholipase D (EC 3.1.4.4) cleaves the ester bond between the phosphatidyl moiety and the head group of phospholipids. In the presence of primary alcohols, the enzyme acts as a transferase, transferring the phosphatidyl moiety to an alcohol [1]. Plant phospholipase D is a family of enzymes, which are grouped into several types and differ in biochemical properties [2]. Plant tissues may have relatively high levels of phospholipase D activity. Numerous observations pointed to its participation in the many physiological processes: seed germination, plant growth and development, fruit maturing, tissue senescence, plant responses to stress factors and phytopathogen invasion [3]. Phospholipase D is assigned as a component of phytohormone signal transduction [3, 4]. The last year it was shown that the enzyme may be involved in microtubule organization and normal plant growth [5, 6]. This paper presents some results of our investigation of phospholipase D involving in the different physiological processes: seed germination, shoot growth and development, responses to viral infection. For this purpose we analysed phospholipase D as a transferase in extracts or paste of the plants tissues and tested the influence of alcohols, inhibiting the hydrolytic activity of the enzyme, upon the physiological processes. So, the research was confined only to the forms of phospholipase D that posses the transphosphatidylation activity.
2. Seed germination, plant growth and development When we analysed phospholipase D activity in the germinating seeds and developing seedlings presented 5 families of dicotyledons and one family of monocotyledons, the enzyme activity was found to change in a complicated manner tending to increase in general [7]. The characters of the activity changes differed even for subspecies and cultivars within a species (subspecies of Glycine max (L.) Merr., cultivars of Rhaphanus sativus L.). These observations were agreeable with results shown in other works where phospholipase D activity changed differently in germinating seeds and developing seedlings of various plant species [1, 8-10]. We supposed that phospholipase D activity during the germination and seedling development depends on the initial activity of the enzyme in the dry seeds, rate of they germination and taxonomic position of the species [7]. Germination of seeds is accompanied by many processes viz. mobilization of storage nutrients, degradation and utilization of some tissues and formation of new ones, as well as the seedling growth itself. Therefore, it is often difficult to elucidate the role of the enzyme in the processes of growth using germinating seeds as a sole model. So we used another model to study the role of phospholipase D in plant growth and development. It was growing shoots of deciduous trees and bushes [11]. Phospholipase D activity in the shoots of ten species of plants belonging to various families and orders of angiosperms as well as one gymnosperm species was
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monitored during the growing season. The activity of phospholipase D increased during bud expansion and shoot growth of Ulmus pumila L., Tilia amurensis Rupr., Pyrus communis L., Larix gmelinii (Rupr.) Rupr., Syringa amurensis Rupr., Acer negundo L., Populus davidiana Dode, Betula costata Trautv. B. platyphylla Sukacz., Quercus mongolica Fisch. Ex Ledeb., being most pronounced in young shoots with highest water content, and usually decreased during shoot maturation [11]. One of the most convenient methods to determine the role of the enzyme in a physiological process is changing its activity in vivo by a genetical approach or using its regulator. The last ten years alcohols especially n-, secand tert-butanol are used for this purpose [5, 6, 12, 13]. There are opposite opinions about the action of prima ry alcohols on phospholipase D activity. Some authors opined that the alcohols competitively inhibit production of phosphatidic acid, a product of phospholipase D activity in vivo [5, 13]. The others considered the primary alcohols at low concentrations as potent activators of phospholipase D [6, 12]. We tested the influence of nbutanol and iso-butanol on growth and development of barley seedlings and their phospholipid content. The both alcohols are primary and they are substrates for the transphosphatidylation activity of phospholipase D, but iso-butanol is a more poor substrate perhaps because of its branched chain [14]. Barley seedlings were grown in the closed glass chambers at 250 C in the dark. The seeds were sown per Petri dishes turned upside down and covered with cheese-cloth so that the ends of the cloth were sunk into water at the bottom of the chambers. After 2 days of incubation the water was replaced by 0.5% (vol/vol) solution of nbutanol or iso-butanol. The seedlings were incubated for other 3 days and then the lengths of their coleoptiles were measured and lipids of the seedlings without grains were extracted. With incubating barley seedlings in 0.5% aqueous n-butanol the growth of their coleoptiles and roots ceased. On the contrary, the incubation of the seedlings in 0.5% iso-butanol influenced upon their further growth to a lesser degree. The 5-day seedlings treated with n-butanol had shorter coleoptiles, contained more total lipids and phospholipids per a unit of fresh weight as compared with the control (table). The seedlings treated with iso-butanol had intermediate values (table). The differences of phospholipid content were no significant among treatments, though phosphatidic acid content was less, and diphosphatidylglicerol content was higher in the seedlings treated with butanol. In the case of the seedlings treated with n-butanol the differences were more pronounced (table). It is interesting, that we couldn’t found phosphatidylbutanol, a product of the transphosphatidylation activity of phospholipase D, in the seedlings grown in the presence of butanol although we used a relatively sensitive reagent, malachite green, for its detection [15]. However there was a spot corresponding to N-acylphosphatidylethanolamine to its chromatographic mobility (data not shown). This phospholipid was found in all samples and its greatest quantities were found in the seedlings treated with butanol (data not shown). Table. The effects of butanol on growth of barley seedlings, their lipid and phospholipid contents
5.9 ± 0.4 n=43 4.30 ± 0.32
Treatments 1 0,5 % isobutanol 3.0 ± 0.2 n=49 6.09 ± 0.08
0,5% nbutanol 1.2 ± 0.1 n=34 8.95 ± 0.70
1.85 ± 0.06
2.75 ± 0.05
3.00 ± 0.49
water (control) Length of coleoptiles, cm 2 Total lipid content, µg per mg fresh weight 3 Total phospholipid content, µg per mg fresh weight 3 Content of phospholipid classes, mole % 3 Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylinositol Phosphatidylserine Diphosphatidylglicerol Phosphatidic acid
52.8 ± 1.9 22.1 ± 0.2 6.9 ± 0.2 8.8 ± 0.4 2.9 ± 0.6 3.9 ± 0.5 2.8 ± 0.6
48.3 ± 0.1 24.2 ± 0.3 6.0 ± 0.3 11.8 ± 0.4 3.8 ± 0.2 4.3 ± 0.1 1.7 ± 0.6
48.2 ± 1.1 24.1 ± 0.1 6.0 ± 0.4 11.6 ± 1.1 2.9 ± 0.3 5.8 ± 0.7 1.5 ± 0.1
1
Barley seeds were ge rminated in water for 2 days. Then the water was replaced by a corresponding solution and the seedlings were grown for 3 days. The data are shown for five -day seedlings. 2 Values are means ± SE of n coleoptiles. 3 Lipid analysis was carried out as describe d by Kotel’nikova et al. [16]. Values are means ± SE of two replicates where each of the replicates included at least 13 seedlings.
J. Gardiner et al. [5] described the inhibitory effect of 1-butanol on seedling growth of Arabidopsis through disturbance of normal microtubule organization. Our results correspond to these data but demonstrate also the possibility of decreasing of lipid mobilization in barley seedlings under the alcohol influence. It is worth to note that 0.1% (vol/vol) 1-butanol stimulated a-amylase production in barley aleurone [13]. We do not consider the effect of butanol at 0.5% concentration on seedling growth as a non-specific toxic effect on the cells because in
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our experiments the seedlings were alive for at least 8 days in the presence of butanol. The problem is whether phospholipase D is involved in the observed effects of butanol. The different effects of n- and iso-butanol on seedling growth correspond to the difference of these alcohols to participate in the transphosphatidylation activity of phospholipase D. However phosphatidic acid content was detectable and phosphatidylbutanol was not found in the seedling treated with n-butanol.
3. Tobacco plant responses to viral infection As it was shown in our recent paper [16] the changes of phospholipase D activity in tobacco (Nicotiana tabacum L.) leaves infected with tobacco mosaic virus (TMV) depended on response to infection. The enzyme activity did not differ from the control in the leaves of the susceptible cv. Samsun within 7 days after inoculation. During the development of a hypersensitive response in the leaves of a resistant cv. Xanthy necrotic phospholipase D activity tended to little increase in both the sites of necrotic lesion formation and in the leaf regions remote from these sites. The changes of phospholipase D activity in the course of the hypersensitive response development were similar to changes of phosphatidic acid content, which is a product of the hydrolytic activity of the enzyme. Therefore a relative increase in phosphatidic acid could result from phospholipase D activity [16]. The evidence presented in the paper [16] is similar to the results on the effect of severe wounding of tissues on the phospholipid metabolism and phospholipase D activity [17]. These results showed that both the damaged sites and regions remote from them were characterized by a phospholipid hydrolysis and higher phosphatidic acid content. This opinion was confirmed by experiments where alcohols were used. To investigate the effect of alcohol on the development of a hypersensitive response, we incubated tobacco leaves of cv. Xanthy nc. inoculated with TMV in water (control), 0.5% or 1.0% solutions of n-butanol or isobutanol at 250 C in the dark. In the case of control leaves necrotic lesions appeared within 1-2 days after inoculation. In contrast to the experiment with the seedling the incubation of the infected tobacco leaves in 0.5% butanol solutions did not prevent local lesion formation. The relatively high concentration of butanol (1%), above which severe leaf injury was observed, suppressed lesion formation. There was no any difference between n-butanol and iso-butanol in their influence upon lesion formation. So the effect of butanol on necrotic lesion formation depended on its concentration, but the nature of alcohol was hardly of any importance for this process. So our results demonstrate that phospholipase D may participate in the cellular events that secure plant growth and development. The change of the enzyme activity during development of a hypersensitive response to viral infection results, perhaps, from the leaf wounding that accompanies necrotic lesion formation.
4. References [1] Heller, M. (1978) Phospholipase D. Adv. Lipid Res. 16, 267-326. [2] Qin, C. and Wang, X. (2002) The Arabidopsis phospholipase D family: characterization of a Ca2+-independent and phosphatidylcholineselective PLD?1 with distinct regulatory domains. Plant Physiol. 128, 1051–1068. [3] Wang, X. (2000) Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions. Prog. Lipid Res. 39, 109-149. [4] Wang, X., Wang, C., Sang, Y., Qin, C., and Welti, R. (2002) Networking of phospholipases in plant signal transduction. Physiol. Plant. 115, 331–335. [5] Gardiner, J., Collings, D.A., Harper, J.D.I. and Marc, J. (2003) The effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis. Plant Cell Physiol. 44, 687-696. [6] Dhonukshe, P., Laxalt, A.M., Goedhart, J., Gadella, T.W.J. and Munnik, T. (2003) Phospholipase D activation correlates with microtubule reorganization in living plant cells. Plant Cell. 15, 2666-2679. [7] Vaskovsky, V.E., Gorovoi, P.G., Khotimchenko, S.V. and Nekrasov, E.V. (1999) Phospholipase D. A short history and new results. Vestnik Dal’nevostochnogo otdeleniya RAN (Bulletin of Far Eastern Branch of Russian Academy of Sciences) No 4, 34-46. (In Russian). [8] Herman, E.M. and Chrispeels, M.J. (1980) Characteristics and subcellular localization of phospholipase D and phosphatidic acid phosphatase in mung bean cotyledons. Plant Physiol. 66, 1001-1007. [9] Lee, M.H. (1989) Phospholipase D of rice bran. II. The effects of the enzyme inhibitors and activators on the germination and growth of root and seedling of rice. Plant Sci. 59, 35-43. [10] Ryu, S.B., Zheng, L. and Wang X. (1996) Changes in phospholipase D expression in soybeans during seed development and germination. J. Am. Oil Chem. Soc. 73, 1171-1176. [11] Nekrasov, E.V. and Kotel’nikova, I.M. (2000) Phospholipase D activity in shoots of arboreal plants during the growing season. Russ. J. Plant Physiol. 47, 456-462. [12] Munnik, T., Arisz, S.A., de Vrije, T. and Musgrave, A. (1995) G Protein activation stimulates phospholipase D signaling in plants. Plant Cell. 7, 2197-2210. [13] Ritchie, S. and Gilroy, S. (1998) Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity. Proc. Natl. Acad. Sci. USA. 95, 2697-2702. [14] Ella, K.M., Meier, K.E., Kumar, A., Zhang, Y. and Meier, G. (1997) Utilization of alcohols by plant and mammalian phospholipase D. Biochem. Mol. Biol. Int. 41, 715-724.
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[15] Vaskovsky, V.E. and Latyshev, N.A. (1975) Modified Jungnickel's reagent for detecting phospholipids and other phosphorus compounds on thin-layer chromatograms. J. Chromatogr. 115, 246-249. [16] Kotel’nikova I.M., Nekrasov, E.V. and Krylov, A.V. (2004) Effect of tobacco mosaic virus on phospholipid content and phospholipase D activity in tobacco leaves. Russ. J. Plant Physiol. 51, 63-69. [17] Ryu, S.B. and Wang, X. (1998) Increase in free linolenic and linoleic acids associated with phospholipase D-mediated hydrolysis of phospholipids in wounded castor bean leaves. Biochim. Biophys. Acta. 1393, 193-202.
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TRIACYLGLYCEROL BIOSYNTHESIS IN MICROSOMES AND OIL BODIES OF THE OLEAGINOUS GREEN ALGA PARIETOCHLORIS INCISA
1
P. SHRESTHA1 , D. COHEN 1,2, I. KHALILOV1 , I. KHOZIN–GOLDBERG1 and Z. COHEN 1
-A. Katz Department for Dryland Biotechnologies, J. Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sde Boker Campus, Israel. 2 -Department of Chemistry, Ben-Gurion University of the Negev, Israel.
1. Introduction: Parietochloris incisa is a fresh water unicellular green alga, capable of accumulating high amounts of arachidonic acid (AA)-rich triacylglycerols (TAG) in cytoplasmic oil-bodies (Bigogno et al., 2002). Under nitrogen starvation, TAG accounts for over 30% of dry weight. AA makes up about 60% of the fatty acids of TAG, comprising over 95% of cellular AA. Labelling studies revealed that phosphatidylethanolamine (PE) is the major donor of AA for TAG. However, very little is known concerning the enzymes involved in the final steps of this unique TAG assembly. DAG is a common precursor to both the acyl-CoA dependent and the independent pathways. Diacylglycerol acyltransferase (DGAT) is responsible for the final acylation of DAG in the acyl-CoA dependent Kennedy pathway. Recently, acyl-CoA independent pathways were discovered involving DAG:DAG and phospholipid:DAG transacylases (Stobart et al., 1997). In the present work, TAG synthesis was studied utilizing radiolabeled DAG or acyl-CoA both in microsomes and oil bodies. 2. Materials and methods Organism and growth conditions: P. incisa was obtained from the Microalgal Biotechnology Lab., J. Blaustein Institute for Desert Research. The algae were cultivated indoors on BG-11 nutrient medium under controlled temperature and light conditions for 14 days as previously described (Bigogno et al., 2002). Cellular fractionation: Due to their very adamant cell walls, the cells were frozen in liquid nitrogen and ground with mortar and pestle. Lipolytic activity of cell-free extracts of the alga was minimized by using high pH grinding buffer (CHES-NaOH, pH 9.0), containing 5 mM EDTA and 1% BSA. The homogenate was centrifuged at 1500 x g for 10 min to remove cell debris and unbroken cells. The supernatant and the floating layer were subjected to a discontinuous sucrose gradient centrifugation at 25000 x g for 1 h. The floating layer of oil bodies above 0.2 M sucrose was collected and the supernatant was centrifuged at 100000 x g for 1 h. The pellet (100000 x g, microsomes) was collected, washed and homogenized in CHES buffer pH 9.0 with 1 mM DTT and stored at -70 ºC until use. The floatation centrifugation step was repeated twice and oil bodies were recovered. All procedures were carried out at 4 ºC. Protein in membrane fractions was quantified by the method of Bradford (1976). Protein content of oil bodies was determined after delipidation with diethylether. Enzymatic assays: Assay mixtures (100 µL) containing substrates, 0.125% BSA, 0.02% Tween and microsomes or oil bodies in 100 mM Tris -HCl pH 7.5, were incubated at 30 ºC for the indicated times. The production of [114 C] TAG from 0.4 mM [1-14 C] 1,2-dioleoylglycerol or 1-stearoyl-2-[14 C] arachidonoyl-DAG (20000 – 30000 DPM) was measured in the presence or absence of unlabeled acyl-CoAs. Alternatively, 20 µM [1-14 C] oleoylCoA (20000 DPM) and unlabeled 0.4 mM DA Gs were utilized (Bouvier-Nave et al., 2000). MgCl2 (150 mM) and 20% glycerol were added, when indicated. [1-14 C] 1,2-diacylglycerols were obtained by the action of phospholipase C (Type V from B. cereus, Sigma) on different phosphatidylcholine (PC) species (Amersham Biosciences, UK). Reactions were terminated by the addition of water and a mixture of chloroform: methanol: acetic acid 50:50:1 (v/v/v). Lipid extraction was followed by TLC separation of the reaction products. Labeled lipids were detected by Phosphoimager (Fujix, BAS 1000). Radioactive spots were scrapped and counted with a scintillation radioactivity counter (Packard, 1600 TR).
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3. Results and Discussion In attempts to characterize the enzymatic activities involved in the biosynthesis of TAG, we isolated oil bodies and microsomes from stationary phase cells. A protocol had been developed that enabled to overcome the endogenous lipolytic activity, the degradation of TAG and the recovery of most of the oil bodies. Fat layer was colored yellow-orange due to the presence of β-carotene. The simultaneous presence of both carotenoid pigment and TAG in chloroplastic (Rabbani et al., 1998) or cytoplasmic (Zhekisheva et al., 2002) oil bodies were described in some other algal species. The acyl lipids of the oil bodies were composed of neutral lipids (97%, mostly TAG) and polar lipids (3-4%); β-carotene and proteins were minor components. Microsomes and oil bodies of P. incisa were able to synthesize TAG when [14 C] 1,2-oleoyl-DAG or 1stearoyl-2-[14 C] arachidonoyl-DAG were supplied as the sole acyl donors. Acyl-CoA independent TAG synthesis was reliant on the presence of MgCl2 (150 mM), and was linear with time (Fig. 1) and protein concentration; pH and temperature optima were observed at 7.5 and 30 ºC, respectively. In microsomes, this activity was not affected by the thiol-specific reagent, p-chloromercuribenzene sulfonic acid (PCMB), or by thiol-protecting reagent, dithiothreitol (DTT), but was almost completely suppressed by Cu +2 (Table 1). Differently from microsomes, the synthesis of TAG in oil bodies was completely abolished by the addition of PCMB and was enhanced by the addition of acyl-CoAs, indicating the presence of two isoforms of the acylCoA independent TAG synthetic enzymes.
146
Table 1. Effect of MgCl2 , acyl-CoAs and thiol reagents on TAG synthesis from [14 C] 1,2-oleoyl-DAG. Complete assay contained 150 mM MgCl2 . Specific activities for microsomes and oil bodies were 0.95 and 17.0 nmol/mg protein/min, respectively (reaction time was 30 minutes).
40
30
200 20
nmol/mg protein (oil bodies)
nmol/mg protein (microsomes)
300
100 10
0
5
10
15
20
25
30
0
time (min)
Figure 1. The utilization of [14 C] 1,2-oleoyl-DAG for acyl-CoA independent TAG synthesis by microsomes (?) and oil bodies (?). For assay conditions, see ‘Materials and Methods’.
Treatments
Microsomes (% of control)
Oil bodies (% of control)
Complete
100
100
- MgCl2
40
35
20 µM oleoyl-CoA
105
159
20 µM arachidonoyl-CoA
105
135
1 mM PCMB
102
3
1 mM DTT
130
10
At low MgCl2 concentration (0-3 mM), the acyl-CoA independent biosynthesis was suppressed in both microsomes and oil bodies (Table 1). However, when unlabeled oleoyl-CoA was added, labeled TAG was obtained, apparently due to the activity of DGAT. DGAT activity was also assayed by incorporation of [14 C] oleoyl-CoA into TAG (Fig. 2A). TAG synthesis has been observed without the addition of DAG, possibly due to presence of the endogenous substrate. Under assay conditions, along with DGAT, polar lipid acyltransferases activities for the synthesis of diacylglyceroltrimethylhomoserine (DGTS), PC and phosphatidylethanolamine (PE) were also observed (Fig. 3). Labeling of DAG may be a result of several reasons: dephosphorylation of phosphatidic acid, reverse action of cholinephosphotransferase and lipolytic degradation of TAG. Alternatively, DAG can be formed by monoacylglycerol (MAG) acyltransferase (MGAT) (Fig. 2B). Indeed, the synthesis of DAG from 2-oleoyl-MAG was observed. However, this DAG was not further acylated to TAG under our assay conditions. 6
TAG
A
nmol/mg protein
5
FFA DAG
Figure 3. TLC separation of the reaction products of DGTS DGAT assay. PE PC
4
No addition + DAG + MAG
3
2
Con
1
+DAG
0
B
20
nmol/mg protein
nmol/mg protein
8
No addition + DAG
6
+ MAG 4
15
10
5
2
0 0
15
30
45
60
75
0
90
time (min)
30
60
90
120
time (min)
Figure 2. Utilization of [14 C] oleoyl-CoA for TAG (A) and DAG (B) synthesis by microsomes with or without addition of 1,2-oleoyl-DAG or 2-oleoyl-MAG. Assay did not contain MgCl2.
Figure 4. Utilization of [ 14 C] oleoyl- CoA for TAG synthesis by delipidated proteins of oil bodies in the abesence of MgCl2.
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Oil bodies, however, failed to synthesize TAG from [14 C] oleoyl-CoA and DAG. Only after successive delipidation, oil bodies proteins catalyzed the TAG synthesis (Fig. 4).
4. Conclusions DAG is a lipid intermediate, which can be derived from the numerous enzymatic pathways, including lipid de novo synthesis, lipid mo difications and phospholipids catabolism. We suppose that this lipid intermediate is supplied from AA-rich phospholipids during TAG accumulation in P. incisa. Under in vitro conditions, microsomes and oil bodies of P. incisa were able to catalyze acyl-CoA independent TAG synthesis from oleoyl and arachidonoyl containing DAGs, probably due to DAG:DAG transacylase activity. The different sensitivity of microsomal and oil bodies activities to PCMB, may be explained by the occurrence of two enzyme forms in different cellular compartments or by different lipid environments. DGAT with lower specific activities was also observed in both cellular fractions.
5. References Bigogno, C., Khozin -Goldberg, I., Boussiba, S., Vonshak, A., and Cohen, Z. (2002) Lipid and fatty acid composition of the green alga Parietochloris incisa. Phytochemistry 60: 497-503. Bouvier-Nave, P., Benveniste, P., Oelkers, P., Sturley, S.L. and Scaller, H. (2000) Expression in yeast and tobacco of plant cDNA encoding acyl CoA: diacylglycerolacyltransferase. Eur. J. Biochem. 267: 85-96. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem . 72: 248-254 Rabbani, S., Beyer, P., Lonting, J.V., Hugueney, P. and Kleining, H. (1998) Induced β-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil. Plant Physiol. 116: 1239-48. Stobart, K., Mancha, M., Lenman, M., Dahlqvist, A. and Stymne, S. (1997) Triacylglycerols are synthesized and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds. Planta 203: 58-66. Zhekisheva, M., Boussiba, S., Khozin -Goldberg, I., Zarka, A. and Cohen, Z. (2002) Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) under nitrogen starvation and high light is correlated with that of astaxanthin esters. J. Phycol. 38: 325-331.
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PLANT LIPID DERIVATIVE SEED TREATMENTS FOR MANAGING FUNGAL SOYBEAN DISEASES NANCY L. BROOKER1 , JAMES H. LONG2 , AND JOSH COLLINS1 1 Pittsburg State University, Department of Biology, 1701 South Broadway, Pittsburg, Kansas, USA 66762 2 Kansas State University, Southeast Agricultural Research Center, 32nd and Pefley, Parsons, Kansas, USA 67357
Introduction Chemically synthesized petroleum-based pesticides in agriculture have been responsible for a number of ecological and safety concerns. Especially under scrutiny are the synthetic fungicides that include a number of compounds with carcinogenic and teratogenic activities. Because of health and environmental concerns, many of these pesticides are currently under evaluation by Federal agencies to assess their safety and environmental impact [1-3]. It is anticipated that many of these pesticides will not be re-registered for future use because of their toxic ity and non-target effects in the environment [2]. As a result of this strong Federal mandate restricting strategies for chemical use and application, and combined with a negative public perception about pesticides in general, this favors the development of newer safer alternative pest control compounds. In order to meet the demands of consumers and growers alike, exploration of alternative methods for managing pests and fungal diseases is underway. One such alternative approach to controlling fungal pests is the use of natural products, specifically plant-derived compounds as natural pesticides. Several natural plant compounds have been identified as having antifungal or anti-microbial activity and they consist of two basic categories of plant compounds; antioxidants and secondary metabolites [4, 5, 6, 7, 8, 9, 10, 11, 12]. Specific compounds that have been identified with anti-microbial activity include: phenolics and polyphenols, terpene/ triterpenoids and essential oils/ lipids, alkaloids, lectins and polypeptides [12]. In order to develop a novel approach to managing fungal soybean diseases, many of these plant-derived compounds have been evaluated for anti-microbial activity using in vitro assays. This study has concentrated on two of these active mo lecules, lipids and terpene/triterpenoid compounds. In plants, lipids are a primary component in defense structures that serve as protective barriers between the plant and its environment [6]. Additionally, terpenoids make-up a large class of secondary metabolites that act as plant defense molecules and include the phytoalexin molecules that play a role in chemical defense of plant cells [6]. Previous studies have shown that both of these classes of compounds possess anti-microbial/anti-fungal activities [6, 7, 8, 9, 10, 11, 12]. Within this study, two novel plant derived compounds, the lipid Sesamol and a phytoalexin analogue were evaluated as natural pesticides for controlling several plant diseases, including Charcoal Rot of Soybean. This disease is caused by the soil borne fungus Macrophomina phaseolina (Tassi) Goid [12]. M. phaseolina is responsible for disease on over 500 economically important hosts and is found to be responsible for soybean yield losses as high as 70% [12]. In 2002, Charcoal Rot Disease in the United States was estimated to cause a loss of 31,964,000 bushels, valued at $174,317,000 [13]. Charcoal Rot Disease is a particularly difficult disease to control due to its persistence in the soil and its increase in inoculum densities due to field cropping history [14, 15, 16, 17, 18]. The Charcoal Rot fungus has an over-wintering sclerotial form (thick-walled resistant spore) and the presence of these structures in plant debris allow for the fungus to stay viable in soil for two or more years in the absence of a host [14, 15, 16, 17, 18, 19]. Additionally, the disease symptoms do not become readily apparent until mid- to late season, even though Macrophomina is known to infect the plant at seedling stage [13]. As a result of these economic and disease management findings, soybean growers in the United States have identified the development of effective sustainable Charcoal Rot Disease control and management strategies as one of their priorities [9, 10, 11, 14]. Additionally, there is a number of early season soil borne diseases that have also been targeted for improved management strategies. These early season fungal pathogens include: Pythium, Phytophthora, Phomopsis and Rhizoctonia and these fungi are responsible for early seed rots and seedling blights in many plants. These diseases are favored by spring moisture and temperature conditions [13, 14]. In preliminary studies, two compounds, sesamol and a phytoalexin analogue were identified in our laboratory as potential anti-microbia l pesticides [9, 10, 11]. Sesamol (3,4- methylenedioxy -phenol) is a lipid compound from sesame seed oil that has been shown to inhibit the growth of several fungi in laboratory studies. The phytoalexin analogue is classified as a triterpenoid compound and has been shown to inhibit the growth of several plant pathogenic fungi in laboratory studies. In order to determine the efficacy of these two novel plant-derived compounds as seed treatments, this soybean field test was developed. Included in this test was a series of commercial seed treatments as internal controls to comparatively assess the effectiveness and range of activity within our novel seed treatments. These commercial seed
149
treatments of Rival and Allegiance fungicides are used at different rates for managing Pythium, Phytophthora, Phomopsis and Rhizoctonia fungal soil diseases. Results of this field test illustrate the commercial potential of plant-derived compounds as novel seed treatments and the range of soil borne diseases they can effectively manage. Experimental Location and Materials
The soybean test field was established at the Kansas State University Southeast Kansas Agricultural Experimental Station near Columbus, Kansas. Soybean seed (variety KS4694), planting equipment and harvesting equipment was generously provided by Kansas State University. Magnacoat seed coating polymer was provided by Gustafson, Inc.
Field Test Methods A randomized block design was used and the experimental site was divided into four replicate blocks of test plots, with each replication consisting of 12 test plots and each test plot consisting of a single seed treatment. Each test plot consisted of four rows measuring 30 feet in length. Soybean seeds were planted at densities of 8 viable seeds per foot of row. Seed Treatments and Preparation Lipid and phytoalexin analogue treatments were applied to seeds at varying rates using commercially available Magnacoat seed coating polymers and colorants per manufacturer’s instructions. In addition to untreated and coating polymer-only treatment controls, two concentrations of Rival/Allegiance commercial fungicides were also applied to seed per manufacturer’s instructions as internal control treatments. Field Data and Sample Collection Percent mortality was calculated based on the number of plants dead at 12 weeks pot-planting as compared to the total number of seed planted per treatment. Viability data was collected at three weeks post-planting (V-2/ V-3 plant developmental stages) and at 12 weeks post-planting (R-6/ R-7 plant developmental stages) to determine the postemergence viability. Plant mortality was determined as the difference between live plant counts taken 2 weeks postgermination compared to live counts taken at R-6/ R-7 developmental stage plants. Results Field assessment of the natural derivative seed treatments yielded a range of activities. Results of this study indicate that Sesamol and the phytoalexin analogue as seed treatments affects the germination frequency of soybean seedlings in a positive manner. In comparison to the mean viability/mortality data in untreated control plots, all plant derivative treated plots had noticeable improvements in germination frequencies ranging from 7.9% to 21.8% (Table 1). In addition, the lipid deriv ative and phytoalexin analogue seed treatments performed on a comparable or higher level, as compared to the commercial Allegiance/Rival seed treatments used as internal controls. Sesamol and phytoalexin analogue seed treatments increased the germination frequency as high as 15.5% as compared to the commercial seed treatments (Table 1). TABLE1. Mean Mortality / Viability Data for Seed Treatments
Treatment Untreated Control
Magnacoat Only Control
Total Mortality/ Treatment A
Mortality Percentage B
Viability Percentage
344
23.9
76.1
286
19.9
80.1
Allegiance
257 17.8 (high rate) Allegiance (low rate) 155 10.8 Sesamol (1000 µg/ml) 186 12.9 Sesamol (500 µg/ml) 39 02.7 Terpenoid (1000 µg/ml) 127 08.8 Terpenoid (500 µg/ml) 250 17.4 Terpenoid (250 µg/ml) 217 15.1 Terpenoid (125 µg/ml) 251 17.4 A The total mortality represents the sum of all four replicate treatment plots.
150
82.2 89.2 87.1 97.3 91.2 82.6 84.9 82.6
C
B
The percent mortality was calculated based on the number of plants dead at 12 weeks of growth (pre-senescence) as compared to the total number of seeds planted per treatment. C
The percent viability was calculated from the total number of seeds planted per treatment as compared to the viability data taken at three weeks of growth. Seed germination rate was >99% and was determined prior to planting.
Discussion and Conclusions Results of this study indicate that the lipid Sesamol and the phytoalexin analogue are effective as antifungal agents in seed treatments. These compounds display an ability to protect seeds from a number of early season soil borne fungi that are particularly devastating to newly emerged seedlings. These early seed rot and seedling blight fungi include: Pythium, Phytophthora , Phomopsis and Rhizoctonia fungi. In addition to protecting the seed and seedlings early in the plant’s life, it also appears that these novel seed treatments may provide a certain degree of residual activity throughout the plant’s life. This residual protection was displayed in the higher viability data taken at R-6/ R-7 developmental stage plants (presenescence) that were treated with the sesamol and the phytoalexin analogue. From this we conclude that these treatments may also have some impact on managing the mid- to late season effects of Macrophomina within the plant. Certainly by applying the plant-derived compounds to the seed coat, this provides a more economical approach to putting the active antifungal ingredients where they will be most useful for the plant’s protection without excess quantity and expense. This in turn is desirable from an ecolo gical perspective, by cutting down on excess chemicals that could leach and run-off from the soil. Thus, these novel seed treatment formulations also reduce non-target effects and further reduce environmental impact and this is in good agreement with public and Federal mandates for safer pesticides.
At this point we can only speculate as to the mechanism(s) of action of these compounds towards fungal growth. Little is known about Sesamol and of the phytoalexin analogue in plants and their associated anti-microbial activities. It has been speculated that these compounds may be acting physiologically in plants as defense molecules or perhaps as signaling molecules to induce physiological plant defense responses. Additionally, the plant derivatives may be functioning as potent systemic antifungal agents within the plant and may be interfering with the membrane integrity/activity within the fungus. In conclusion, seed treatment germination data from this study support the role of novel plant compounds as plant protective agents. Future studies will concentrate on the mode(s) of action and the protective roles these compounds play in the rhizosphere of plants.
Acknowledgements. Financial support for this investigation was provided by grants to NLB from the Kansas Soybean Commission. We are grateful to Kelly Kusel, Joyce Erikson, Ryan Lord and Kelly Borden for their excellent technical assistance.
References [1] National Agricultural Chemicals Association. 1991. Annual Report 1991. Washington, D.C. [2] Ragsdale, N.N., Henry, M.J. and Sisler. 1993. Minimizing Non-target Effects of Fungicides. National Agricultural Pesticide Impact Assessment Program. American Chemical Society, 524:332-341. [3] National Resource Council. 1987. Regulating Pesticides in Food. National Academy Press, Washington, D.C. [4] Trione, E.J. and Ross, W.D. 1988. Lipids as Bioregulators of Teliospore Germination and Sporidial Formation in the Wheat Bunt Fungi Tilletia sp. Mycologia, pp. 38-45. [5] Cohen, Y., Gisi, U. and Mosinger, E. 1991. Systemic Resistance of Potato Plants Against Phytophthora infestans Induced by Unsaturated Fatty Acids. Physiological and Molecular Plant Pathology, 38:255-263. [6] Kabara, J.J. 1986. Fatty Acids and Esters Anti- Microbial/Insecticidal Agents. ACS Symposium Series, 325:220-23813. [7] Branen, A.L., Davidson, P.M. and Katz, B. 1980. Anti- Microbial Properties of Phenolic Antioxidants and Lipids. Food Technology, 5:42-63. [8] Kato, N. 1981. Anti-microbial Activity of Fatty Acids and Their Esters Against A Film-Forming Yeast in Soy Sauce. Journal of Food Safety, 2:121126. [9] Barnes, B. J., Norman, H. A. and Brooker, N. L. 1997, Inhibition of Three Soybean Fungal Plant Pathogens by Lipid Derivatives and Natural Compounds. In Physiology, Biochemistry and Molecular Biology of Plant Lipids, Editors, John P. Williams, Mobashsher U. Khan and Nora W. Lem, Kluwer Academic Publishers, Dordrecht, pp. 236-238. [10] Brooker, N.L., LeGrande, C., Long, J. and Norman, H.A. 1998.Inhibition of Three Soybean Fungal Plant Pathogens by Lipid Derivatives and Natural Compounds in Advances in Plant Lipid Research, Editors Juan Sanchez, Enrique Cerda-Olmedo and Enrique Martinez-Force, Universidad de Sevilla Publishers, Sevilla, pp.553-556. [11] Brooker, N.L., Long J.H. and Stephan S.M. 2000. Field Assessment of Plant Derivative Compounds For Managing Fungal Soybean Disease. Biochemical Society Transactions 28:919-922. [12] M. Cowan, 1999. Plant Products as Anti-microbial Agents Clinical Microbiology Reviews, 10(12) 564-582. [13] Compendium of Soybean Diseases. 1982. Second Edition, James B. Sinclair, Editor, American Phytopathological Series. pp. 30-33. [14] 2002 U.S. Crop Data Estimates. 2002. USDA Agricultural Statistics Service, http:/www.usda.gov/nass/ [15] Long, J.H., Todd, T., Sweeney, D. and Granade, G. 1994. The Effect of Crop Rotation on Charcoal Rot and Soybean Grain Yield. Report of Progress, Kansas State University 1994 Agricultural Research, Southeast Kansas Branch Station, pp. 85-86. [16] Wantanabe, T., Smith, R.S., Jr. and Snyder, W.C. 1970. Populations of Macrophomina phaseolina in Soil as Affected by Fumigation and Cropping. Phytopathology, 60:1717-1719. [17] Short, G.E., Wyllie, T.D. and Bristow, P.R. 1980. Survival of Macrophomina phaseolina in Soil and in Residue of Soybean. Phytopathology, 70:1317. [18] Dhingra, O.D. and Sinclair, J.B. 1975. Survival of Macrophomina phaseolina Sclerotia in Soil: Effects of Soil Moisture, Carbon:Nitrogen Ratios, Carbon Sources, and Nitrogen Concentrations. Phytopathology, 65:236-240. [19] Ayanru, D.K.G. and Green, R.J. 1974. Alteration of Germination Patterns of Sclerotia of Macrophomina phaseolina on Soil Surface. Phytopathology, 64:595-601.
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LIPID AND OXILIPIN PROFILE DURING STORAGE OF POTATO TUBERS FAUCONNIER MARIE-LAURE1 , WELTI RUTH2 , DELAPLACE PIERRE1 , MARLIER MICHEL3 , DU JARDIN PATRICK1 . 1
Plant Biology Unit, Gembloux Agricultural University, Gembloux, Belgium. Division of Biology, Kansas State University, Manhattan, Kansas, USA. 3 General and Organic Chemistry Unit, Gembloux Agricultural University, Gembloux, Belgium 2
1. Abstract In our study, potato tubers (cv Bintje) were stored at 20 °C where they sprout rapidly and undergo accelerated aging. Analysis of the fatty acid hydroperoxides (HPOs) formed during the storage period revealed that 9-Shydroperoxide of linoleic acid (9-HPOD) was the main oxylipin formed. The fraction of HPO formed by autooxidation was low. Between 45 and 60 days of storage, an increase in 9-HPOD concentration was observed, correlated with an increase in colneleic acid concentration and with multiple sprouts development. Analysis of phospholipids and galactolipids by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the decrease in the amount of linoleic acid in complex lipids correlates well with the amount of 9-HPOD and colneleic acid produced. Complementary analysis revealed that a small fraction of the colneleic acid was esterified in phospholipids in the 2-position. Although HPO content was low after 120 days of aging, during this period, membrane integrity decreased drastically. Thus, we conclude that the presence of fatty acid hydroperoxides is not responsible for membrane damage in potato tubers during aging. 2. Introduction Fatty acid hydroperoxides (HPOs) are formed by autooxidation or by the action of enzymes such as lipoxygenase (LOX) or α-dioxygenase on polyunsaturated fatty acids [1]. HPOs are key intermediates in the LOX pathway; they can be converted enzymatically to a variety of compounds called oxylipins. At least seven enzymatic pathways use HPOs as substrates: hydroperoxide lyase, allene oxide synthase, peroxygenase, epoxy alcohol synthase, reductase pathway, divinyl ether synthase, and finally, LOX that can acts on HPO [2]. Colneleic acid and colnelenic acid are oxylipins formed by the action of divinyl ether synthase (DES) on the 9hydroperoxide of linoleic acid (9-HPOD) and the 9-hydroperoxide of linolenic acid (9-HPOT), respectively [3]. Although our knowledge of oxylipins’ role in the defence of plants against pathogens and stress is increasing rapidly, almost nothing is known about the role of these molecules in aging and senescence. Even though LOX is often proposed to be responsible for peroxidative damage to membrane lipids during aging and senescence, its role is far from clear. 3. Results 3.1. Physiological changes during potato tuber aging Potato tubers are organs of vegetative propagation that can maintain viability up to three years under optimal storage conditions: low temperature and high relative humidity. Under our experimental conditions (20°C), potato tubers sprout rapidly and undergo accelerated aging. During storage of potato tubers, four stages of physiological development can be described [4]: (1) dormancy, during which there is no sprouting even under favourable conditions, which corresponded to the period from 0 to 15 days under our conditions, (2) apical dominance, in which only one sprout develops, which corresponded to the period from 15 to 45 days, (3) the multiple sprout stage, in which apical dominance is released and more than one bud sprouts at the same time, which began around 45 to 60 days, and (4) the daughter tuber stage, in which sprouts are replaced by daughter tubers appearing directly on the mother tuber; this stage did not occur during the 210 days of aging in our experiments.
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3.2. HPOs during aging of potato tubers The extraction and HPLC method that we have developed allow the rapid and single-step determination of individual HPOs, colneleic and colnelenic acid. HPOs were extracted from potato tuber powder with diethyl ether. HPOs were identified on the basis of retention time and UV spectra by comparison with standards synthesised, purified by HPLC, and identified after derivatisation by GC-MS. The HPO concentration during storage, presented in Figure 1 (nmol/g fresh weight), was calculated using 15-HEDE as internal standart. The 9isomers of HPO, in particular 9-HPOD, were the main species formed in potato tubers. The maximal concentration of 9-HPOT was reached at 45 days, and the maximal concentration of 9-HPOD was reached at 60 days. Following these peaks, the HPO concentration dropped and remained low for all the isomers until the end of the experiment (210 days). It can be pointed out that colneleic acid content is high at the beginning of the storage period (day 0) while 9-HPODE content is low. The comportment of potato tuber at the very beginning of the storage period can considered as erratic. The phenomenon can be due to postharvest stress that increase respiration and cause modifications to different metabolic pathways. The phenomenon can be according to us differentiated from aging due to storage which is the aim of our study.
Figure 1: Fatty acid hydroperoxide concentrations during aging of potato tubers.
Analysis of auto-oxidation products and the chiral analysis of HPO furnished interesting results on the origin of the HPO. Table 1 shows the percentage of 12- and 16-HPOT formed by auto-oxidation compared to the 9- and 13-HPOD and HPOT that can be either formed enzymatically or by auto-oxidation. The low levels of 12- and 16-HPOT indicated that auto-oxidation was only responsible for a small part of the HPO present in the tuber. During aging, the auto-oxidation of fatty acids increased, but the phenomenon was still quite limited. In senescing leaf, non-enzymatic lipid peroxidation has been demonstrated to play a dominant role in lipid peroxidation [5], but this was clearly not the case in potato tubers even after 210 days of storage at 20°C. Chiral analysis of 9- and 13-HPOD after HPLC purification of the sample from 0 days of storage confirmed the importance of the enzymatic formation of these species. There was a large enantiomeric excess of the S form; 67% of 13-HPOD was in the S form, and 91% of the 9-HPOD was in the S form. Thus, it is apparent that lipoxygenase and more particularly LOX-1 were mainly responsible for the synthesis of HPO in the tubers. Indeed, the 9- isomers of HPOs were the major HPOs formed, consistent with the specificity of LOX-1 [6].
Table 1: Percentage of 9-, 12-, 13- and 16-HPO formed during storage of potato tuber (percentage of the total HPO concentration). Storage
9-HPOD and
13-HPOD and
12 and 16
time (days)
9-HPOT (%)
13-HPOT (%)
HPOT (%)
0
84.9
12.7
2.4
60
81.8
15.1
3.1
210
69.1
22.4
8.5
153
3.3. Oxylipins during aging of potato tubers HPLC analysis of a crude diethyl ether extract of potato tuber revealed only the presence of HPOs, colneleic acid, colnelenic acid, and slight traces of hydroxy fatty acids, whatever the storage time or the wavelength of the UV detector (diode array). Colneleic and colnelenic acid content in the tuber during aging are shown in Figure 2 (nmol/g fresh weight). Colneleic acid was present in al rger quantities than colnelenic acid, which is in agreement with the higher concentration of 9-HPOD, the precursor of colneleic acid, compared to 9-HPOT, the precursor of colnelenic acid. The time course of the changes in colneleic acid levels during the storage was quite similar to that of the changes in 9-HPOD levels. Both the colneleic acid and 9-HPOD levels showed a clear increase around 45 days of storage, but colneleic acid was always five to ten times less concentrated than 9HPOD. The phenomenon could be explained by two possible mechanisms: 9-HPOD was converted by other enzymatic systems to other oxylipins, or colneleic acid was the main species formed but it was converted to other species. It has previously been shown that colneleic acid is the main oxylipin in potato cells but hydroxy fatty acids and trihydroxy fatty acids were also identified upon elicitor treatment [7]. Colneleic acid is clearly involved in plant defence against pathogens [7, 8] and is also an inhibitor of LOX activity [9], but this work is the first indication for a role of colneleic acid during normal life cycle of the tuber, and for a potential role of colneleic acid in sprout development.
Figure 2: Colneleic and colnelenic acid concentrations during aging of potato tubers.
3.4. Phospholipid and galactolipid content of potato tubers during aging In order to investigate the origin of the fatty acids that are substrates for the lipoxygenase pathway, analysis of lipids, and more particularly phospholipids and galactolipids, was undertaken by ESI-MS/MS, a rapid and sensitive method for quantitative determination of membrane phospholipids and galactolipids and their individual molecular species [10]. Lipid profiling by ESI-MS/MS requires only simple sample preparation and small amounts of plant material. Analysis of lipid extracts by ESI-MS/MS allowed the identification and quantification of the principal phospholipids and galactolipids during the storage of potato tubers. Figure 3 shows the concentrations (nmol/g fresh weight) of the main classes of phospholipids and galactolipids during the storage period. Levels of the major non-plastidic phospholipids, PC, PE, and PI, decreased rapidly between 0 and 45 days of storage and remained low at 60 days. Between 60 days and 90 days, when multiple sprouts were growing, the levels of PC, PE, and PI increased 4 to 5-fold, then remained relatively steady from 90 to 210 days. The major plastidic galactolipids, MGDG and DGDG followed a similar pattern, but with a lesser 2 to 3fold increase occurring between 60 and 90 days. Levels of the major plastidic phospholipid, PG, underwent much smaller changes with only a small increase toward the end of the storage period. Dehydration of the tuber during storage at 20°C accounted for just a small fraction of the observed increases in lipid content between 60 and 90 days, and certainly did not account for the drop in lipid content between 0 and 45 days. The drop in lipid content between 0 and 45 days is likely to be directly related to the large increases in 9-HPOD and 9-HPOT, colneleic acid, and colnelenic acids, whose levels peaked at 45 to 60 days. Indeed, the amount of these compounds present at 45 to 60 days was about 100 nmol/g fresh weight. This correlates well with the amount of loss of complex polar lipids between 0 and 45 days, when the total of these species dropped 113 nmol/g fresh weight; most of the hydrolysed lipids were diacyl species containing about 47% 18:2 and 15% 18:3 [11]. On the other hand, the physiological basis for the increases in PC, PE, PI, PA, MGDG, and DGDG levels between 60 and 90 days is less clear. The relative amounts of the lipid classes also varied during aging. In decreasing prevalence, the main classes of lipids are PC > DGDG > PI at 0 days of storage, while PC > PE > PI after 210 days of storage. These alterations in the relative amounts of PC, PE, and DGDG are in agreement with those
154
observed during 38-month storage of the potato variety Désirée at 4°C by Zabrouskov and Knowles [12], who found that PC and DGDG were the main lipid classes in young tubers while PC and PE were dominant in older samples (38 months). These workers also observed increases in PC and PE levels during storage [11]. PA, lysoPC, lysoPE, and lysoPG may be formed by degradation of the other phospholipid classes. These lipids were present at high levels in potato tuber. Initially, PA accounted for 9.1 ± 1.1 mole % of the total lipid, while lysoPC, lysoPE, and lysoPG accounted for 12.2 ± 2.5%, 4.1 ± 0.3%, and 0.4 ± 0.1% of the total lipids, respectively. In contrast to the other diacyl phospholipids, except PG, PA levels remained constant for the initial 60 days of storage. Interestingly, lysoPC and lysoPE levels dropped during this period, like their diacyl counterparts. These data sugges t that the lipase(s) that produce the free fatty acids used as LOX substrates must have removed the fatty acids from both the sn1 and sn2 positions of polar lipids, without accumulation of lysoPC or lysoPE In fact, lipases found in potato tuber vacuoles, the patatins, have low specificity; recently it was demonstrated that a recombinant patatin-like Arabidopsis enzyme is capable of hydrolysing acyl groups from both sn1 and sn2 positions of phospholipids [12]. Between 60 and 90 days of aging, PA, lysoPC, and lysoPE levels all increased 2 to 3-fold. LysoPC and lysoPE levels dropped again between 90 and 210 days of storage. These changes resulted in a continuous drop throughout the aging period in the mole percentage of lysoPC and lysoPE in the total lipids, so that at 210 days, lysoPC accounted for only 1.9 ± 0.2% and lysoPE for 0.7 ± 0.1% of the total lipid.
Figure 3: Phospholipid and galactolipid levels during aging of potato tubers.
ESI-MS/MS analysis allows us to determine the amounts of individual lipid molecular species (figure 4). In each head group class, species are identified in terms of the total number of acyl carbons and the total number of double bonds. Molecular species containing 34 carbons and two double bonds are major molecular species of PC, PE, PI, PA, PG, and DGDG, while molecular species containing 36 carbons and four double bonds are prominent species of PC, PE, PA, and DGDG. Since potato tubers contain high levels (47%) of 18:2 and very low levels (0.5%) of 18:1 [24], the 34:2 species must be largely a combination of 16:0 and 18:2 (rather than 18:1-18:1), while the 36:4 species must be largely an 18:2-18:2 combination (rather than 18:1-18:3). The most abundant lysophospholipid acyl species was 18:2. Results shows that the ratio of the 36:4 to 34:2 species of PC, PE, PI, and PA tended to be lower at 60 days than at either 0 or 210 days. This suggests that there may have been some preferential hydrolysis of 36:4 (18:2-18:2) as compared to other molecular species of PC, PE, PI, and PA at the time of formation of 9-HPOD and colneleic acid, and that perhaps 36:4 (18:2-18:2) species were preferentially hydrolysed, releasing the LOX substrate, 18:2. Similarly, the 18:2 species of lysoPC and lysoPE dropped to greater extent than the other lyso molecular species between 0 and 60 days, suggesting that these 18:2 lysophospholipids also may have undergone some preferential hydrolysis. However, essentially all
155
molecular species of PC, PE, PI, MGDG, and DGDG were decreased at 60 days compared to 0 days and any discrimination among molecular species by the enzymes that catalysed the lipid degradation that occurred between 0 and 60 days was a subtle phenomenon.
Figure 4: Phospholipid and galactolipid molecular species during aging of po tato tubers. Note the differences in the scales among the three graphs. Species with masses significantly greater than 0.2 nmol/g fresh weight are shown. A. Aging for 0 days. B. Aging for 60 days. C. Aging for 210 days.
In a previous study [11], we determined that membrane integrity dropped drastically between 120 and 240 days of aging. In that study we showed that that the double bond index (DBI) of fatty acid species of potato tubers fluctuated during aging of tubers. ESI-MS/MS analysis of phospholipids and galactolipids confirmed that there were no major shifts toward either more unsaturated or saturated molecular species. Thus, it is unlikely that changes in unsaturation contributed significantly to the alterations in membrane integrity that were observed previously. Since levels of fatty acid hydroperoxides were also relatively low at the end of the storage period, it is also unlikely that the presence of these species contributed to the loss of membrane integrity. Our ESIMS/MS data demonstrate that incorporation into complex lipids was not a major fate of hydroperoxy fatty acids. No increases were detected during aging in complex lipid species with masses that might be expected for species containing hydroperoxides. For example, 18:2-HPOD PC, would have the same nominal mass as 38:2 PC, and the species that we have identified as “38:2 PC” could potentially include some 18:2-HPOD PC. However, this species was present in low amounts (1.4 mole % of PC species), and there was no increase in the mo le percent of the species identified as “38:2 PC” during aging. LysoPCs and lysoPEs, which can destabilize membrane structure, were also at their lowest levels at the end of the storage period. On the other hand, the mole percentages of PE and PA increased from 12% and 9% of the total polar lipids to 18% and 16%, respectively, while PC levels dropped slightly, from 21% to 19% during the aging. These changes might have led to formation of non-bilayer lipids that destabilized membrane structure, since both PE and PA have the capacity to form non-bilayer phases [13, 14].
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3.5. Colneleic acid in phospholipids Complementary analyses were performed to determine if colneleic acid could be find in phospholipids. We demonstrate that a small fraction of the colneleic acid is esterified in phospholipids. This colneleic acid was released by chemical hydrolysis and phospholipase A 2 but not by a lipase with 1-acyl specificity. Phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositol contain molecular species with nominal masses consistent with identification as palmitoyl colneleoyl species. Exact mass analysis of its fragments confirmed the identity of palmitoyl, colneloyl phosphatidylinositol. 4.
Concluding remarks
Our results demonstrate that, during aging and sprouting in potato tubers, the LOX pathway is activated. The LOX pathway seems to be finely regulated as high enzymatic activities and product accumulation is restricted to a narrow window of time during the storage period, with levels of products peaking at 45 to 60 days of aging. Galactolipases and phospholipases liberate free fatty acids, including 18:2, which is peroxidised to HPOs by LOX. HPOs are then converted by DES to colneleic acid, which is degraded to 9-oxo -nonanoic acid. We postulate that the LOX pathway plays a role in the mobilisation of carbohydrates to allow sprout growth. A role for colneleic acid in tuber aging, which is indicated here for the first time, remains to be determined. The role of 9-oxo -nonanoic acid also requires further investigation. We have previously demonstrated that membrane integrity drops after 120 days of tuber aging. However during that late period of aging, fatty acid HPO content is low. Thus, oxygenated lipid species are not responsible for the loss of membrane integrity. 5. References [1] Blée, E. (2002) Impact of phyto-oxylipins in plant defense. Trends Plant Sci., 7, 315-322. [2] Feussner, I. and Wasternack, C. (2002) The lipoxygenase pathway. Annu. Rev. Plant Biol., 53, 275-297. [3] Grechkin, A.N. (2002) Hydroperoxide lyase and divinyl ether synthase. Prostaglandins and other Lipid Mediators, 68-69, 457-470. [4] Ellishèche, P. (1996) Aspects physiologiques de la croissance et du developpement. In La pomme de terre (Rouselle, P., Robert, Y. and Crosnier, JC., eds.), pp 72-124. INRA, Paris. [5] Berger, S., Weichert, H., Porzel, A., Wasternack, C., Kühn, H. and Feussner, I. (2001) Enzymatic and non-enzymatic lipid peroxidation in leaf development. Biochim. Biophys. Acta. 55821, 1-11. [6] Mulliez , E., Leblanc, JP., Girerd, JJ., Rigaud, M. and Chottard, JC. (1987) 5-lipoxygenase from potato tuber. Improved purification and physiological characteristics. Biochim. Biophys. Acta 916, 13-23. [7] Göbel, C., Feussner, I., Schmidt, A., Schell, D., Sanchez-Serrano, J., Hamberg, M. and Rosahl S. (2001) Oxylipin profiling reveals the preferential stimulation of the 9-lipoxygenase pathway in elicitor-treated potato cells. J. Biol. Chem. 276, 6267-6273. [8] Weber, H., Chételat, A., Caldelari, D. and Farmer, E.E. (1999) Divinyl ether fatty acid synthesis in late-blight-diseased potato leaves. Plant Cell, 11, 485-493. [9] Corey, E., Nagata, R. and Wright, S. (1987) Biomimetic total synthesis of colneleic acid and its function as lipoxygenase inhibitor.Tetrah. Lett. 28, 4917-4920. [10] Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H.E., Rajashekar, C.B., Williams, T.D. and Wang, X. (2002) Profiling membrane lipids in plant stress responses. J. Biol. Chem. 277, 31994-32002. [11] Fauconnier, ML., Rojas-Beltran, J., Delcarte, J, Dejaeghere, F., Marlier, M. and du Jardin, P. (2002) Lipoxygenase pathway and membrane permeability and composition during storage of potato tuber (Solanum tuberosum L. cv Bintje and Désirée) in different conditions. Plant Biol. 4, 77-85. [12] Zabrouskov, V. and Knowles, N.R. (2002) Changes in lipid molecular species and sterols of microsomal membranes during aging of potato (Solanum tuberosum L.) seed-tubers. Lipids. 37, 309-315. [13] Verleij, A.J., DeMaagd, R., Leunissen-Bijvelt, J., and DeKruijff, B. (1982) Divalent cations and chlorpromazine can induce nonbilayer structures in phosphatidic acid-containing membranes. Biochim. Biophys. Acta. 684, 255-262. [14] Cullis, P.R., and DeKruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559, 399-420.
6. Abbreviations DBI: double bond index DES: divinyl ether synthase DGDG: digalactosyldiacylglycerol ESI-MS/MS: electrospray ionisation tandem mass spectrometry GC -MS: gas chromatography-mass spectrometry 9-HPOD: 9-hydroperoxide of linoleic acid 9-HPOT: 9-hydroperoxide of linolenic acid 13-HPOD: 13 hydroperoxide of linoleic acid 13-HPOT: 13-hydroperoxide of linolenic acid HPO: fatty acid hydroperoxide LOX: lipoxygenase LysoPC: lysophosphatidylcholine LysoPE: lysophosphatidylethanolamine
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LysoPG: lysophosphatidylglycerol MGDG: monogalactosyldiacylglycerol PA: phosphatidic acid PC: phosphatidylcholine PE: phosphatidylethanolamine PG: phosphatidylglycerol PI: phosphatidylinositol
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SEASONAL ALTERATIONS IN FATTY ACID COMPOSITION OF PHOSPHOAND GLYCOLIPIDS FROM ZOSTERA MARINA AND LAMINARIA JAPONICA Fatty acids of polar lipids from marine macrophytes S.N. GONCHAROVA, N.M. SANINA AND E.Y. KOSTETSKY Far Eastern Nat ional University Sukhanov st. 8, Vladivostok, 690600, Russia
1 . Introduction Marine macrophytes are the numerous group of poikilothermic organisms. Functioning of biological membranes of poikilotherms first of all depends on the environmental temperature. Sea macrophytes belong to the most temperature sensitive poikilothermic organisms (Quinn and Williams 1983; Harwood and Jones 1989). To survive, poikilotherms are needed to maintain liquid crystalline state in their biomembrane lipid matrix. In other words, thermoadaptation of marine macrophytes is related to the effective compensation of lipid matrix viscosity (Joyard et al., 1998; Murata and Siegenthaler 1998). It is regulated by the changes in qualitative and quantitative fatty acid composition of major membrane lipids. Despite of the high thermal sensitivity of marine macrophytes, only fragmental data are available on fatty acid composition of polar lipid classes provided optimal viscosity of membrane lipid matrix in these plants (Williams et al., 1996; Sanina et al., 2003). However, there is a wide range of data on fatty acid composition of total lipids (Khotimchenko et al., 2002; Li et al., 2002). Such information is constantly supplemented by new data (Goncharova et al., 2000; Sanina et al., 2003, 2004), while a little is known about seasonal variations in the composition of fatty acids both of total and individual polar lipids of marine macrophytes (Sanina et al., 2003). Therefore, we analyzed and compared the fatty acids composition of individual classes of major phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG)) and glycolipids (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulphoquinovosyldiacylglycerol (SQDG)) of sea grass Zostera marina and brow alga Laminaria japonica collected in winter and summer. 2. Materials and Methods 2 species of marine macrophytes Zostera marina L. (division Magnoliophyta, class Liliopsida, order Najadales) and Laminaria japonica Aresch. (division Phaeophyta, class Phaeophyceae, order Laminariales) were harvested in the Sea of Japan at 23° C and 2.9° C. Freshly collected macrophytes were thoroughly cleaned to remove epiphytes, small invertebrates and sand particles, then heated for 2 min in boiling water to inactivate enzymes. Total lipid extracts from about 10 kg of macrophytes were obtained according to Folch et al. (1957). The lipid composition was analyzed by silica thin-layer chromatography (TLC). Crude phospholipids and glycolipids were isolated from total lipid extract by column chromatography on silica gel by elution with acetone, acetone/benzene/acetic acid/water (200:30:3:10, by vol.) and a gradient of chloroform and methanol. Then, lipids were purified by preparative silica TLC using chloroform/methanol/water (65:25:4, by vol.). Their purity was checked by two-dimensional silica TLC. Individual classes of phospholipids and glycolipids were methylated according to the method of Carreau and Dubacq (1978). The sum of methyl esters was isolated by TLC using toluene followed by elution with chloroform and resolubilization in hexane. GLC analysis of methyl esters was carried out using Shimadzu GC-9A chromatograph equipped with a flame-ionization detector and a silica capillary column [25 m x 0.25 mm] with Carbowax 20M; the carrier gas was helium. Temperature was 200°?. Percentages of fatty acids were estimated as described (Carreau and Dubacq, 1978).
159
3. Results and discussion The received results are shown in Tables 1 and 2. The sea grass Z. marina contained three fatty acids, 16:0, 18:2n -6 and 18:3n-3 in winter and summer. The high content of 16:3n-3 (15.0-30.5%) occurred in MGDG only. The level of polyenoic C20 fatty acid 20:5n-3 was low and reached maximal value, 4.9% in PC in winter). The major fatty acids of brown alga L. japonica were 14:0, 16:0, 16:1, 18:1n-9 and polyunsaturated fatty acids (PUFAs) C18 (18:2n -6, 18:3n-3, 18:4n-3) and C20 (20:4n -6, 20:5n-3) in both seasons. This general picture of fatty acid distribution was typical for total lipids of sea grasses and brown algae. The level of C18 and C20 PUFAs in polar lipids depended on taxonomic position of macrophytes (Vaskovsky et al., 1996; Khotimchenko et al., 2002; Li et al., 2002). TABLE 1. Fatty acid composition of phospho- and glycolipids from Z. marina harvested in winter (W) and summer (S) (% of the total fatty acids). Fatty acids with contents of less than 3% are excepted. Fatty acid
MGDG
DGDG W
PC
S
PE
W
S
W
S
14:0
0.3
0.6
-
0.2
0.7
0.3
16:0
1.7
5.8
4.1
10.8
21.0
20.4
PG
W
S
W
S
0.1
0.1
28.1
0.9
16.9
33.1
1.5
28.1
16:1
0.8
1.6
0.6
1.1
1.1
0.5
0.5
0.3
8.7
14.7
16:3n-3
30.5
15.0
5.4
1.8
0.2
0.2
0.2
-
0.2
0.1
18:1n-9
0.4
3.1
1.4
1.9
1.1
1.6
0.5
0.9
0.6
2.9
18:2n-6
0.8
10.0
3.1
10.3
23.3
37.4
26.6
32.8
9.6
4.1
18:3n-3
56.7
58.8
78.3
70.0
42.3
35.6
45.9
30.8
39.8
34.1
20:5n-3
2.7
1.2
3.3
1.4
4.9
1.9
1.7
-
3.1
3.4
SFA
2.5
7.7
5.1
12.3
23.4
22.5
20.3
34.9
29.3
29.0
MUFA
1.7
4.7
2.7
3.0
2.9
2.1
1.9
1.2
12.6
17.6
PUFA
94.5
87.5
92.1
85.0
72.3
75.2
77.0
63.8
54.8
41.7
Unsaturated/
38.5
12.0
18.6
7.3
3.2
3.4
3.9
1.9
2.3
2.0
324
242
504
249
247
194
318
160
164
145
Saturated UI
SFA, MUFA, PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; UI, unsaturation index. TABLE 2. Fatty acid composition of phospho- and glycolipids from L. japonica harvested in winter (W) and summer (S) (% of the total fatty acids). Fatty acids with contents of less than 3% are excepted. Fatty acid
MGDG
DGDG
SQDG
PC
PE
PG
W
S
W
S
W
S
W
S
W
S
W
14:0
5.8
5.0
3.4
9.0
1.1
3.6
18.5
12.7
3.0
4.4
1.5
16:0
2.9
5.5
1.9
20.0
25.5
45.2
12.4
12.4
9.5
29.3
27.0
16:1
1.1
4.0
7.1
20.5
2.6
4.3
2.6
4.4
4.6
6.1
12.1
18:0
0.9
0.5
0.6
3.3
1.5
3.6
0.4
0.7
1.9
3.8
0.3
18:1n-9
4.2
9.6
1.2
14.1
17.0
21.9
2.6
12.1
3.9
9.2
13.1
18:2n-6
6.3
11.1
1.1
10.9
5.1
7.7
8.3
12.4
2.8
5.4
9.5
18:3n-6
3.3
8.0
-
1.8
1.4
0.9
0.7
0.4
0.2
0.7
0.2
18:3n-3
5.4
8.7
4.5
5.2
11.4
3.0
2.7
1.3
3.0
2.8
29.0
18:4n-3
54.3
20.3
38.0
3.2
9.3
0.9
0.8
0.4
1.6
0.8
0.9
20:4n-6
1.5
9.9
2.6
2.1
2.0
3.0
25.0
29.1
44.8
26.9
3.2
20:5n-3
10.9
15.9
33.2
3.4
9.9
1.4
17.7
7.5
18.5
2.3
1.4
SFA
9.8
11.5
5.9
35.0
28.1
53.2
32.0
26.6
15.5
40.0
29.5
MUFA
5.9
14.0
12.2
37.7
19.6
30.0
5.2
17.6
9.0
19.9
25.4
PUFA
83.1
74.5
81.5
27.3
46.6
16.8
62.0
54.0
73.5
39.2
44.7
Unsaturated/
9.1
7.7
15.9
1.8
2.4
0.9
2.1
2.7
5.3
1.5
2.4
324
387
504
120
182
79
247
214
318
169
157
Saturated UI
SFA, MUFA, PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; UI, unsaturation index.
160
Comparative analysis of major fatty acids from Z. marina and L. japonica have shown that the sums of saturated and monoenic fatty acids increased in phospho - and glycolipids in both species in summer. As a rule, percentage of PUFAs of n -3 series increased and of n -6 series decreased in winter in all lipids. Unsaturated index (UI) and the ratio between unsaturated and saturated fatty acids decreased in polar lipids of both macrophytes in winter compared to summer. MGDG and DGDG were the most unsaturated lipids among membrane lipids of Z. marina and L. japonica in both seasons. The values of their UI reached 324 and 504 in winter, respectively. The considerable percentage of unsaturated fatty acids in galactolipids perhaps is connected with their function in electrons transport at photosynthesis (Simidjiev et al., 2000). The higher percentage of PUFAs in winter seems to be important during low-temperature acclimatization and for the protection of photosynthetic machinery from low-temperature photoinhibition (Gombos et al., 1994). The lowest UI was observed for photosynthetic lipids PG and SQDG. Also, PG was characterized by the significant contents of 16:1 (8.7- 14.7% in Z. marina and 12.1% in L. japonica ). Both these peculiarities of PG and SQDG are typical for other plants (Joyard et al., 1998). The received results could be helpful in interpretation of thermotropic behavior of the major phospho- and glycolipids of marine macrophytes studied. Acknowledgements. The research was supported by Award VL-003X1 of CRDF and NATO CLG ? 978844 . References Carreau, J.P. and Dubacq, J.P. (1978) Adaptation of macro-scale method to the micro-scale for fatty acid methyl transesterification of biological lipid extracts. J. Chromatogr. 151, 384-390. Folch, J., Less, M., Sloane- Stanley, G.H. (1957) Isolation and purification of total lipids from tissues. J. Biol. Chem. 226, 497-509. Gombos, Z., Wada, H. and Murata, N. (1994) The recovery of photosynthesis from low temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance. Proc. Natl. Acad. Sci. USA. 91, 8787-8791. Goncharova, S.N., Kostetsky, E.Y., Sanina, N.M. (2000) Role of lipids in molecular thermoadaptation mechanisms of seagrass Zostera marina. Biochem. Soc. Trans. 28, 887-890. Harwood, J.L. and Jones, A.L. ( 1989) Lipid metabolism in algae. Adv. Bot. Res. 16, 1-53. Joyard, J., Marechal, E., Miege, Ch., Block, Ch., Dorne, A.-J. and Douce, R.. (1998) Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts in P.- A. Siegenthaler and N. Murata (eds). Lipids in photosynthesis: structure, function and genetics. Kluwer Academic Publishers, Dordrecht. pp. 22-46. Khotimchenko, S.V., Vaskovsky, V.E. and Titlyanova, T.V. (2002) Fatty acids of marine algae from the Pacific Coast of North California. Botanica marina. 45, 17-22. Kostetsky, E. Y., Goncharova, S. N., Sanina, N. M., Shnyrov, V. L. (2004) Season influence on lipid composition of marine macrophytes. Botanica Marina, 47, 134-139. Li, X., Fan, X., Han, L. and Lou, Q. (2002) Fatty acids of some algae from the Bohai Sea. Phytochemistry. 59, 157-161. Murata, N. and Siegenthaler, P-A. (1998) Lipids in photosynthesis: An Overview in P.- A. Sigenthaler and N. Murata (eds.). Lipids in photosynthesis: structure, function and genetics. Kluwer Academic Publishers, Dordrecht. pp. 1-16. Quinn, P. J. and Williams, W. P. (1983) The structural role of lipids in photosynthetic membranes. Biochim. Biophys. Acta. 737, 223-266. Sanina, N.M., Goncharova, S.N., Kostetsky, E.Y. (2004) Fatty acid composition of individual polar lipid classes from marine macrophytes. Phytochemistry. 65, 721-730. Sanina, N.M., Goncharova, S.N., Kostetsky, E.Y. (2003) Seasonal changes in thermotropic behavior of phospho- and glycolipids from Laminaria japonica. In N. Murata et al. (eds.), Advanced Research on Plant Lipids. Kluwer Academic Publishers, Dordrecht, pp.385-388. Simidjiev, I., Stoyalova, S., Amenitsch, H., Javorfi, T., Mustardy, L., Laggner, P., Holzenburg, A. and Garab, G. (2000) Self- assembly of large, ordered lamellae from non- bilayer lipids and integral membrane proteins in vitro . Biochemistry. 97, 1473-1476. Vaskovsky, V.E., Khotimchenko, S.V. , Xia, B. and Hefang, L. (1996) Polar lipids and fatty acids of some marine macrophytes from the yellow sea. Phytochemistry. 42, 1347-1356. Williams, W.P., Sanderson, P.W., Cunningham, B.A., Wolfe, D.H., Lis, L.J. (1993) Phase behaviour of membrane lipids containing polyenoic acyl chains. Biochim. Biophys. Acta. 1148, 285-290.
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SEASONAL CHANGES IN PHYSICO-CHEMICAL PROPERTIES OF POLAR LIPIDS FROM SEAGRASS ZOSTERA MARINA N.M. SANINA, S.N. GONCHAROVA, E.Y. KOSTETSKY Far Eastern National University 27 Oktiabrskaya St., Vladivostok. Russia 690950
2 . Introduction Marine macrophytes belong to poikilotherms which seem to be the most vulnerable organisms in conditions of global climate changes. In turn, their productivity determines the successful survival of both marine and terrestrial heterotrophic organisms. Thermoadaptive capacity of marine macrophytes including widespread seagrasses depends on the efficacy of intra - and intermolecular rearrangements in their mebrane lipid matrix. It was shown some general and specific seasonal changes in lipid composition of taxonomically different macrophytes (Goncharova et al., 2004; Kostetsky et al., 2004). Also it was revealed that thermotropic behavior and fatty acid composition of major glyco - and phospholipids of brown alga Laminaria japonica substantially changed with seasons (Sanina et al., 2003). The purpose of present work was the study and the comparison of seasonal alterations in the crystal-liquid crystal-isotropic phase transitions and fatty acid composition of polar lipids of other marine macrophyte, seagrass Zoster marina, which is known as valuable raw material for production of pectin enterosorbent, zosterin (Zaporozhets et al., 1991). 3 . Materials and Methods Z. marina (Embryophyta) was harvested in Possiet Bay (the Sea of Japan) in winter and summer seasons from seawater of 3°C and 20°C, respectively. Freshly collected seagrass was thoroughly cleaned to remove epiphytes, small invertebrates and sand particles and then heated for 2 min in boiling water to inactivate enzymes. Total lipid extracts from about 10 kg of seagrasses were obtained according to the method of Folch et al. (1957). Crude glyco- and phospholipids were isolated from total lipid extract by column chromatography on silica gel by elution with acetone, acetone/benzene/acetic acid/water (200:30:3:10, by vol.) and a gradient of chloroform and methanol. Then, lipids were purified by preparative silica TLC using chloroform/methanol/water (65:25:4, by vol.). Chromatographically pure lipids were solubilized in chloroform and introduced into standard aluminium pans. Vacuum-dried samples of approx. 10 mg were sealed into pans and placed in a DSM-2M differential scanning calorimeter (Puschino, Russia). Samples were either heated or cooled at 16 °C/min in a temperature range between –100 °C and 80°C at a sensitivity of 5 mW. The peak in the plot of heat capacity versus temperature was recorded as the phase-transition temperature, Tmax. Temperature ranges of isotropic transitions of the same phospholipids were defined by means of POLAM-P-312 polarizing microscopy (Russia) with a heated stage, at a magnification of x100. Analysis of acyl chains linked to phospho- and glyco-lipids was carried out by GLC as described by Sanina et al. (2004). 3. Results and Discussion DSC thermograms of phospo - and glycolipids from Z. marina , collected in winter, are shown in Fig. 1 together with respective thermograms received earlier for “summer” lipid samples (Goncharova et al., 2000). Thermotropic behavior of two non -photosynthetic phospholipids
163
(PLs), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) similarly changed in result of the seasonal temperature drop. It was mainly occurred as the substantial decrease of T max by approx. 12 and 20°C, respectively. Therefore the synchronism of their DSC transitions was not disturbed in winter. This peculiarity was earlier marked for L. japonica (Sanina et al., 2000). It is appeared that the same level of fluidity should be maintained in
Figure 1. DSC thermograms of major lipids isolated from Zostera marina, harvested in winter (solid line) and in summer (dotted line). PC (A), PE (B), PG (C), MGDG (D), DGDG (E).Vertical bar represents 0.5 mW. Scanning rate, 16 °C/min. Sample weight, 10 mg
both major phospholipids located in different leaflets of membrane bilayer. The cooperativity of thermal transition of both PLs was reduced in winter. Other phospholipid, phosphatidylglycerol (PG) together with glycolipids (GLs) characterizes the photosynthetic membranes (Garab et al., 2000). Their seasonal changes in thermotropic behavior differed from ones of PC and PE that was also agreed with previous data on L. japonica (Sanina et al., 2000). So, the profile and the position of thermogram of PG were not changed, while the cooperativity was reduced in winter compared to summer. The stable thermotropic behavior seems to be connected with sufficiently low T max of PG in summer already. The same seasonal behavior was observed in thermal transition of DGDG. The cooperativity of its main peak at -2°C was sharply reduced, while T max shifted a little (till -6°C). Low cooperativity of thermal transition of monogalactosyldiacylglycerol (MGDG) was maintained in both seasons. However, there was the redistribution of poorly resolved peaks on the respective thermograms. In winter, both low- and high -temperature maxima of heat absorption were dropped, thereby the middle one at -10°C became more pronounced. Hence, temperature parameters of DSC t ransitions of photosynthetic lipids of Z. marina are less changeable compared with non -photosynthetic ones. Seasonal changes in thermal transitions were accompanied by the following rearrangements in fatty acid composition of polar lipids (Table 1). At lower seasonal temperature, unsaturation of acyl chains was classically elevated in all studied lipids excepting PG. The increased unsaturation could induce the lowering of the phase transition cooperativity (Williams et al., 1993). Also, that could be the reason of decreased Tmax of PC and PE. The interchange of linoleic and linolenic fatty acids mainly underlied physical state adjustments PC and PE. In PE, the adequate alteration in percentage of 16:0 also was substantial. PG was the most saturated lipid in both seasons. Nevertheless, it was characterized by one of the lowest Tmax that is probably connected with the repulsion between charged polar groups of this anionic lipid. In both GLs, linoleic acid decreased by 7-9% in winter. In contrast, 16:3n-3 and linolenic
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acids increased by 15 and 8% in MGDG and DGDG, respectively. The content of 16:0 decreases less. TABLE 1. Fatty acid composition of glyco- and phospholipids from Zostera marina harvested in winter (W) and summer (S) (% of the total fatty acids). Fatty acids with contents of less than 2% are excepted. Fatty acid 14:0 16:0 16:1 16:3n3 18:1n9 18:2n6 18:3n3 20:5n3 SFA MUFA PUFA Unsaturated/ Saturated UI
MGDG W S 0.3 0.6 1.7 5.8 0.8 1.6 30.5 15.0 0.4 3.1 0.8 10.0 56.7 58.8 2.7 1.2 2.5 7.7 1.7 4.7 94.5 87.5 38.5 12.0
DGDG W S 0.2 4.1 10.8 0.6 1.1 5.4 1.8 1.4 1.9 3.1 10.3 78.3 70.0 3.3 1.4 5.1 12.3 2.7 3.0 92.1 85.0 18.6 7.3
W 0.7 21.0 1.1 0.2 1.1 23.3 42.3 4.9 23.4 2.9 72.3 3.2
324
504
247
242
249
PC
S 0.3 20.4 0.5 0.2 1.6 37.4 35.6 1.9 22.5 2.1 75.2 3.4
W 0.1 16.9 0.5 0.2 0.5 26.6 45.9 1.7 20.3 1.9 77.0 3.9
194
318
PE
S 0.1 33.1 0.3 0.9 32.8 30.8 34.9 1.2 63.8 1.9
W 28.1 1.5 8.7 0.2 0.6 9.6 39.8 29.3 12.6 54.8 2.3
160
157
PG
S 0.9 28.1 11.4 2.9 4.1 34.1 3.4 33.1 21.1 43.3 1.9 155
Temperature range of DSC peaks at the range of 30-50°C correlated with temperature of isotropic transition observed under polarizing microscope. As shown earlier, their position on temperature scale may determine the capacity of poikilotherms to survive at the acute elevation of environmental temperature (Sanina et al., 2002, 2003). Acknowledgements. The research was supported by NATO CLG ? 978844 and Award VL-003X1 of CRDF.
References Folch, J.., Less, M., Sloane- Stanley, G.H. (1957) Isolation and purification of total lipids from tissues. J. Biol. Chem. 226, 497-509. Garab, G., Lohner, K., Laggner, P., Farkas, T. (2000) Self-regulation of the lipid content of membranes by non-bilayer lipids: a hypothesis. Trends in Plant Sci. 5, 489-494. Goncharova, S.N., Kostetsky, E.Y., Sanina, N.M. (2004) The Effect of seasonal shifts in temperature on the lipid composition of marine macrophytes. Russ. J. Plant Physiol. 51, 169-175. Goncharova, S.N., Kostetsky, E.Y., Sanina, N.M. (2000) Role of lipids in molecular thermoadaptation mechanisms of seagrass Zostera marina. Biochem. Soc. Trans. 28, 887-890 Kostetsky, E. Y., Goncharova, S. N., Sanina, N. M., Shnyrov, V. L. (2004) Season influence on lipid composition of marine macrophytes. Botanica Marina, 47, 134-139. Sanina, N.M., Goncharova, S.N., Kostetsky, E.Y. (2004) Fatty acid composition of individual polar lip id classes from marine macrophytes. Phytochemistry. 65, 721-730 Sanina, N.M., Goncharova, S.N., Kostetsky, E.Y. (2003) Seasonal changes in thermotropic behavior of phospho- and glycolipids from Laminaria japonica. In N. Murata et al. (eds.), Advanced Research on Plant Lipids. Kluwer Academic Publishers, Dordrecht, pp.385-388. Sanina, N.M., Kostetsky, E.Y. (2002) Thermotropic behavior of major phospholipids Zaporozhets, T.S., Besednova, N.N., Liamkin, G.P., Loenko, I.N., Popov A.A. (1991) Antibacterial and therapeutic effectiveness of a pectin from sea grass Zostera. Antibiot. Khimioter. 36, Williams, W.P., Sanderson, P.W., Cunningham, B.A., Wolfe, D.H., Lis, L.J. (1993) Phase behaviour of membrane lipids containing polyenoic acyl chains. Biochim. Biophys. A cta. 1148, 285-290.
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PRODUCTION OF AN ISOMER OF CONJUGATED LINOLENIC ACID, PUNICIC ACID, IN RAPESEED AND RICE J. KOHNO-MURASE1 ; M. IWABUCHI1 ; K. KOBA 2 ; J. IMAMURA 1 1 Plantech Research Institute, 1000 Kamo shida-cho, Aoba-ku, Yokohama, 227-0033, Japan. 2 Siebold University of Nagasaki, Nagasaki 851-2195, Japan 1. Introduction The conjugated fatty acids occur as diene, triene, and tetraene in which the most common cojugated polyenoic acids are octadecanoic acids, termed CLNAs. Five CLNAs isomers occur as major seed oils of several plants: alfa-eleostearic (cis-delta9, trans-delta11, trans-delta13), calendic (t rans-delta8, trans-delta10, cis-delta12), punicic (cis-delta9, trans-delta11, cis-delta13), jacaric (cis-delta8, trans-delta10, cis-delta12), and catalpic (trans-delta9, trans-delta11, cis-delta13) acids (1). Tung oil contains high levels of alfa -eleostearic acid is known to be as materials of mainly quick-drying enamels and varnishes. There is also growing evidence showing that supplementation with CLNA has cytotoxic effects on tumor cells and that uptake of CLNA has an effect on lipid metabolism (2-3). Recently cDNAs encoding enzymes that catalyze the formation of the conjugated double bonds in CLNA have been identified (4-8). These enzymes were termed conjugases and were shown to be divergent forms of delta12-oleate desaturase (FAD2). Punicic acid is major storage lipid in Punica granatum and Trichosanthes kirilowii seeds, which accumulate punicic acid up to over 80% (w/w) and about 40% of the fatty acid in seed lipid respectively. In this study we isolated cDNAs that encode enzymes associated with the formation of one of the conjugated linolenic acid (CLNA) isomers, punicic acid (cis-delta9, trans-delta11, cis-delta13) from T. kirilowii and P. granatum. Expression of them in Brassica napus seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid ca. 4 % (w/w) of the total seed oils. We also expressed the cDNA in rice in seed-specific manner and produced rice accumulating punicic acid in the seeds. Feeding experiments with mice using the pomegranate oil accumulating punicic acid showed that weight gain of perirenal and epididymal adipose tissues was reduced considerably. 2. Experimental Procedures FAD2-related cDNA isolation Total RNA was isolated from mature seeds of T. kirilowii and P. granatum as described (8). To isolate cDNAs that encode polypeptide related to FAD2, degenerate primers were designed to target conserved amino acid sequences in FAD2-related enzymes. The amplified fragments were cloned into pGEM -T Easy plasmid vector (Promega) and sequenced using a PRISM DyeDeoxy Terminator Cycle Sequencing System (Applied Biosystems). Finally full-length cDNAs of FAD2-related cDNAs were isolated by PCR amplification with Pyrobest DNA polymerase (Takara Shuzo). Expression of TkFac and PgFac in plants To produce crops accumulating punicic acid in the seeds, TkFac and PgFac were introduced in Brassica napus and Oryza sativa by Agrobacterium-mediated transformation and expressed them under control of seed specific napin and globulin seed specific promoters, respectively. Feeding experiments To analyze the effect of dietary punicic acid on body fat in mice, twenty four male CD-1mice (6wk old) were randomly assigned to four groups of six animals each according to the experimental fats: 6% of soy bean oil by weight +0%, 0.1%, 0.5% and 1.0% pomegranate oil and total fatty acid concentration is 7% adjusted by addition of Perilla oil. Animals were maintained on the respective diets ad libitum for 4wks. During the feeding period, body weight and food consumption were recorded every other day. After 16 h of food deprivation, animals were anesthetized with diethyl ether, and the liver and other tissues, including perirenal and epididymal white adipose were excided and weighted. 3. Results and Discussion PCR amplification and the sequence analysis resulted in isolation of two types of full-length cDNAs fragments closely related to FAD2 in each experiment for T. kirilowii (TkFac and TkFad2) and P. granatum (PgFac and PgFad2). The phylogenetic analysis indicated that TkFad2 and PgFad2 were grouped within a delta12-oleate desaturases, and on the other hand, TkFac and PgFac were grouped within a conjugase branch (8). This analysis suggested that TkFad2 and PgFad2 were delta12 desaturase and TkFac and PgFac were conjugase. To examine whether TkFac and PgFac are associated with the formation of punicic acid, the full-length cDNAs encoding TkFac and PgFac were expressed in Arabidopsis plants under control of the constitutive CaMV 35S promoter or
166
the seed-specific promoters. From the results we confirmed that TkFac and PgFac are conjugase associated with the formation of the conjugated trans-delta11, cis-delta13 double bonds of punicic acid. To produce crops accumulating punicic acid in seeds we expressed TkFac gene under control of napin promoter in B. napus. The analysis showed that punicic acid was accumulated in all the transgenic plants observed. Maximal concentration of punicic acid in seed oils was 3.8% in TkFac-1 (Table 1). Punicic acid accumulation was accompanied by changes in relative amounts of other fatty acids. For example, increase of oleic acid level and decrease of linoleic and linolenic acid levels . This trend has been observed transgenic plants expressing other Fad 2 related enzymes, such as delata12-epoxygenase (9). These facts suggested that this increase in oleic acid and decrease in linole ic and linolenic acids might be caused by inhibition of the endogenous delat12-oleate desaturase activity at the translational or post-translational levels by the presence of exogenously expressed conjugase enzyme. We also produced transformed rice with PgFac gene expressed with globulin promoter. Similarly, punicic acid accumulation was observed in the transgenic rice seeds accompanied with increase of oleic acid contents (data not shown). We analyzed the effect of dietary punicic acid on body fat and liver lipid in mice. The experimental diet containing pomegranate oil as source of punicic acid were prepared and fed to mice. a Table 1. Fatty acid composition of T1 seed of B.napus expressing TkFac
control
b
TkFac-1
16:0
18:0
18:1
18:2
18:3
20:0
20:1
22:0
22:1
Punicic acid
7 .8
1.3
46.6
30.6
4.7
0.6
0.9
0.4
0
0
6.3
0.9
49.0
24.7
9.2
0.4
1. 1
0.3
0
3.8
a: Values are represented as weight % of the total fatty acids b: Average values of six non-transgenic B. napus
After four weeks feeding of this experimental diet, the tissues were excised and weighted. As the result, the weight of epididymal adipose tissue, that fat tissue surrounding testis was lower in mice fed punicic acid than in control mice. This trend also was observed in the weight of perirenal adipose tissue. These results indicate that punicic acid is effective to reduce visceral lipids and can improve human health through prevention for life-style related disease such as diabetes, high-lipid blood syndrome and hypertension. This work was supported by the Research and Development Program for New Bio -industry Institutive of the Bio-oriented Technology Research Advancement Institution. 4. References [1] Smith, C.R. Jr. (1970) Progress in the Chemistry of Fats and Other Lipids. In R.T. Holman (ed), Pergamon Press, Oxford pp.137-177. [2] Suzuki, R., Noguchi, R., Ota, T., Abe, M., Miyashita, K., and Kawada, T. (2001) Cytotoxic effects of conjugated trienoic fatty acids on mouse tumor and human monocytic leukemia cells. Lipids 36, 477-482 [3] Koba, K., Akahoshi, A., Yamasaki, M., Yamada, K., Iwata, T., Kamegai, T., Tsutsumi, K., and Sugano, M. (2002) Dietary conjugated linolenic acid in relation to CLA differently modifies body fat mass and serum and liver lipid levels in rats. Lipids 37, 343-350 [4] Cahoon, E. B., Carlson, T. J., Ripp, K. G., Schweiger, B. J., Cook, G. A., Hall, S. E., and Kinney, A. J. (1999) Biosynthetic origin of conjugated double bonds: production of fatty acid components of high -value drying oils in transgenic soybean embryos. Proc. Natl. Acad. Sci. U. A. S. 96, 12935-12940 [5] Cahoon, E. B., Ripp, K. G., Hall, S. E., and Kinney, A. J. (2001) Formation of conjugated delta8, delta10-double bonds by delta 12oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid. J. Biol. Chem. 276, 2637-2643 [6] Qui, X., Reed, D. W., Hong, H., MacKenzie, S. L., and Covello, P. S. (2001) Identification of a Delta 4 fatty acid desaturase from thraustochytrium sp. Involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. Plant Physiol. 125, 847-855 [7] Hornung, E., Pernstich, C., and Feussner, I.. (2002) Formation of conjugated Delta 11 Delta 13-double bonds by Delta 12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur. J. Bioche,. 269, 4852-4859 [8] Iwabuchi, M., Kohno-Murase, J., and Imamura, J. (2003) Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds J Biol. Chem. 278, 4603-4610 [9] Singh, S., Thomaeus, S., Lee, M., Stymne, S., and Green, A. (2001) Transgenic expression of a delta 12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid. Planta 212, 872-879
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PARTICLE BOMBARDMENT-MEDIATED TRANSFORMATION OF ELAEIS OLEIFERA IMMATURE ZYGOTIC EMBRYOS WITH ANTISENSE PALMITOYLACP THIOESTERASE GENE BHORE, SUBHASH J1, 3 ; CHA, THYE SAN2 ; SHAH, FARIDA H1, 3 1 School of Bioscience and Biotechnology, Faculty of Science and Technology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia. 2 Department of Biological Sciences, Faculty of Science and Technology, KUSTEM, Mengabang Telipot, 21030 Kuala Terengganu, Malaysia. 3 Current address: Melaka Institute of Biotechnology, Pejabat Pos Ayer Keroh, 75450 Melaka, Malaysia. Email:
[email protected]
1. Abstract The palm oil derived from the fleshy mesocarp tissue of the American oil palm, Elaeis oleifera contains 28 % saturated fatty acids (of the total fatty acids). Among the saturated fatty acids palmitic acid (C16:0 ) is the most abundant accounting for 25 % of the total fatty acids. Our aim is to minimize the C16:0 content. The palmitoylacyl carrier protein thioesterase (PATE) gene, one of the key genes involved in plastidial fatty acid biosynthesis is known to regulate the accumulation of the C16:0 . In oil palm, E. oleifera mesocarp, C16:0 content can be minimized by post-transcriptional regulation of PATE gene expression. Molecular mechanisms of inactivating genes, such as antisense mediated gene silencing can be implemented in mesocarp tissue specific manner enabling the inactivation of PATE gene expression in the developing mesocarp without affecting gene expression in other tissues of the E. oleifera plant. In this study, by particle bombardment-mediated method of plant transformation 12 weeks old [Weeks After Pollination (WAP)] immature zygotic embryos (IZEs) of E. oleifera were transformed with a construct carrying 619 bp long antisense PATE gene driven by oil palm mesocarp specific promoter (OPMSP). Selection of the transformed IZEs was accomplished using hygromycin (HYG). Plantlets were regenerated from the selected (HYG resistant) IZEs. A total of 56 HYG resistant plantlets were regenerated successfully, of which 5 were verified to be transformants by polymerase chain reaction (PCR) amplification of the transformed expression cassette, and nucleotide sequencing of the PCR products. Southern hybridization of genomic DNA of five positive plantlets further confirmed their transgenic nature. We postulate that the post-transcriptional PATE gene silencing in mesocarp tissue of E. oleifera may increase the level of palmitoleic (C16:1 ), stearic (C18:0 ) and oleic (C18:1 ) acids up to some extent at the expense of C16:0 . The successful integration of antisense PATE is reported. In addition to this, construction of novel, effective and efficient transformation vectors for PATE gene silencing is also reported. 2. Introduction Oil palm is one of the world’s most significant sources of edible and industrial fats and oils. There are two oil palm species namely, Elaeis guineensis Jacq, and E. oleifera. Because of high oil yield, African oil palm, E. guineensis Jacq. (Tenera) is highly favored for the commercial basis cultivation. However, oil palm E. oleifera is a species of economical interest and is an important source of different trait that can be exploited in traditional plant breeding. Palm oil obtained from E. oleifera contains 28 % saturated fatty acids (of the total fatty acids). Among the saturated fatty acids, C16:0 is predominantly accumulated (25 %) in the E. oleifera fruit mesocarp although plants are known to synthesize at least 200 different types of fatty acids (Rajanaidu et al., 1997; Thelen and Ohlrogge, 2002; van de Loo et al., 1993). The PATE is one of the key genes involved in the fatty acid biosynthesis pathway (Ohlrogge et al., 1997).The high level of C16:0 in palm oil is in part because of C16:0-ACP substrate specific activity of PATE enzyme (Othman et al., 2000). Our goal is to suppress the expression of PATE gene in mesocarp of E. oleifera (and E. guineensis Jacq. Tenera) to minimize the level of C16:0 for oil quality improvement. Palmitic acid content in palm oil can be minimized by post-transcriptional regulation of the PATE gene, which can be achieved by using different types of constructs, such as antisense, intron-spliced inverted repeats, and/or inverted-repeats of the PATE gene. To achieve our goal, we have successfully isolated PATE cDNA (GenBank Acc. No AF507115), and mesocarp tissue specific gene promoter (Shah and Cha, 2000). For post-transcriptional silencing of PATE we have transformed oil palm E. oleifera IZEs with an antisense PATE construct driven by OPMSP. In this paper, the successful integration of antisense PATE driven
168
by OPMSP, and construction of novel transformation vectors for effective post-transcriptional silencing of PATE gene is reported. 3. Materials and Methods 3.1 Plant Materials Twelve-week-old (WAP) IZEs were used as target tissue for the bombardment of plasmid constructs. The IZEs were isolated aseptically from 12-week-old (WAP) fresh fruits collected from field grown E. oleifera plant (Palm No 16). Fruits were generously supplied by the Plant Breeding Department, United Plantation Berhad, Perak, Malaysia. 3.2 Plasmid vectors and bacterial strains Bacterium, E. coli strain DH5-α was used to harbor the binary plasmid, pPSP’AP-VF6, which was constructed using binary cloning vector pCAMBIA 1301. The HYG gene driven by the CaMV 35S promoter and terminator sequence was used as the selection marker gene (Figure 1). The antisense PATE gene (619 bp long from 5’ region of the gene; GenBank Accession No AF507115) was driven by the mesocarp specific sesquiterpene synthase gene promoter, which we reported previously (Shah and Cha; 2000). Bacteria were cultivated overnight at 37 °C in LB medium supplemented SacII
XhoI 3’ 35S
XhoI HYG
r
EcoRI CaMV 35S
BglII OPMSP
BstEII aPATE
SphI
3’nos
RB
LB
Figure 1. A linear map of the T-DNA of pPSP’AP-VF6. LB Left T-DNA border, 3’ 35S CaMV 35S terminator, HYG r Hygromycin resistant gene. CaMV 35S cauliflower mosaic virus 35S promoter, OPMSP oil palm mesocarp specific promoter, aPATE antisense palmitoyl- acyl carrier protein (ACP) thioesterase, 3’ nos NOS terminator, RB right T-DNA border.
with 50 µg/ml kanamycin then collected by centrifugation at 3000 RPM for 5 min and plasmid, pPSP’AP-VF6 was extracted using Plasmid DNA extraction kit (Promega).
3.3 Determination of hygromycin concentration in selection medium To determine the optimum selection concentration of HYG, we carried out a preliminary experiment. The controls IZEs were cultured on HYG free medium and on the same medium containing HYG at different concentrations (2-16 mg/l). After 42 days of culture, IZEs on medium containing 12 –16 mg/l HYG were turned completely brown, but the few were surviving on the medium containing 8-10 mg/l HYG. Therefore, a selection process of IZEs on MS medium containing 8 mg/l HYG was used in the subsequent transformation experiments. 3.4 Transformation, selection and regeneration The IZEs of E. oleifera, which were isolated aseptically from fresh fruits (of palm No 16) were arranged at the center in petry plates containing semi solid modified MS medium (Fig. 2A, B, and C). Biolistic-mediated transformation of IZEs with binary plasmid DNA, pPSP’AP-VF6 was completed by using the combination of all optimized physical parameters reported by Parveez et al (1997) with minor modifications. After 7 days of bombardment IZEs were shifted onto selection medium in two groups. One group of IZEs was on MS medium, supplemented with 8 mg l-1 HYG, while another group was on same medium (MS) but was supplemented with 12 mg l-1 HYG. More than 65 % of the IZEs became dark brown in color, and died by the end of six weeks incubation under 14 hour photoperiod (photon flux 150 µmol/m2 /s1 at 28 °C). Hygromycin resistant IZEs were transferred onto MS basal semi solid medium (without HYG), which was not supplemented with plant growth regulators (Murashige and Skoog 1962). The germinating IZEs were allowed to generate shoots and roots in the absence of plant growth regulators. 3.5 DNA extraction and molecular analysis Genomic DNA was extracted from leaves of individual plantlets resistant to HYG, and non-transformed control plantlets (regenerated from IZEs) using the SDS method described by Sambrook et al (1989). PCR amplification was carried out using the following conditions: 40 cycles of 1 min at 94 °C, 1 min at 63 °C, 1 min and 30 sec at 72 °C. The pair of primer used for amplification of the transformed expression cassette was 5’.. TACAGGAA TTC CCA ACA TGT CCA GAG GC..3’ and 5’..CTC GGT AAC CATC TTT GGT CTT TCA TTC CC..3’. For the southern hybridization, DNA samples were digested with EcoRI, separated on an 1 % agarose gel and transferred onto Hybond-N+ blotting membrane under alkaline conditions. The PATE gene probe DNA was prepared by using 629 bp long fragments of PATE (GenBank Accession No AF507115) by labeling it with [32 P]. After 18 hours hybridization, autoradiography was completed as described by Sambrook et al., (1989). 4. Results and discussion The IZEs were bombarded with gold particles coated with plasmid, pPSP’AP-VF6. Two controls were used, one where IZEs were not bombarded at all, and second where IZEs were bombarded with only gold particles
169
(without plasmid DNA coating) (Fig. 2D, and E). After six weeks of selective cultivation, HYG resistant IZEs were looking green, however non-resistant IZEs became brown in color and died (Fig. 2F). The HYG resistant IZEs were recovered and a total of 56 plantlets were regenerated from the selected IZEs (Fig. 2 G). Genomic DNA was extracted from each of the regenerated HYG resistant plantlets. PCR amplification revealed that 5 out of 56 regenerated plantlets had the expected 1942 bp band, as did the plasmid control, while no such band was present in the non-transformed control plantlets. PCR analysis was repeated for the 5 PCR +ve plantlets. The positive results suggested that antisense PATE gene was present along with its OPMSP in these plantlets. The negative results in the remaining 51 plantlets could be due to temporal expression of the HYG gene, which could have helped IZEs to survive on the selection medium, or because the PATE gene was not stably integrated into the plant genome and subsequently lost during the process of growth and development. The [32 P]-labeled probe was used to hybridize with digested DNA of five PCR-positive plantlets and nontransformed control plantlet DNA. On autoradiogram, different banding patterns were observed for the transgenic plantlets, while
A
Figure 2. (A) Fresh fruits of E. oleifera, fruits of uniform size were used to isolate immature zygotic embryos (IZEs). (B) Horizontally cut fruit. (C) Immature zygotic embryos of the E. oleifera , arranged for bombardment of the construct pPSP’AP VF6. (D) Non-transformed and non selected IZEs. (E) IZEs that were bombarded with gold particles without construct (on nonselection medium). (F) Selection of the IZEs transformed with plasmid construct, pPSP’AP -VF6 was accomplished using hygromycin. (G) Regenerated plantlets of E. oleifera.
B
C
F
D
G
single band with smearing was observed in the non-transformed control plantlet/s (Shah et al., another paper in this 16th Plant Lipid Symposium; 2004). These results confirm the integration of the PATE gene in the genome of these transgenic plantlets. Based on the banding patterns, it appears that 1-3 copy inserts were present of the antisense PATE gene. These results are in accord with particle bombardment-mediated transformation reported in other plants. Compared with our previous investigations in E. guineensis Jacq. Tenera where embryogenic and non-embryogenic calli were used for the bombardment, use of plant growth regulators (and prolonged in vitro time) lead to the induction of phenotypic abnormalities in more than 30% plantlets regenerated (Tan et al., 2004). Therefore, in our experiment we purposely avoided the use of PGRs and did not observed visible phenotypic abnormalities in the plantlets regenerated directly from IZEs, and this is a positive feature of transformation using IZEs. Our goal is to suppress the expression of PATE gene, in mesocarp A.
OPMSP / 35S
B.
Intron
aPATE
OPMSP / 35S
sPATE
Intron
OPMSP / 35S
sPATE
aPATE
OCS aPATE
OCS
C. OCS
Figure 3. Diagrammatic representation of the 6 different expression cassettes (3 driven by OPMSP and another 3 driven by CaMV 35S promoter). (A) Expression cassette with antisense PATE. (B) Expression cassette with intron-spliced inverted repeat of PATE. (C) Expression cassette with inverted repeat of PATE. 35S, cauliflower mosaic virus 35S promoter (CaMV 35S); OPMSP, oil palm mesocarp specific promoter; a/sPATE, antisense/sense PATE; OCS, Octopine synthase terminator
of E. oleifera to minimize the level of C16:0 . Therefore, efforts are underway to transform oil palm E. oleifera (along with E. guineensis Jacq. Tenera) with novel construct designs such as, intron-spliced inverted repeats, and inverted repeats of PATE gene (Fig. 3). PATE is driven by OPMSP in one set of experiment, and constitutive,
CaMV 35S promoter in another set of experiment. The major hurdle is of slow growth of the plantlets and long generation time of oil palm. Therefore, evaluation of the fatty acid profile in the fruit mesocarp will require a longer time. Acknowledgements The authors are grateful to the Ministry of Science and Technology of Malaysian Government for funding (Grant No IRPA: 09-02-02-0161), to the United Plantation Berhad, Perak, Malaysia for supplying fresh fruit bunches of Elaeis oleifera for this study, and to Mr. Raai for his help in photography.
5. References 1. 2.
Ohlrogge john B., and Jaworki Jan G. (1997) Regulation of fatty acid synthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 109-136. Othman A., C. Lazarus, T. Fraser and K. Stobart. (2000) Cloning of a palmitoyl- acyl carrier protein thioesterase from oil palm. Biochemical Society Transactions 28:619-622.
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3.
Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. 4. Parveez GKA., Chowdhury MKU, Saleh Norihan M. (1997) Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device. Industral Crops and products, 6: 41-50. 5. Rajanaidu N., B.S. Jalani and A. Kushairi. (1997) Genetic improvement of oil palm, in: M. S. Kang (Ed.), Crop improvement for the 21st century, Published by Research signpost, India, pp 127-137. 6. Sambrook J., E.F. Fritsch, T Maniatis. (1989) Molecular Cloning: A Laboratory Manual, 2nd, Cold Spring Harbor press, Cold Spring Harbor, NY. 7. Shah F.H., and Cha T.S. (2000) A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase., Plant Science, 154:153-160. 8. Tan C.L, Bhore S.J, and Shah F.H. (2004) Quantitative and qualitative assessment of phenotypic variation in oil palm (Elaeis guineensis Jacq. Tenera) plantlets regenerated from transformed and non-transformed calli. Indian J. Plant Physiology. In press. 9. Thelen Jay J. and John B. Ohlrogge. (2002) Metabolic Engineering of Fatty Acid Biosynthesis in Plants. Metabolic Engineering, 4: 12-21. 10. Van de Loo, F. J., Fox, B. G., and Somerville, C. (1993) Unusual fatty acids. In “Plant Lipids” (T. Moore, Ed.), pp. 91-126, CRC, Boca Raton, FL.
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SEED-SPECIFIC HETEROLOGOUS EXPRESSION OF A NASTURTIUM FAE GENE IN ARABIDOPSIS RESULTS IN AN EIGHT-FOLD INCREASE IN ERUCIC ACID CONTENT ELZBIETA MIETKIEWSKA 1,3 , E. MICHAEL GIBLIN1 , SONG WANG1 , DENNIS L. BARTON2 , JOAN DIRPAUL1 , VESNA KATAVIC2 , AND DAVID C. TAYLOR1 1
National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 2 CanAmera Foods, P.O. Box 479, 125 Willow Court, Osler, SK, S0K 3A0 3 Plant Breeding and Acclimatization Institute, Mlochow Research Center, Poland
1.
Introduction
A major goal of our research is to obtain, by genetic manipulation, Brassica napus L. cultivars with higher proportions (above 66 mol%) of erucic acid (22:1) in their seed oil than in present Canadian HEA cultivars. We have selected Tropaeloum majus, garden nasturtium, as a source of new strategic genes based on the fact that this plant is capable of producing significant amounts of erucic acid (70-75% of total fatty acid) and accumu lates trierucin as the predominant TAG in its oil (Taylor et al. 1992). Very long-chain fatty acids (VLCFAs) are synthesized by a membrane-bound fatty acid elongation complex (elongase, FAE) using acyl-CoA substrates. The first reaction of elongation involves condensation of malonyl-CoA with a long chain substrate producing a β-ketoacyl-CoA. Subsequent reactions are reduction of βhydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a second reduction to form the elongated acylCoA. The β-ketoacyl-CoA synthase (KCS) catalyzing the condensation reaction plays a key role in determining the chain length of fatty acid products found in seed oils and is the rate-limiting enzyme for seed VLCFA production (Lassner et al., 1996). Here we report isolation of a nasturtium FAE gene and demonstrate the involvement of its encoded protein in the elongation of monounsaturated and saturated fatty acids. 2.
Results and Discussion
2.1. Isolation of T. majus FAE homolog Based on sequence homology among plant fatty acid elongase genes, a full-length cDNA clone was amplified by PCR using a degenerate primers approach and the sequence submitted to GenBank (Accession number AY082610). The T. majus FAE cDNA encodes a polypeptide of 504 amino acids that is most closely related to an FAE2 from roots of Zea mays (69 % amino acid identity; Schreiber et al., 2000), (Figure 1). The T. majus FAE polypeptide also shared strong identity with FAEs from Limnanthes douglasii (67%; Cahoon et al., 2000) and from seeds of jojoba (Simondsia chinensis) (63%; Lassner et al., 1996). Homology of the nasturtium FAE to two Arabidopsis β-ketoacyl-CoA synthases AraKCS (Todd et al., 1999) and AraCUT1 (Millar et al., 1999) involved in cuticular wax synthesis was on the level of 57% and 53%, respectively. These homologs all exhibit the capability to elongate saturated fatty acids to produce saturated VLCFAs. The FAE1 polypeptides involved in the synthesis of VLCFAs in Arabidopsis (James et al., 1995) and Brassica seeds (Clemens and Kunst, 1997) showed approximately 52-54% identity with the T. majus FAE. LimFAE SimFAE NasFAE ZeaFAE AraKCS AraFAE BraFAE AraCUT
54.8 50
40 30 Nucleotide Substitutions (x100)
172
20
10
0
Figure 1. Dendrogram of the β-ketoacyl-CoA synthase gene family based on the amino acid sequences. The alignment was carried out by the Clustal W method using Lasergene analysis software (DNAStar, Madison, WI). The dendogram contains the sequences of the Limnanthes (LimFAE, GenBank Acc# AF247134), jojoba (SimFAE, GenBank Acc# U37088), nasturtium (NasFAE, GenBank Acc# AY0826190), corn (ZeaFAE, GenBank Acc# AJ292770), Arabidopsis (AraFAE, GenBank Acc# U29142; AraKCS, GenBank Acc# AF053345: and AraCUT, GenBank Acc# AF129511) and Brassica (BraFAE, GenBank Acc# AF009563).
1.2. Tissue specific expression and copy number estimate of T. majus FAE Northern blot analyses were performed to investigate the expression profile of the FAE gene. Total RNA was isolated from different nasturtium tissues including roots, leaves, floral petals and mid-developing embryos. A strong hybridization signal with FAE-specific probe was observed only with RNA isolated from developing embryos (Figure 2A).
A
B R
L
P
E
1
2
3
4
Figure 2. Northern and Southern analyses of T. majus FAE. A. Northern analysis of FAE gene expression in T.majus. Total RNA was isolated from roots (R), leaves (L), petals (P) and embryos (E). B. Southern blot analysis of the FAE gene in T.majus. Genomic DNA was digested with restriction enzymes: EcoRI (lane 1), AccI (lane 2), NcoI (lane 3) and HindIII (lane 4).
A Southern blot hybridization was performed with nasturtium genomic DNA digested with several restriction enzymes including EcoRI, AccI, NcoI and HindIII. The FAE gene has no internal EcoRI, AccI or NcoI sites, while one internal HindIII site exists. Autoradiography revealed the presence of one stronglyhybridizing fragment in all cases except with HindIII for which two strongly hybridizing fragments were evident (Figure 2B). In addition a minimum of 4 weakly hybridizing fragments were detected. Thus, we have concluded that T. majus FAE belongs to a multigenic family consisting of 4 to 6 members. A similar multigenic family has been found for a rapeseed FAE1 gene member (Barret et al., 1998). 1.3. Seed specific expression of T. majus FAE in Arabidopsis plants To establish the function of elongase homolog, the cDNA was introduced into Arabidopsis background (ecotype Wasilewskija) under the control of the napin promoter. From vacuum-infiltration experiments, 25 kanamycinresistant T1 plants were selected. The T2 progeny were collected individually from each plant and the fatty acid composition determined. Significant changes in fatty acid composition in comparison to the wild type (empty vector) were found. On average, the proportion of erucic acid (22:1 ∆13) increased from 2.1% in wild type to 9.6% in T2 transgenic seeds at the expense of 20:1 ∆11 (data not shown). Homozygous T3 lines were analyzed to examine the range of VLCFA proportional re-distribution induced by expression of the nasturtium FAE gene. The 12 best T3 lines are shown in Figure 3.
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20:1c11
22:1c13
18.0 16.0 14.0 % wt/wt
12.0 10.0 8.0 6.0 4.0 2.0
23 -3 23 -8
20 -1 20 -4
16 -1 16 -5
15 -2 1510
8-7
8-2
26
24
RD 10 -6 RD 15 -8 ntW S
0.0
[Transgenic] Line
Figure 3. Fatty acid composition of transgenic Arabidopsis seeds. Proportions of 20:1 c 11 and 22:1 c13 in seed oils from non-transformed A. thaliana ecotype Wassilewskija (ntWS), 2 plasmid-only transgenic control lines (RD10-6 and RD15-8), and the twelve best A. thaliana T3 homozygous transgenic lines expressing the T. majus FAE gene under control of the napin promoter.
The erucic acid content was increased by up to 7 to 8-fold in lines 15-2, 15-10, 16-1, 20-1 and 23-8. Small increases in the proportions of 24:1 ∆15 were also observed. There was also a relatively significant increase in the proportion of saturated VLCFAs. The content of 22:0 and 24:0 in the best T3 transgenic lines increased up to 2.2 and 0.7% in comparison to 0.2 and 0.08% respectively, in the wild type background, (data not shown). Compared to other FAE genes heterologously-expressed in Arabidopsis the current expression of the nasturtium FAE gene has resulted in the highest increase in erucic acid proportions observed thus far in A. thaliana. For instance, introducing the jojoba FAE into Arabidopsis resulted in an increase in 22:1 proportions from about 2% in the control up to 4% in the transgenic seeds (Lassner et al., 1996). The nasturtium FAE homolog described herein, may have a larger engineering impact when strongly expressed in a seed-specific manner in H.E.A Brassicaceae. 3. References Barret, P., Delourne, R., Renard, M., Domergue, F., Lessire, R., Delseny, M. and Roscoe, T.J. (1998) A rapeseed FAE1 gene is linked to the E1 locus associated with variatio n in the content of erucic acid. Theor. Appl. Genet. 96, 177-186. Cahoon, E.B., Marillia, E-F., Stecca, K.L., Hall, S.E., Taylor, D.C. and Kinney A.J. (2000) Production of fatty acid components of meadowfoam oil in somatic soybean embryos. Plant Physiol. 124, 243-251. Clemens, S. and Kunst, L. (1997) Isolation of a Brassica napus cDNA (accession no. AF009563) encoding a 3-ketoacyl-CoA synthase, a condensing enzyme involved in the biosynthesis of very long chain fatty acids in seeds (PGR 97-125). Plant Physiol. 115, 313-314. James, D.W., Lim, E., Keller, J., Plooy, I. and Dooner, H.K. (1995) Directed tagging of the Arabidopsis fatty acid elongation 1 (FAE1) gene with the maize transposon activator. Plant Cell, 7, 309-319. Lassner, M.W., Lardizabal, K. and Metz, J.G. (1996) A jojoba ß-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants. Plant Cell, 8, 281-292. Millar, A.A., Clemens, S., Zachgo, S., Giblin, E.M., Taylor, D.C. and Kunst, L. (1999) CUT1 , an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell, 11, 825-838. Schreiber, L., Skrabs, M., Hartmann, K., Becker, D., Cassagne, C. and Lessire, R. (2000) Biochemical and molecular characterization of corn (Zea mays L.) root elongaeses. Biochem. Soc. Trans. 28, 647-649. Taylor, D.C., Magus, J.R., Bhella, J., Zou, J-T., Mackenzie, S.L., Giblin, E.M., Pass, E.W. and Crosby, W.L. (1992a) Biosynthesis of triacylglycerols in Brassica napus L. cv. Reston; target: trierucin. In Seed Oils for the Future (MacKenzie, S.L. and Taylor, D.C., eds), American Oil Chemists' Society, Champaign, IL, pp. 77-102. Todd, J., Post-Beittenmiller, D. and Jaworski, J.G. (1999) KCS1 encodes a fatty acid 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J. 17, 119-130.
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CHARACTERIZATION OF A MUTANT IN ARABIDOPSIS ACBP2 G. MISHRA 1 , S. RAMALINGAM1 , M.S.F. LIE KEN JIE2 AND M -L. CHYE1 1 Department of Botany, 2 Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
1. Abstract Cytosolic acyl-CoA binding proteins (ACBPs) are 10-kDa proteins that bind long-chain acyl-CoAs and are involved in their storage and intracellular transport. We have characterized Arabidopsis cDNAs and their genes encoding larger membrane-associated ACBPs, ACBP1 and ACBP2 (Chye, 1998; Chye et al. 1999, 2000; Li and Chye, 2003). His -tagged ACBP1 binds oleoyl-CoA while recombinant ACBP2 binds palmitoyl-CoA (Chye et al., 2000). ACBP2 has three distinct domains: an N-terminal membrane domain, an acyl-CoA binding domain and a C-terminal domain of ankyrin repeats. We have previously shown that the membrane domain targets ACBP2:GFP to the endoplasmic reticulum and predominantly to the plasma membrane (Li and Chye, 2003) while the ankyrin repeats are involved in protein-protein interactions (Li and Chye, 2004). To further elucidate the role of ACBP2, we have isolated a knock-out mutant by a reverse genetics approach. Using gas chromatography in fatty acid analysis, we detected greater accumulation of 16:0 in mutant than wild-type which may be related to loss of ACBP2 function in binding palmitoyl-CoA. The ACBP2 mutant, phenotypically similar to the wild -type under normal growth conditions, exhibited distinct differences in plant growth and bolting under cold-stress. Unlike wild-type, it showed an absence of lateral roots when grown on ½ Murashige and Skoog medium. Root growth of the ACBP2 mutant reverted to normal when the medium was supplemented with auxin, suggesting that the mutant could possibly be deficient in auxin signaling or polar transport. 2. Experimental procedures Plants were grown in soil at 23 o C under 16 h light/8 h dark cycles. For cold-stress treatment, 10-day old seedlings grown at 23 o C were transferred to 10 o C for 35 days. DNA pools and seed stocks of independent T-DNA-transformed A. thaliana of ecotypes Columbia and Wassilewskija were obtained from the Arabidopsis Biological Resource Center, Ohio State University. FAMEs were prepared from wild-type or the ACBP2 mutant according to Browse et al. (1986a). Lipid extraction was carried on leaves (Browse et al., 1986b) for thin layer chromatography (Lepage, 1967). The different classes of lipids analyzed were phosphoinositol, phosphatidylcholine, sulpholipids, digalactosyldiacylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid (PA), monogalactosyldiacylglycerol and neutral lipids. 3. Results and discussion 3.1. Isolation of an Arabidopsis ACBP2 knock -out mutant We identified an ACBP2 knock-out mutant line from CS19943, a Thomas Jack pool consisting of ten TDNA insertion lines. The location of the T-DNA insertion in ACBP2 was confirmed by PCR using an ACBP2 gene-specific primer ML205, located 1520 bp upstream from the insertion site, and reverse primer Oligo 113 on the T-DNA left border. These primers gave a 1.8-kb PCR band that hybridized to the 32 Plabeled ACBP2 full-length cDNA probe on Southern blot analysis. Further PCR using ACBP2 gene-specific forward primer ML251 and reverse primer ML252, at the 5’- and 3’-untranslated regions of ACBP2, respectively, gave an expected 2.2-kb band from wild-type A. thaliana Columbia that was absent in the mutant, indicative that the plant is homozygous for the mutation. The site of insertion was mapped to intron 5 of ACBP2. Southern blot analysis of DNA from wild-type and mutant using a 32 P-labeled T-DNA left border probe, showed a 2.2-kb AccI hybridizing band only in mutant DNA, confirming the presence of a single T-DNA insert in ACBP2. RT-PCR and western blot analyses indicated the absence of ACBP2 mRNA and protein in the mutant.
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3.2. The ACBP2 mutant has an altered fatty acid composition As ACBP2 binds palmitoyl-CoA (Chye et al., 2000), a study on the fatty acid composition of the ACBP2 knock-out mutant was carried out (Table 1). Gas chromatography analysis of fatty acid methyl esters derived from mature green leaves showed an accumulation of palmitic acid and a reduction of linolenic acid in the ACBP2 mutant when compared to wild-type (WT). TABLE 1. Total fatty acid composition of leaf samples from wild-type and the ACBP2 mutant grown at 23 ºC. All values in mol % are mean of fifteen independent samples. Values in brackets show standard error. WT ACBP2 mutant
16:0 14.8 (0.2) 16.3 (0.4)
16:1 traces
16:2 3.9
16:3 12.5
18:0 0.5
18:1 3.5
18:2 16.7
traces
4.3
11.9
1.3
4.2
16.5
18:3 47.6 (0.5) 45.0 (0.4)
To further investigate the lipid classes that exhibit altered fatty acid composition in leaf samples, gas chromatography analysis was carried out on fatty acid methyl esters derived from different lipids separated through thin layer chromatography. We observed an accumulation of palmitic acid and a reduction of linolenic acid in the PA subclass (Table 2) whereas no other significant changes in other subclasses were observed. TABLE 2. Fatty acid composition of PA subclass from wild-type and the ACBP2 mutant grown at 23 ºC. All values in mol % are mean of fifteen independent samples. Values in brackets show standard error. WT ACBP2 mutant
16:0 43.9 (2.8) 51.7 (1.9)
16:1 traces
16:2 4.5
16:3 1.3
18:0 3.8
18:1 3.4
18:2 16.4
traces
8.9
0.4
1.9
2.2
14.2
18:3 26.7 (1.5) 22.6 (0.3)
3.3. Phenotypic analysis of the ACBP2 mutant The ACBP2 knock-out mutant was examined for phenotypic changes since we detected a change in fatty acid content, suggesting that ACBP2 is not a redundant gene. No observable differences were observed between mutant and wild-type germinated and grown at 23 o C. When mutant and wild -type were coldstressed, a reduction of 35% in leaf size was observed at 10 o C in the mutant (Figure 1). Also, the ACBP2 mutant bolted at 23 days compared to 15 days for wild-type. The mean bolt length was 15.6 cm for wildtype and 3.2 cm for the ACBP2 mutant (Figure 2). In contrast, no difference in bolt lengths was observed at 23 o C. The bolt length in wild-type grown at 23 o C was 30 cm compared to 15.6 cm for those grown at 10 o C. Application of gibberellic acid (GA) on the cold-stressed wild-type Arabidopsis and the ACBP2 mutant revealed that GA had no effect on the mutant whereas it had a significant effect in increasing the bolt length of wild-type (Table 3). Wild-type Arabidopsis flowered at 20 days of cold treatment at 10 o C, much earlier than the mutant, which flowered at 28 days of cold treatment and flowers were fewer than on wild-type. When the plants were relieved of this cold-stress and were grown for a week at 23 o C, a reversal in bolting and flowering occurred in the mutant. Figure 1. A 35% reduction in leaf size was observed for the cold-stressed mutant (right) but not wild-type (left).
TABLE 3. Bolt length measurements (cm) of cold-stressed wild-type and mutants on day 2, 4 and 6 after spray with 100 ppm GA. Plants were sprayed following 25 days of cold treatment. The mean of seven plants in each category was used in tabulation.
Plant WT s unsprayed Days
Figure 2. Phenotype under cold stress. Plants germinated and grown for 7 days (23 o C) were transferred to 10o C for 35 days.
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WT sprayed with GA
Mutant unsprayed
Mutant sprayed with GA
2
6.3
11.0
3.3
4.0
4
7.5
13.2
4.4
5.0
6
9.3
15.4
5.7
6.6
Absence of lateral roots was observed in the ACBP2 mutant when grown on ½ MS medium for 14 days (Figure 3). Since absence of lateral roots could be due to the lack of auxin, the effect of indoleacetic acid (IAA) supplementation to the growth medium was investigated. We observed that supplementation of 10 ppm IAA to the ACBP2 mutant reverted it to wild-type phenotype (Figure 4).
Figure 3. Seeds were surface-sterilized and germinated on ½ MS medium at 23 o C (16h light/ 8h dark cycles) for 14 days. Photograph taken 12 days after germination shows an absence of lateral roots in the ACBP2 mutant.
Figure 4. Root growth of mutant on IAA supplemented medium. ACBP2 mutant seeds were germinated on ½ MS medium in the absence or presence of IAA (10 mg/l for 14 days (16h light/ 8h dark cycles).
Our results suggest that the ACBP2 mutant accumulates 16:0 in the PA subclass, displays lateral root inhibition, and shows a short bolt phenotype and GA -insensitivity at cold temperature. When the mutant was complemented (Clough and Bent, 1998) with the full-length ACBP2 cDNA expressed from the CaMV 35S promoter using a derivative from pSOV (Mylne and Botella, 1998), ACBP2 activity was restored in the complemented plant. We conclude the ACBP2 mutant is likely involved in auxin signaling and in GA signaling under cold stress. 4. Acknowledgments We thank J. R. Botella for plasmid pSOV, W. K. Yip for discussions on GA treatment and J. Browse and M. Miquel for suggestions on lipid analysis. We acknowledge H. K. Wing in GC and the ABRC, Ohio State University, for provision of T-DNA tagged lines. This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (Project HKU7232/00M) and HKU Faculty of Science Seed Fund (2002). GM was supported by a postgraduate studentship from the University of Hong Kong. 5. References Browse, J., McCourt, P.J. and Somerville, C.R. (1986a) Fatty acid composition of leaf lip ids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal. Biochem. 152, 141-145. Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. (1986b) Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the ‘16:3’ plant Arabidopsis thaliana. Biochem. J. 235, 25-31. Chye, M.-L. (1998) Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain. Plant Mol. Biol. 38, 827-838. Chye, M.-L., Huang, B.-Q. and Zee, S.-Y. (1999) Isolation of a gene encoding Arabidopsis membrane-associated acyl- CoA binding protein and immunolocalization of its gene product. Plant J. 18, 205-214. Chye, M.-L., Li, H.-Y. and Yung, M.-H. (2000) Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14 [C]palmitoyl- CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats. Plant Mol. Biol. 44, 711-721. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. Lepage, H. (1967) Identification and composition of turnip root lipids. Lipids 2, 244-250. Li, H.-Y. and Chye, M.-L. ( 2003) Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2. Plant Mol. Biol.51, 483492. Li, H.-Y. and Chye, M.-L. (2004) Arabidopsis acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive elementbinding protein, AtEBP, via its ankyrin repeats. Plant Mol. Biol.51, 483-492. Mylne, J. and Botella, J.R. (1998) Binary vectors for sense and antisense expression of Arabidopsis ESTs. Plant Mol. Biol. Rep. 16, 257-262.
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IS SER 282 CRUCIAL FOR THE FUNCTION OF BRASSICA NAPUS FAE1 CONDENSING ENZYME? V. KATAVIC1* , D.L. BARTON1 , E.M.GIBLIN2 , W.D.REED 2 , A. KUMAR1 1 , D.C. TAYLOR2 1 CanAmera Foods, P.O.Box 479, 125 Willow Court, Osler, Saskatchewan, Canada, S0K 3A0; 2 National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
1. Abstract Is there an absolute requirement for the serine 282 to yield a functional fatty acid elongase 1 condensing enzyme? To gain some insight we generated several mutated FAE1 polypeptides: using a site directed mutagenesis approach we have introduced point mutations in the FAE1 coding sequence which led to the substitution of serine 282 with aliphatic (threonine; asparagine; glutamine; cysteine; glycine; alanine; valine or isoleucine) or aromatic (tyrosine or tryptophan) amino acids. The mutated FAE1 polypeptides were expressed in yeast and the fatty acid methyl esters from yeast lysates were analyzed by gas chromatography. The GC analyses revealed that amino acid substitutions with asparagine, glutamine, valine, isoleucine, tyrosine or tryptophan resulted in complete loss of enzyme activity, while the fatty acid elongase activity assays showed that mutated FAE1 peptides with threonine, alanine, glycine or cysteine had either reduced, similar or higher enzyme activity than in the FAE1 condensing enzyme with serine 282. Overall, our data demonstrate that there is not an absolute requirement for serine at position 282 to yield a functional FAE1 condensing enzyme; substitutions with small aliphatic amino acids, either polar or non-polar, resulted in functional FAE1 polypeptides. 2. Introduction Fatty acid elongase 1 (FAE1) is a seed-specific condensing enzyme, 3-ketoacyl-CoA-synthase, which catalyses the first enzymatic reaction in very long chain monounsaturated fatty acid (VLCMFA) biosynthesis in high erucic acid (HEA) Brassicaceae. Genes and coding sequences for FAE1s from different Brassicaceae were isolated and manipulated extensively by expression in homologous species as well as in heterologous host systems (Han et al., 2001; Katavic et al., 2001). However, our knowledge about the mechanisms and kinetic properties of FAE1 condensing enzymes is limited mainly due to limitations resulting from their membrane-bound nature. We have shown that seed-specific expression of Arabidopsis thaliana FAE1 in high erucic acid B. napus cv. Hero has led to increased proportions of erucic acid (up to 11%) and total VLCFAs (up to 10%) in the oil, while the seed- specific expression of A. thaliana FAE1 in low erucic acid (LEA) canola cv. Westar complemented the mutation and partially restored the capacity to biosynthesize VLCMFAs in the developing seed (Katavic et al., 2001). Low erucic acid rapeseed and canola (low erucic acid and low glucosinolates rapeseed) cultivars were developed through traditional breeding and selection to meet the requirement for a high quality edible vegetable oil (Downey, 1964). Several groups have focused their research on elucidating the mutation(s) involved in the loss of FAE1 condensing enzyme activity in LEA B. napus cultivars (Han et al., 2001; Roscoe et al., 2001). Recently, we have provided the experimental evidence that the low erucic acid trait in LEA and canola B. napus cultivars can be attributed to a single amino acid substitution in the FAE1 condensing enzyme which prevents the biosynthesis of eicosenoic and erucic acids. We have shown that the activity of a non-functional FAE1 of B. napus could be restored by substituting Phe282 by serine which is found in equivalent positions of all known FAE1 condensing enzymes (Katavic et al, 2002). As a continuation of our work we have focused on determining whether serine 282 in functional FAE1 condensing enzyme is a catalytic residue. To test our hypothesis which centers on hydropathy of the segment containing the adjacent Asn283 and possible significance of maintaining a theoretical hydrophilic transition point in the sequence at Ser282 we used a site directed mutagenesis approach to generate several mutated
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polypeptides, substituting serine 282 with either aliphatic or aromatic amino acids. Here we report and discuss the results of analyses of mutated FAE1 polypeptides by heterologous expression in yeast. 3. Results 3.1. Hydropathy analysis of functional FAE1 condensing enzyme Previously, we have shown that the presence of hydrophobic phenylalanine 282 in the non-functional FAE1 condensing enzyme instead of the hydrophilic serine 282 in the functional FAE1 induces dramatic changes in the hydrophilicity values for amino acids in the transition region from a highly hydrophobic domain around res idue 278 to a highly hydrophilic domain around residue 286, shifting the asparagines 283 from the hydrophilic loop in the functional condensing enzyme to a hydrophobic domain in the non-functional FAE1 enzyme (Katavic et al., 2003). Based on these findings we hypothesized that substitution of serine 282 with any amino acid that induces the shift of asparagines 283 from the hydrophilic loop to the hydrophobic domain will abolish FAE1 enzyme activity, while the substitution with any amino acid that does not induce this hydrophobic shift will result in an active FAE1 peptide. 3.2. Expression of mutated FAE1 peptides in yeast and GC analyses To test our hypothesis we introduced specific point mutations into the FAE1 nucleotide sequence resulting in the substitution of serine 282 with either the threonine, aparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, tyrosine or tryptophan. We cloned the mutated FAE1 polypeptides into a yeast expression vector (pYES2.1/V5-His -TOPO) and expressed them in yeast cells. The results from yeast FAME analyses by GC revealed that the substitution of serine 282 with either aparagine, glutamine, valine, isoleucine, tyrosine or tryptophan resulted in complete loss of enzyme activity (Fig. 1).
26:0
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Figure 1. GC chromatographs showing fatty acid profiles of yeast cells transformed with chimeric FAE1 gene. FAME were prepared from yeast lysates expressing non-functional FAE1 from LEA B. napus cv. Westar (WSF), functional FAE1 with serine 282 (S282) and mutated
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FAE1 peptides S282Y, S282W, S282N, S282Q, S282V, S282I, S282T, S282C, S282G and S282A. As a negative control we used pYES2.1 /V5-His-TOPO plasmid (pYES2.1). * FAME profiles for S282Y, S282W, S282N, S282Q, S282V and S282I were all similar to WSF.
Elongation products (pmol/min/mg protein)
3.3. Elongase activity in yeast cells upon expression of mutated FAE1 clones To determine elongase activity in yeast cells expressing different mutated FAE1 clones we performed elongase activity assays using yeast homogenates prepared from induced yeast cells and radio-labeled 18:1-CoA and malonyl-CoA as substrates. Results are reported as the elongation products produced in reactions (pmol/min/mg protein) and are the mean ±SD of three determinations (Fig. 2). 20 : 1 ?11
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S282
S282T
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Figure . 2. Elongase activity assayed in lysates from yeast cells upon expression of FAE1 condensing enzyme. Lysates were prepared from yeast cells expressing non-functional FAE1 from LEA B. napus cv. Westar (WSF), functional FAE1 with serine 282 (S282) and mutated FAE1 peptides S282T, S282N, S282Q, S282C, S282G, S282A, S282V, S282I, S282Y and S282W. As a negative control we used pYES2.1/V5-His-TOPO plasmid (pYES2.1). Protein samples were incubated at 30ºC with shaking at 100 r.p.m. for 60 min with 18 µM [114 C] oleoyl-CoA (0.37 GBq.mol-1 ) and 1 mM malonyl Co-A in the presence of 1 mM ATP, 1 mM CoA-SH, 0.5 mM NADH, 0.5 mM NADPH and 2 mM MgCl2 in a final volume of 500 µL. After incubation, reaction mixtures were saponified, transmethylated and analyzed by HPLC equipped with a flow-through scintillation counter. Results are reported as the elongation products produced in reaction (pmol/min/mg protein) and are the mean ±SD of three determinations.
4. Conclusions In our effort to elucidate the role of serine 282 in functional FAE1 condensing enzyme and determine whether it is crucial for enzyme activity we have produced several mutated FAE1 polypeptides by substituting the amino acid residue serine with neutral, polar or non-polar aliphatic and aromatic amino acids and analyzed their function upon expression in yeast cells. Our analyses have shown that substitution S282G resulted in an enzyme with slightly higher total 20:1 + 22:1 elongase activity, which could be due to the relatively small size of its side chain. It is possible that a very small amino acid allows for proper folding of the protein molecule. Substitutions with larger aromatic amino acids tyrosine and tryptophan or with aliphatic amino acids with longer (isoleucine) or branched side-chains (asparagine, glutamine, valine) resulted in a non-functional enzyme. Substitutions of S282T or S282A resulted in slightly or significantly lower enzyme activity, respectively. We expected the mutated FAE1 polypeptide with threonine 282 to show activity similar to serine 282 because both enzymes are present in nature (although the genomic DNA clone encoding for putative 3-ketoacyl condensing enzyme with threonine 282 was thus far isolated from only one plant species). However, our results indicate that the FAE1 with threonine 282 has lower enzyme activity than FAE1 enzyme with a serine amino acid residue at the same position. Surprisingly, the mutated FAE1 clone S282C showed consistently higher condensing enzyme activity compared to the naturally-occurring serine 282. Overall, our results demonstrate that serine 282 in the FAE1 is not crucial for enzyme function because substitution with several small neutral either polar or non-polar aliphatic amino acid residues resulted in active enzymes. 5. References [1] Han, J. Lühs, W., Sonntag, K., Zähringer, U., Borchardt, D.S., Wolter, F.P., Heinz, E. and Frentzen, M. (2001) Plant. Mol. Biol. 46, 229-239. [2] Katavic, V., Friesen, W., Barton, D.L., Gossen, K.K., Giblin, E.M., Luciw, T., An, J., Zou, J-T., MacKenzie, S.L., Keller, W.A., Males, D. and Taylor, D.C. (2001) Crop Science 41, 739-747. [3] Downey, R.K. (1964) J. Am. Oil. Chem. Soc. 41, 475-478. [4] Roscoe, T.J., Lessire, R., Puyaubert, J., Renard, M. and M. Delseny, M. (2001) FEBS Letters 492, 107-111. [5] Katavic, V., Mietkiewska, E., Barton, D.L., Giblin, M.E., Reed, D.W. and Taylor, D.C. (2002) Eur. J. Biochem. 269, 5625-5631. [6] Katavic, V., Mietkiewska, E., Barton, D.L., Giblin, M.E., Reed, D.W. and Taylor, D.C. (2003) Advanced Research on Plant Lipids, Kluwer Academic Publishers, Dordrecht.
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INITIAL STUDIES ON THE FATTY ACID DESATURASE GENES IN THE UNICELLULAR GREEN ALGAE PARIETOCHLORIS INCISA
I. KHALILOV, I. KHOZIN–GOLDBERG AND Z. COHEN A. Katz Department for Drylands Biotechnologies, J. Blaustein Institute for Desert Research, BenGurion University of the Negev, Sde Boker Campus, Israel
1. Introduction The microalga Parietochloris incisa (Trebouxiophyceae, Chlorophyta) is the richest plant source of the polyunsaturated arachidonic acid (AA). Under nitrogen starvation, AA constitutes almost 60% of total fatty acids, reaching a content of over 20% of dry weight. Sequence analysis of the chloroplast Small-subunit (SSU) 16S rRNA and the nuclear SSU 18S rRNA genes showed the close similarity of this alga to several Chlorella species. Based on the phylogenetic similarity, we attempted to identify and clone for the first time the fatty acid desaturase genes of P. incisa, utilizing the sequences of low-temperature inducible Chlorella vulgaris gene for ω3 desaturase (Suga et al., 2002). 2. Materials and methods Algal culture - P. incisa was grown on the BG11 medium under constant light and temperature. Cells were harvested at the logarithmic and stationary phase, after 3 and 14 days, respectively and used for DNA and RNA isolation. DNA, RNA isolation and manipulations - Total RNA was isolated from 300 mg wet weight of cells by using the RNeasyR Plant Kit (Qiagen, Md, USA). mRNA was purified from total RNA by using the GenEluteTM mRNA Miniprep Kit (Sigma, St. Louis, USA). RT -PCR reactions were carried out by AccessQuickTM RT-PCR System (Promega, Madison, USA) with purified mRNA as a template. Genomic DNA was obtained as described by Dawson et al. (1997). PCR reactions on genomic DNA were carried out with FailSafe TM PCR PreMix Selection Kit (Epicentre, Madison, USA). The resulting products were ligated into pGEM -T Easy vector (Promega, Madison, USA) and transformed into competent JM 109 E. coli cells. After purification of plasmids, nucleotide sequences were determined by the dideoxy chain termination method using Applied Biosystems PRISM 3100 Genetic analyzer. Primers: For 18S SSU rRNA sequence, amplification primers designed by Kosher and White (1992) and Handa et al. (2003) were utilized. The partial sequence of ω3 desaturase cDNA was amplified by RT-PCR using following pairs of primers: forward (DES1) 5’-ACTCACCACGCTAACCACGG - 3’ and reverse (DES1) 5’CGTAGTCACGGTCAATGGTG – 3’; forward (DES2) 5’- ACCATGTTCTGGGCCCTCTT - 3’ and reverse (DES2) 5’- TGCGTGCCAATGTCGTGGTG – 3’. Primers were designed according to histidine rich regions of ω3 desaturase protein of green algae Chlorella vulgaris (THHANHG and HHDIGTH).
3. Results 3.1 Phylogenetic analysis The partial 18S rRNA gene sequence from P. incisa was aligned with several green algae sequences registered in the NBCI database using the CLUSTALW program. The P. incisa gene has 94-98% identity with other species of Trebouxiophyceae (Fig. 1) and a very high affinity to Lobosphaera tirolensis and Myrmecia bisecta. Created phylogenetic tree supports the cyto-morphological reclassification of this species from Myrmecia incisa to the genus Parietochloris (Watanabe et al., 1996). On the basis of molecular diagnostics data P. incisa has high similarity to several species Chlorella species.
3.2 Cloning of the ω3 desaturase
Alignment analysis of the amino acid sequences of Chlorella vulgaris and higher plants ω3 desaturases revealed the presence of highly conserved histidine-box mo tives common to the fatty acid desaturases. These conserved His -box motifs, were utilized for design of primers. A 650 bp cDNA fragment putatively encoding amino acid
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Fig. 1. Phylogenetic position of green algae Parietochloris incisa created on basis partial gene 18S rRNA by using the CLUSTALW program (http://www.ebi.ac.uk/clustalw).
Sequence, characteristic of the ω3 fatty acid desaturase, was obtained using the RT-PCR approach (Fig. 2). Comparison of the deduced amino acid sequence of the fragment to ω3 desaturases from available algae and Arabidopsis thaliana showed 70% identity to Chlorella vulgaris, 66% identity to Dunaliella salina, and about 60% identity to the ω3 desaturases of Arabidopsis thaliana (FAD7 and FAD8)(Gibson et al., 1994). The identity is particularly high in the histidine rich regions, previously identified as essential for ω3 desaturases. The partial intron containing genomic DNA fragment was also obtained (data not shown). The predicted hydrophobicity plot for the deduced amino acid sequence also revealed a profile characteristic of a fatty acid desaturase, with the histidine-boxes located in hydrophilic areas and separated by hydrophobic regions (data not shown). References: Dawson, H.N, Burlingame, R., Cannons, A.C. (1997) Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Curr. Microbiol. 35, 356-362. Gibson, S., Arondel, V K., Iba, K. and Somerville, C. (1994) Cloning of a temperature-regulated gene encoding a chloroplast omega-3 desaturase from Arabidopsis thaliana. Plant Physiol. 106, 1615–1621. Handa, S., Nakahara, M., Tsubota, H., Deguchi, H. and Nakano, T. (2003) A new aerial alga, Stichococcus ampulliformis sp. nov. (Trebouxiophyceae, Chlorophyta) from Japan. Phycol. Research 51, 203–210. Kocher, T.D. and White, T.J. (1992) Evolutionary analysis via PCR. In Erlich H.A. (Ed.) PCR Technology: Principles and Applications for DNA Amplification. Oxford University Press, New York, pp. 137-47. Suga, K., Honjoh, K., Furuya, N., Shimizu, H., Nishi, K., Shinohara, F., Hirabaru, Y., Maruyama, I., Miyamoto, T., Hatano, S. and Iio, M. (2002) Two low-temperature-inducible Chlorella genes for ∆12 and ω-3 fatty acid desaturase (FAD): isolation of ∆12 and ω-3 fad cDNA clones, expression of ∆12 fad in Saccharomyces cerevisiae, and expression of ω-3 fad in Nicotiana tabacum. Biosci. Biotechnol. Biochem. 66, 1314–1327. Watanabe, S., Hirabayashi, S., Boussiba, S., Cohen, Z., Vonshak, A. and Richmond, A. (1996) Parietochloris incisa comb. Nov. (Trebouxiophyceae, Chlorophyta). Phycol Research 44, 107–108.
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P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
---------------------------------------------------------------------------MVAALTTQLVCSAAIVRSARPSGLSRALPLRRR----------- 33 -----------------------------------------------------------MANLVLSECGIRPLPRIYTTPRSNFLSNNN---KFRPSLSSSSYKTSSSPLSFGLNSRDG 57 MASSVLSECGFRPLPRFYPKHTTSFASNPKPTFKFNPPLKPPSSLLNSR---YGFYSK-- 55
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
------------------------------------------------------------VTLRVANIAAPAEDMLVGQPAVVPPFEGGKRLANAPPFTLQDMRDAIPAECFEKDTFRS 92 -----------------------------------------------------------FTRNWALNVSTPLTTPIFEESPLEEDNKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKS 117 -TRNWALNVATPLTT---LQSPSEED-TERFDPGAPPPFNLADIRAAIPKHCWVKNPWMS 110
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
------------------------------------------LVGX--AHQSFSMHKTLN AAHLALDVGIVAALAIGAYTIGNPLVWPLYWFLQGTMFWALFVVGHDCGHQSWSNNKTLN -----------------------------------------------------------LSYVVRDVAIVFALAAGAAYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNDPKLN MSYVVRDVAIVFGLAAVAAYFNNWLLWPLYWFAQGTMFWALFVLGHDCGHGSFSNDPRLN
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
NLVGNITHSSILVPYHGWRISHRTHHANHGHVENDESWHPVSKRIYNQMESMAKIGRLAF DFVGNIVHSSIMVPYHGWRISHRTHHANHGHVENDESWHPIVKSNYEKLDKWAKLGRLLF ----------YWVPYHGWRISHRNHHANHGHVENDESWHPTRKSVYDKMDDLGRIGRLTL SVVGHLLHSSILVPYHGWRISHRTHHQNHGHVENDESWHPMSEKIYNTLDKPTRFFRFTL SVAGHLLHSSILVPYHGWRISHRTHHQNHGHVENDESWHPLPESIYKNLEKTTQMFRFTL ***********.** ************* : *. ::. :: *: :
76 212 50 237 230
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
PLPLFAYPFYLWQRSPGKTGSHYDPKCDLFVPQ-EAPMIRTSNAFMLGMLAILGACTYAL PFPLFAYPFYLLNRSPGKNGSHYDPKSDLFTAS-EGPLVETSNKFQCAWIGFLAGCTVAL PWSMFAYPFYLWKRSPGKTGSHYDPNCDLFVTKTEKEQCITSNVFLVAWLGVLAACKTQL PLVMLAYPFYLWARSPGKKGSHYHPDSDLFLPK-ERKDVLTSTACWTAMAALLVCLNFTI PFPMLAYPFYLWNRSPGKQGSHYHPDSDLFLPK-EKKDVLTSTACWTAMAALLVCLNFVM * ::****** ***** ****.*..*** .. * **. . ..* . :
135 271 110 296 289
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
GPLAMFNLYFIPYVINVVWLDAVTYLHHHGPHDENEKIPXYRGEEWSYLRGGLSTIDRDF GPLAMLNLYVLPYWVFVVWLDVVTYLHHHGPSDPEEEVPWYRGEEWSYFRGGLSTIDRDY GIPAMINLYVMPYWFFVVWLDVVTYLQHHGSHDPEEKLPWFRGEEWSYLRGGLTTLDRDY GPIQMLKLYGIPYWINVMWLDFVTYLHHHG---HEDKLPWYRGKEWSYLRGGLTTLDRDY GPIQMLKLYGIPYWIFVMWLDFVTYLHHHG---HEDKLPWYRGKEWSYLRGGLTTLDRDY * *::** :** . *:*** ****:*** ::::* :**:****:****:*:***:
195 331 170 353 346
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
GIFNHIHHDI--------------WHANRIPRGRHGGPG-----SMRPSGPI-------GIFNKLHHDIGTHVVHHLFPQIPHYHLQKATEAVKPIMGDYYREPEKSPGPIPTHLFEPL GIFSRIHHSIG-------------IHVVHQFFPQIPHYQ--------------------GLINNIHHDIGTHVIHHLFPQIPHYHLVEATEAAKPVLGKYYREPDKS-GPLPLHLLEIL GWINNIHHDIGTHVIHHLFPQIPHYHLVEATEAAKPVLGKYYREPKNS-GPLPLHLLGSL * :..:**.* * .
228 391 196 412 405
P.incisa Ch.vulgaris Dun.salina At FAD7 At FAD8
---------------------------------RRSFARDHFVDNTGDIVFYQQDKNLKL------- 418 ---------------------------------AKSIKEDHYVSDEGEVVYYKADPNLYGEVKVRAD 446 IKSMKQDHFVSDTGDVVYYEADPKLNGQRT---- 435
16 152 177 170
Fig. 2 Sequence alignments of deduced amino acid sequences of ω3 desaturases. Sequence alignments were generated using the CLUSTALW (1.82) program. GenBank accession No for Chlorella vulgaris CvFAD3 (AB075527), for Dunaliella salina ω3 desaturase (AF 083613), Arabidopsis thaliana ω3 desaturases FAD7 (NM 111953), FAD 8 (NM 120640). Conserved histidine regions on gray background were utilized for the design of primers.
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IDENTIFICATION OF TWO NOVEL FAMILIES OF MEMBRANE-BOUND NON HEME IRON OXYGENASES INVOLVED IN PLANT STEROL BIOSYNTHESIS: A VIGS APPROACH. S. DARNET, M. BARD*, A. HOEFT AND A. RAHIER Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, 28 rue Goethe, 67083 Strasbourg cedex, France,. and *Purdue University Indianapolis, IN 46202, USA
1. Introduction Sterols are ubiquitous and essential membrane components found in all eukaryotes. They regulate membrane fluidity and permeability and interact with lipids and proteins within the membranes. They are also precursors of a vast array of bioactive compounds involved in important cellular and developmental processes, and plant sterols are particularly linked to brassinosteroids synthesis [1]. The structure of sterols and their biosynthetic pathway differ significantly between fungi, animals and plants. In higher plants, the conversion of cycloartenol (D1) (Figure 2) to functional phytosterols (S1-S3) involves the removal of the two methyl groups at C4 by an enzymatic complex including the sterol-4α-methyl-oxidase (SMO) catalysing the initial step of the demethylation process [2]. In plants, the physiological outcome of two biosynthetically distinct C4demethylation steps as well as the role of transient 4,4-dimethylsterol and 4α-methylsterol intermediates with distinct sterol nucleus, remain to be elucidated. Moreover, mutants of plant deficient in SMO activity have not been reported yet, probably reflecting that blockade of the SMO step compromises the viability of plants due to the accumulation of C4-methylated sterols. In addition, study of the crucial role of C4-demethylation in plants is hampered by the lack of inhibitors of the steps involved, as well as by the absence of identification of the committed genes. Thus, the cloning and precise functional identification of genes involved in plant C4demethylation, could give important clues for the role of C4-methylsterols in plants. We report the cloning of genes belonging to two families of plant SMOs, (SMO1 and SMO2). In order to identify the precise functions of these genes we used a VIGS approach in Nicotiana benthamiana with a viral RNA containing cDNA fragments from SMO1 or SMO2. Results indicate clear and distinct biochemical phenotypes, consisting in specific accumulations of substrates of corresponding SMOs. 2. Identification of cDNAs encoding SMOs from plants. By combining homology and sequence motif searches with knowledge relating to sterol biosynthetic genes across species, five SMO cDNAs from Arabidopsis thaliana (AtSMO) belonging to two families have been cloned [3,4]. All AtSMOs amino-acid sequences were characterized by the presence of three histidine-rich motifs, HX3 H X8or9 HX2 HH X73or75 HX2 HH, which exhibit a topology and spacing of amino acids within the histidine motifs typical of the ERG3 –ERG25 family, PFAM01598. Moreover, the spacing and topology of the histidine motifs are clearly distinct from those found in the extended family of membrane-bound fatty acid desaturases/hydroxylases [5]. One possible function for these tripartite motifs would be to provide the ligands for a presumed catalytic diiron center, as previously proposed for other enzymes possessing these motifs and catalyzing desaturations or hydroxylations [5]. From the multiple alignments of the full-length amino acid sequences of AtSMOs, and sterol-desaturase-like enzymes, we constructed a phylogenetic tree (Figure 1). This tree clearly confirmed the existence of two families of putative plant SMOs isoenzymes, SMO1 and SMO2. AtSMO1 and AtSMO2 families contain 3 and 2 isoforms, respectively, and sequences comparison show that the plant sequences are orthologous to the fungal and mammalian sequences. In addition, cDNA fragments corresponding to each family were isolated in N.benthamiana.
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Figure 1. Dendogram of SMO and sterol-desaturase-like proteins.
3. Expression of AtSMOs in the yeast erg25 ergosterol auxotroph lacking SMO activity. Two groups of complementation were obtained with plant SMO cDNAs: The SMO2 group of complementation restored growth, but only low levels of ergosterol biosynthesis. The SMO1 group did not complement erg25-25c [3,4]. This data suggest that only one family of SMOs is functional for the oxidation of 4,4-dimethylzymosterol, the physiological substrate of the yeast SMO accumulating in erg25 [6,7]. It could reflect a strict and distinct substrate specificity of the plant SMO1 isoenzymes unable to oxidize 4,4-dimethylzymosterol, the physiological substrate of yeast SMO [7] in contrast to the S.cerevisiae SMO and to the plant SMO2 isoenzymes. 4. Silencing endogenous Nicotiana benthamiana sterol C4 methyl oxidases. To elucidate the precise functions of SMO1 and SMO2 genes families, we have reduced their expression by using a virus-induced gene silencing (VIGS) approach [8] in Nicotiana benthamiana. The genetic inhibition of the plant SMOs should result in the accumulation of the substrates of the targeted enzymes. Two cDNA fragments of 387bp and 498bp corresponding respectively to NtSMO1 and NtSMO2 were cloned from Nicotiana tabacum and were inserted into the viral TTO1 vector, and N.benthamiana inoculated with the viral transcripts [5]. The sterol profile of infected plants was quantified by GC-MS and unequivocally identified by coincidental retention time and an electron impact mass spectrum identical to that of authentic standards [4]. Remarkably, following silencing with SMO1, a substantial accumulation 4,4-dimethyl-9β,19cyclopropyl-sterols (i.e 24-methylenecycloartanol D2) was obtained, while qualitative and quantitative levels of 4α-methylsterols were not affected (Figure 2). In the case of silencing with SMO2, an important accumulation of 4α-methyl-∆7 sterols (i.e 24-ethylidenelophenol M2 and 24-ethyllophenol M6) was found, with no change in the levels of 4,4dimethylsterols. Moreover, as these respective biochemical phenotypes are observed in several distinct infected plants but not in control plants infected with TTO-CCS (capsanthin-capsorubin synthase) or non-infected plants, they can confidently be ascribed to the presence of the NtSMO1 and NtSMO2 cDNAs [4]. Taken together, the obtained sterol profiles indicate that 4,4-dimethyl-9β,19-cyclopropylsterol, such as (D2), were the preferred substrates of SMO1, and 4α-methyl-∆7 -sterols, such as (M2), of SMO2. These specific accumulations of 4,4dimethylsterols or 4α-methylsterols are consistent with a poor overlapping in the substrate specificities of these two families of tobacco SMOs leading to the clear distinct biochemical phenotypes observed in this study. This data compared remarkably well with the clear distinct substrate specificities previously determined for the native microsoma l maize SMOs [2]. The present data clearly ascribe the function of SMO1 as a 4,4-dimethyl-9β,19-cyclopropylsterol-4αmethyl-oxidase and SMO2 as a 4α-methyl-∆7 -sterol-4α-methyl-oxidase indicating that in photosynthetic eukaryotes, two distinct families of sterol C4-methyl-oxidases control respectively the level of 4,4-dimethyland 4α-methylsterol precursors [4].
185
Figure 2. Accumulation of SMO substrates induced by SMO1 or SMO2 silencing.
The major biosynthetic pathway from cycloartenol (D1) to 24-methyl and 24-ethyl sterols is represented by open arrows. Dashed arrows represent minor pathways including several biosynthetic steps. Full arrows indicate pathways revealed after down regulation of SMO1 or SMO2. D denotes 4,4dimethylsterols, M-4α-methylsterols and S 4-desmethylsterols. The boxes represent the C4 -demethylation multienzymatic complexes including sterol 4α-methyl oxidase (SMO), 4α-carboxysterol-C3dehydrogenase/C4-decarboxylase (4α-CD) and sterone reductase (SR). Values shown are the content of each compound (% of total sterols) for representative plants infected with TTO1-SMO1, with TTO1 SMO2, and with TTO1-CCS (control), respectively. 5. Conclusion. Plant sterol biosynthesis is profoundly different from that found in animals and fungi, where a single C4 methyloxidase gene is involved in the removal of both methyl groups at C4 [6]. To our knowledge, plant mutants affected in SMO activity have not been reported yet. In this respect, the present study could give important clues for the elucidation of the physiological roles of 4,4-dimethyl- and 4α-methyl-sterols, and of the biological significance of the existence of two distinct C4-demethylation complexes in photosynthetic eukaryotes 6. References. [1 ] Clouse, S.D. (2002) Arabidopsis mutants reveal multiple roles for sterols in plant development. Plant Cell 14, 1995-2000.
[2] Pascal, S., Taton, M. and Rahier, A. (1993) Plant sterol biosynthesis: identification and characterization of two distinct microsomal oxidative enzymatic systems involved in sterol C4 -demethylation. J. Biol. Chem. 268, 11639-11654. [3] Darnet, S., Bard, M. and Rahier, A. (2001) Functional identification of sterol- 4α-methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol-4α-methyl oxidation. FEBS Lett. 508, 39-43. [4] Darnet, S., and Rahier, A. (2004) Plant sterol biosynthesis: identification of two distinct families of sterol 4α-methyl oxidases. Biochem. J. 378, 889-898. [5] Shanklin, J. and Cahoon, E.B. (1998) Desaturation and relared modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 611-641. [6] Bard, M., Bruner, P.A., Pierson, C.A., Lees, N.D., Biermann, B., Frye, L., Koegel, C. and Barbuch R. (1996) Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase. Proc. Natl. Acad. Sci USA 93, 186-190. [7] Darnet, S., and Rahier, A., (2003) Enzymological properties of sterol- C4-methyl-oxidase of yeast sterol biosynthesis. Biochem. Biophys. Acta 1633, 106-117. [8] Baulcombe, D.C. (1999) Fast forward genetics based on virus-induced gene silencing. Curr. Opin. Plant Biol. 2, 109-113.
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SILENCING OF A PLDα LEADS TO CHLOROSIS IN TRANSGENIC TOMATO LEAVES D. KLAUS, B. TER RIET, B. BARGMANN, M. HARING & T. MUNNIK Swammerdam Institute for Life Science, Section of Plant Physiology,
University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, Netherlands, E-mail:
[email protected] 1. Indroduction Phospholipase D (PLD) has been implicated in a variety of plant processes,including lipid catabolism, cell signalling and regulation of the microtubule cytoskeleton [1,2]. PLD hydrolyses phospholipids at the phosphodiester bond and transfers the phosphatidyl group to water, generating PA (Fig. 1). Choline
Phospholipase D
P
P
OH P
H 2O
Choline Phosphatidylcholine
Phosphatidic Acid
Figure 1. PA formation via PLD
In tomato (Lycopersicon esculentum) the PLD gene family consists of three α-, two β-, one δ and two ζclasses [3; unpublished]. LePLDα1, LePLDα2 and LePLD α3 show high identity and conserved amino-acid characteristics compared with Arabidopsis and rice class α PLDs [4,5]. These PLDs contain a Ca 2+ / phospholipid binding (C2) domain near the N terminus, which is found in other proteins that are involved in signal transduction and membrane trafficking. PLDα2 and -α3 transcripts are highly expressed in flower parts, particularly in petals and stamens [3]. 2. Results To silence the tomato PLDα2 isoform in tomato, an RNAi approach was used. This resulted in chlorotic leaves under certain light con-ditions in independent transformants (T3 generation). The phenotype seems to correlate with specific wavelengths (Fig. 2), and is probably caused by differences in leaf or chloroplast morphology. Leaves of silenced plants appear to be thinner and their veins turn up much brighter. In fully expanded chlorotic leaves we also detected a smaller size of the chloroplasts (data not shown). When DNA, RNA and protein contents were determined by southern, northern and western blot analysis, respectively, the silencing did not seem to affect the PLD α2 transcript, because the protein was still present (Fig. 3). In contrast, a closely related isoform PLD α3 (87.9% identity) was silenced. However the PLD α1 isoform (73.8% identity) was also not affected. PLD generates PA and as shown in figure D, the amount of PA was dramaticaly decreased up to 30% in the silenced leaves. Because the silenced leaves dis played chlorosis, a wavelength scan of the chlorophyll was performed and the chlorophyll content was measured (Fig. 4). As anticipated, both are decreased in the silenced plants. Determination of the total lipid composition only revealed a small decrease in MGDG (62 mol% to 56 mol%) in the silenced leaves (Data not shown).
187
NS
S
NS
S
NS
S
S
NS
Pia Plus (400W) 300-320µE 23.5°C
NS
S
S
NS
Agro (400W) 250-270µE, 25.5°C
NS
S
NS
S
S
NS
HPI-T (400W) 130-150µE 24°C
Fig. 2 The absence of specific wavelengths induces chlorosis in the leaves of PLDα-silenced plants.
4 weeks old plants were transferred to different light conditions. 4 days later, silenced plants showed chlorotic leaves. This phenotyp is discovered in independent transformants. Western
Northern
PA formation in overnight 3232P-labelled
(% (% of total PL) PL) P-labbeledleaf leafdiscs discs of total
leaf S NS S
Probe: PLDα2
leaf NS S
flower NS S Antibody PLDα2
7
6
5
PLDα 3
4
PLDα1
3
2
PLDα3
1
0
3-4 (NS)
Fig. 3 Northern and Westernblot analysis
1,2
3-9 (S)
6-2 (NS)
Fig 4 PA formation
Wavelength scan of silenced and not silenced chlorophyll Chlorophyll Carotenoids a+b
OD
1 0,8 0,6
3-4 (NS)
3.77 ±0.83
0.61 ±0.11
3-9 (S)
1.70 ±0.71
0.31 ±0.13
6-2 (NS)
2.82 ±0.35
0.44 ±0.05
6-5 (S)
1.53 ±0.30
0.27 ±0.04
0,4 blanco
0,2 0 400
line 3-4 (NS) line 3-9 (S)
500
6-5 (S)
600
700
800
900
wavelength (nm)
Fig. 4 Wavelength scan of chlorophyll and chlorophyll content
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3. Conclusions Silencing of a PLD α results in chlorotic leaves under certain light conditions, presumably when specific wavelengths are present or absent. One possible explanation is, that our PLD α is involved in light-dependent activation of the phytochromes or their interactions. Tomato contains five phytochromes phyA, B1, B2, C, E and one cryptochrome, cry1 [6, 7]. Interactions between phytochromes and chryptochrome occur in several different ways, depending on plant development and light conditions. They are very complex and, so far, poorly understood. In a protoplast system, it has been shown that phytochrome and chryptochrome effects occur via distinct pathways involving cGMP and Ca 2+ [8]. Silencing of our PLDα may effect a photoreceptor activity or the interactions among the photoreceptors. The reduced size of the chloroplasts in fully expanded chlorotic leaves as well as the reduced chlorophyll content indicates, that our PLD α might also be involved in chloroplast development and/or function. Recently, McGee et al. [5] detected a PLD α in the chloroplasts of rice, but so far we were not able to detect this in our tomato plants. To further investigate in which process(es) our PLDα is involved, we will determine its localisation by immunolocalization- and GFP-studies and specify its response to certain light conditions.
References: [1] Meijer H.J.G & Munnik, T. (2003) Phospholipid-based signalling in plants. Annu. Rev. Plant Biol. 54, 265-306 [2] Dhonukshe P., Laxalt A.M, Goedhart J., Gadella T.W.J. & Munnik T. (2003) Phospholipase D activation correlates with microtuble reorganization in living plant cells. Plant Cell 15, 2666-2679 [3] Laxalt A.M., ter Riet B., Verdonk J.C., Parigi L., Tameling W.I.L, Vossen J., Haring M., Musgrave A. & Munnik T. (2001) Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDb1 on elicitation with xylanase. Plant J. 26(3), 237-247 [4] Qin C. & Wang X. (2002) The Arabidopsis Phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD1 with distinct regulatory domains. Plant Physiol. 128, 1057-1068 [5] McGee J.D., Roe J.L., Sweat T.A., Wang X., Guikema J.A. & Leach J.E. (2003) Rice Phospholipase D isoforms show differential cellular location and gene induction. Plant Cell 44(10), 1013-1026 [6] Weller J.L., Schreuder M.E.L, Smith H., Koornneef M. & Kendrick E. (2000) Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato. Plant J. 24(3), 345-356 [7] Weller J.L, Perrotta G., Schreuder M.E.L., van Tuinen A., Koornneef M., Guiliano G. & Kendrick R.E (2001) Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2. Plant J. 25, 427-440 [8] Christie J.M & Jenkins G.I. (1996) Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell 8, 1555-1567
189
IDENTIFICATION OF A FATTY ACID ?11-DESATURASE MICROALGA THALASSIOSIRA PSEUDONANA
FROM
THE
T. TONON, D.H. HARVEY, R. QING, Y. LI, T.R. LARSON AND I.A. GRAHAM CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, United Kingdom
1. Introduction Besides the common fatty acids (FAs) 16:0, 16:1?9, 18:0 and 18:1? 9 found in most living organisms, trace amounts of more unusual fatty acids can be found in a wide range of species. For instance, 16:1?11 has been reported in several species of Pavlova, in the Eustigmatophyte Nannochloropsis oculata, and in the diatom Thalassiosira pseudonana [1,2]. This FA accounts for a very small portion of the total FAs in these microalgae, and its specific role in the algal cells is unknown. However, 16:1? 11 is a very important biosynthetic precursor for sex pheromones in insects. For instance, in the corn earworm Helicoverpa zea, which produces a pheromone mixture of Z11-16:Ald and Z9-16:Ald in a 30:1 ratio, the most abundant desaturase-encoding transcript is HzeaLPAQ (also called HzPGDs1) which encodes a ?11-desaturase that does not possess a cytochrome b5 extension, and therefore requires free cytochrome b5 for activity. Many acyl-CoA ?11-desaturases with different specificities have been isolated from insects [3], but none from other species. Here we present the characterisation of a cytochrome b5 desaturase exhibiting ?11-desaturase activity, isolated from the marine microalga T. pseudonana. This desaturase is closely related to the front-end class of fatty acid desaturases involved in the biosynthesis of polyunsaturated fatty acids (PUFAs). 2. Materials and methods The draft genome of the diatom T. pseudonana has been sequenced to approximately nine times coverage by the whole genome shotgun method. Sequence data were produced by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) and the raw sequence data were downloaded and installed on a local server. Batch tblastn searches were carried out using protein sequences of 13 known desaturases as query (AY332747, AF489588, AF489589, AY082392, AY082393, AF139720, AY278558, AF309556, U79010, AF084558, AF084559, AF031477, AF078796). All non-redundant sequences with an E value less than 0.001 were retrieved and assembled into contigs which were translated into amino sequences in three frames in the orientation indicated by tblastn result. Twelve putative desaturase gene models were then constructed manually based on sequence homology. These 12 T. pseudonana sequences, arbitrarily designated TpdesA to TpdesL, were aligned with the 13 functionally characterised desaturases from other species mentioned above, using ClustalX version 1.8. The alignment was then reconciled and further adjusted. Only nine near full-length Thalassiosira sequences were retained for further analysis. A dataset of 250 conserved residue positions was used for construction of the phylogenetic tree. Distance analysis used the program protdist of the Phylip 3.5c package with a PAM250 substitution matrix and a tree was then built from the matrix using fitch (Fitch-Margoliash method). Bootstrap analyses were carried out with 1000 replicates using the neighbour-joining algorithm. For the temporal expression experiment, total RNAs were extracted at different stages of growth for cDNA synthesis. After reverse transcription, PCR was carried out using TpdesG and TpdesN specific primers. Amplification of the 18S rRNA gene was also performed as a marker for constitutive expression. To functionally characterise TpdesN, the entire coding was amplified from genomic with the Expand High Fidelity PCR system (Roche), gel purified, restricted with EcoRI and BamHI and cloned into the corresponding sites behind the galactose-inducible GAL1 promoter of pYES2 (Invitrogen) to yield the plasmid pYDESN. This vector was then transformed into Saccharomyces cerevisiae, and transformants were selected on minimal medium plates lacking uracil. For the feeding experiment, cultures were grown at 22°C in the presence of 2% (w/v) raffinose and 1% (w/v) Tergitol NP-40 (Sigma). Expression of the transgene was induced when OD600nm reached 0.2-0.3 by supplementing galactose to 2% (w/v). At that time, the appropriate fatty acids were added to a final concentration of 50 µM for PUFAs and 500 µM for saturated FAs. Incubation was then carried out at 20°C for three days. For fatty acid analysis , microalgae or yeast cells were harvested by centrifugation. Total fatty acids were extracted and transmethylated as previously described [4]. Most fatty acid methyl esters (FAMEs) were identified by comparison of retention times to a 37 FAME mix (Supelco). PUFA FAMEs were also identified by comparison to a sample of standard Menhaden oil (Supelco), transmethylated as per the samples. Dimethyl
190
disulphide (DMDS) adducts were used to determine the double bond position in identified and unidentified monounsaturated FAMEs. Picolinyl esters were also made from FAMEs to confirm their identities. Picolinyl esters were injected and separated by GC-MS using the same conditions as for DMDS adducts [5]. 3. Results and discussion From the T. pseudonana genomic DNA database, 12 unique contigs corresponding to putative desaturase sequences were assembled. They all showed significant sequence similarity to query sequences, with nine Thalassiosira sequences containing near full length open reading frames compared to other known desaturases (data not shown). Interestingly, the predicted amino acid sequence of these nine T. pseudonana desaturases have a characteristic fused cytochrome b5 haem-binding domain (HP[G/A]G) at their N-terminus and three histidine boxes (H[X]3-4H, H[X]2-3HH and Q[X]2-3HH) with the replacement of the first histidine by glutamine in the third histidine box in all but two of the predicted proteins (TpDESA and TpDES B). These are common characteristics of a large subgroup of front-end acyl group desaturases [6]. As a first step to establishing function of the many putative desaturase sequences, we focused on the TpdesG and TpdesN contigs because they were very simila r at the amino acid level and did not group with any of the characterized PUFA desaturases (data not shown). A temporal expression study showed that TpdesN was constitutively transcribed during algal cultivation, while no expression was detected for TpdesG (Figure 1). Growth phase:
early exponential
late exponential
early stationary
Incubation time:
142 h
237 h
311 h
Nitrate degraded:
20%
60%
100%
M
1
2
3
4
1
2
3
4
1
2
3
4
M
18S rRNA (675 bp)
TpdesN (519 bp) TpdesG (414 bp)
Figure 1. RT-PCR expression analysis of TpdesG and TpdesN. Cells were harvested at different stages of growth for total RNA extraction and cDNA synthesis. PCR was performed on cDNA derived from reverse transcribed RNA using TpdesG, TpdesN and 18s rRNA specific primer pairs. PCR was carried out on undiluted (lane 1) and five-fold serial dilutions (lane 2-4) of each cDNA. The 18S rRNA gene was used as a control of cDNA synthesis.
The TpdesN genomic sequence was found to be full-length and intronless. Functional characterisation of TpdesN in yeast was carried out to establish the specificity of the corresponding enzyme. Supplementing yeast cultures of TpdesN transformants with PUFAs as potential substrates for desaturation revealed no new products. There was also no evidence of ?12-desaturase activity with the endogenous 18:1?9. However, an increase in the peak area of a FAME eluting in the range of sixteen carbon FAMEs was seen and GC-MS based analysis revealed this to be 16:1?11 fatty acid (Figure 2). Small amounts of this FA were also present in wild type yeast. However, quantitative comparison of FA levels in the empty vector pYES2 and pYDESN transformants showed that the proportions of 16:1?11 increased in the presence of TpdesN in both unfed cells and cells that had been fed with different saturated FA. No other changes in either peak area or new peaks were detected in pYDESN transformants, indicating that TpDESN is specifically involved in conversion of 16:0 to 16:1?11.
191
16:1? 9
Detector response
18:1? 9
16:0
16:1? 11 18:0 I.S.
pYDESN pYES2 Retention time
Figure 2. GC analysis of FAMEs from yeast transformed with the empty plasmid pYES2 (dotted line) or the plasmid containing TpDESN (full line). Invsc1 yeast strain transformed with both plasmid were induced for three days at 20°C without supplementation before sampling for fatty acid analysis. I. S., internal standard (17:0). The experiment was repeated three times and results of a representative experiment are shown.
Therefore, although the TpDESN primary sequence is very similar to front-end desaturases, it should not be considered a member of this family of desaturases because it uses a saturated FA as substrate. Identification of such a novel enzyme expands the functional repertoire of the membrane-bound desaturases and it should provide useful comparative information for understanding phylogenetic relationships between these enzymes. Moreover, given the FA profile of T. pseudonana cells and the complexity of the desaturase gene family, it is likely that other assembled contigs will encode ∆4, ∆5 and ∆6 desaturases. It will now be very interesting to functionally characterise these remaining putative desaturase genes and study the relationship between regioselectivity and primary amino acid sequence. A crystal structure for these enzymes is still not available due to technical difficulties in obtaining sufficient quantities of purified membrane-bound protein. Molecular genetic approaches involving site-directed mutagenesis have provided new insight into structure-function relationships, including for example that residues in close proximity to the histidine motifs have been found to be involved in shifting the ratio of desaturation/hydroxylation activities [7]. Detailed comparative analyses and computer modeling of these diverse desaturases from T. pseudonana may further guide site-directed mutagenesis studies aimed at defining key residues controlling substrate specificity and regioselectivity of the introduced double bond. Acknowledgements: Financial support for this work was provided by the Department for Environment, Food and Rural Affairs, grant no. NF 0507. Renwei Qing is a visiting scholar from Sichuan University of China supported by the China Scholarship Council, grant no. CSC 22851086. [1] Dunstan, G.A., Volkman, J.K., Barrett, S.M. and Garland, C.D. (1993) Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture. J. Appl. Phycol. 5, 71-83. [2] Brown, M.R., Dunstan, G.A., Norwood, S.J. and Miller K.A. (1996) Effects of harvest stage and light on the biochemical composition of the diatom Thalassiosira pseudonana. J. Phycol. 32, 64-73. [3] Roelofs, W.L., Liu, W., Hao, G., Jiao, H., Rooney, A.P. and Linn Jr, C.E. (2002) Evolution of moth sex pheromones via ancestral genes. Proc. Natl. Acad. Sci. USA 99, 13621-13626. [4] Tonon, T., Harvey, D., Larson, T.R. and Graham, I.A. (2002) Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochemistry 61, 15-24. [5] Tonon, T., Harvey, D., Qing, R., Larson, T.R. and Graham, I.A. (2004) Identification of a fatty acid ?11-desaturase from the microalgae Thalassiosira pseudonana. FEBS Lett. 563, 28-34. [6] Sperling, P., Ternes, P., Zank, T.K. and Heinz, E. (2003) The evolution of desaturases. Prost. Leuko. Essent. Fatty Acids 68, 73-95. [7] Broun, P., Shanklin, J., Whittle, E. and Somerville, C. (1998) Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids. Science 282, 1315-1317.
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BIOCHEMICAL CHANGES AND CARBON SUPPLY IN LINSEED DEVELOPING EMBRYOS S. TROUFFLARD1 , I. PROST2 , B. THOMASSET 3 , J-N. BARBOTIN1 , A. ROSCHER1 , S. RAWSTHORNE4 , J -C. PORTAIS5 1- Université de Picardie Jules Verne, UMR-CNRS 6022, 33, rue Saint-Leu, F-80039 Amiens Cedex, France 2- Université Paul Sabatier, UMR-CNRS 5546, 24, Chemin de Borde-Rouge, 31326 Castanet Tolosan, France 3- Université Technologique de Compiègne, UMR-CNRS 6022, 60203 Compiègne, France 4- John Innes centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom 5- Laboratoire Biotechnologie-Bioprocédés, UMR-CNRS 5504-UR INRA 792, 135 Avenue de Rangueil, 31077 Toulouse, France Introduction Plant storage reserves are localised in seeds. The composition of these reserves depends on both the plant species and the seed’s developmental stage. Developing oilseed embryos are known to accumulate mainly oil, as well as starch and proteins. Metabolic aspects of storage product accumulation have already been studied in oilseed rape (3, 6). It has been shown that according to the developmental stage of the embryos, carbon flow into lipids and the composition of storage products change. We initiated the same kind of approach in linseed. Some information on fatty acid synthesis in linseed has been reported (2), but the metabolic basis of storage product accumulation in linseed is still not well understood. Preliminary investigations on linseed are presented here and include (i) measurement of the temporal pattern of storage product accumulation in linseed embryos, (ii) a study of the nature of carbon substrates that might be implicated in fatty acid synthesis, and (iii) preliminary results of the influence of light on this synthesis.
Materials and methods
Plant material Linseed plants were grown in a greenhouse at 18°C under a 16 hour photoperiod in daylight supplemented by sodium light. The age of the embryos was determined by tagging each new flower every day. The age of the embryos is then expressed as days after flowering (DAF). Embryos were harvested between 10 and 50 DAF to determine the protein, lipid and starch content. Starch content was determined enzymatically, protein content was estimated using a dye-binding method (1) and lipid content was measured by pulsed NMR.
Plastid isolation Plastid preparation was as described by Fox et al. (5). Recovery of plastids from initial homogenates and their latency were expressed according to the activity of NADP-glyceraldehyde phosphate dehydrogenase (NADPGAPDH) or alkaline pyrophosphatase (AP). Plastid feeding experiments and measurement of incorporation of carbon into fatty acids and starch were performed according to Eastmond and Rawsthorne (3). Except where described, all feeding experiments were carried out under very low intensity room lighting in opaque eppendorf tubes placed in sample racks. For feeding experiments with light, embryos aged from 18 to 25 DAF were used
193
to do the plastid preparations and incubations were carried out in tubes exposed to an artificial light source (500 W tungsten/halogen lamp set to provide 300 umoles.m-2 .s -1 of photosynthetically active radiation).
Chemicals Chemicals and reagents were as described in Eastmond and Rawsthorne (3). Radiolabelled substrates were purchased from NEN Life Science products, (Houndslow, UK).
Results and Discussion
Storage product accumulation The temporal pattern of storage product accumulation in linseed embryos was determined (Fig 1). The oil content rises rapidly from the early stage of development to the mid-to-late stage of development (15 to 30 DAF) while protein accumulation starts later and occurs at a rate of about half that of the oil. Both storage products reach their maximum at about 30 DAF. There was very little starch accumulation measurable in linseed embryos, with the maximum value that we report here representing only one twentieth of that in rapeseed (3). Apart from this difference in starch content the oil and protein accumulation profiles for linseed and rapeseed are similar, although relative protein content is 2.5 times greater in the former.
2.0
0.05
1.8 0.04
1.4 1.2
0.03
1.0 0.8
0.02
Starch (mg/embryo)
Protein and Lipid (mg/embryo)
1.6
0.6 0.4
0.01
0.2 0.0
0.00 10
15
20
25
30
35
40
45
Days after flowering
Protein Starch Lipid
Figure 1: Storage product accumulation (lipids, proteins and starch) during the development of linseed embryos
Carbon supply for storage product accumulation Feeding experiments on isolated linseed plastids were done to study plastidial capacity for the import/use of e xogenous substrates (lipid and starch precursors), and to then compare these rates to those of lipid accumulation in planta. Plastids were prepared from embryos at between 18 and 24 DAF when lipid accumulation is both rapid and increases linearly (Fig. 1). Of the possible substrates which may be a source of
194
carbon for starch or fatty acid synthesis, we supplied
14
C-labelled malate, acetate, pyruvate and glucose-6-
phosphate (Glc-6-P). The results were expressed according to the activity of the plastidial enzy me marker: NADP-GAPDH (Table 1). Table 1: Incorporation of 14 C-substrate into fatty acids and starch in linseed plastids. Substrates were supplied at a concentration of 2mM. Values are the mean +/- SE of measurements made on three separate plastid preparations, or are the mean of two replicated or one single experiment when the SE is not given. Substrates for fatty acid
Calculated in vitro rate
Estimated in planta rate
in vitro / in pla nta
synthesis
nmoles of acetate.h-1 .embryo -1
nmoles of acetate.h-1 .embryo -1
%
14
3.2 ± 0.92
14
[2- C] pyruvate
11.8 ± 1.17
[U-14 C] malate
2.8 ± 0.48
[1- C] Glc-6-P
14
2.7 10.1 166
1.2
[1- C] acetate
1.2 ± 0.51
1
Bicarbonate (ATP, light)∗
0.16
0.14
Substrates for starch synthesis
Calculated in vitro rate -1
14
[1- C] Glc-6-P
Estimated in planta rate -1
nmoles of hexose.h .embryo
nmoles of hexose.h-1 .embryo-1
3.7
0.066
9307
The calculated rate of starch synthesis from Glc -6-P by intact plastids is about 93 times the in planta rate. Given the low rate of starch accumulation in whole linseed embryos (Fig. 1), this result suggests that there might be substantial starch turn-over during embryo development. Of the substrates tested for fatty acid synthesis, the highest rate was supported by pyruvate, although even in this case the rate of fatty acid synthesis in vitro accounted only for 10% to the in planta rate. Compared to pyruvate, other metabolites and especially acetate were used only poorly. To determine if the use of NADP-GAPDH as a means of expressing the rate of fatty acid synthesis leads to unexpected errors in the comparison of our in vitro data to in planta rates, the feeding results were expressed using a different plastidial enzyme marker as a reference: alkaline pyrophosphatase. The recovery and the latency values obtained from using this enzyme were slightly different from those obtained with NADPGAPDH (Table 2). Even when the rate of fatty acid synthesis was expressed per unit of alkaline pyrophosphatase, the calculated in vitro rate was still low compared to the in planta rate. However, it is intriguing that different plastidial marker enzymes result in different plastid recovery rates. ∗Also, despite the photosynthetic potential (see below), no significant light fixation of CO2 could be measured. Table 2: Activity of two plastidial marker enzymes, NADP-dependent glyceraldehydes 3-phosphate dehydrogenase (NADP-GAPDH) and alkaline pyrophosphatase (AP), in two independent plastid preparations and in the whole embryos from which the plastids were derived.
NADPH-GAPDH Activity in whole embryos
AP Recovery %
Activity in whole embryos
(unit of enzyme
(unit of enzyme
activity/embryo)
activity/embryo)
Recovery %
0.011
11.7
0.029
5.8
0.031
4.7
0.025
5.8
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Influence of light and substrate concentration on fatty acid synthesis Developing linseed embryos are chlorophyllous and light has been shown to influence fatty acid synthesis from acetate by isolated plastids (2). In order to investigate the photosynthetic nature of the developing embryos in more detail, transmission electron microscopy images were made at different stages of development (Fig 3).
Figure 3: Transmission electron microscopy images at different stages of linseed embryo development.
Chloroplast-like organelles with granal stacks are visible at the earliest stage of development that we studied (14 DAF). The mid-to-late and late stages of development reveal a progressive disappearance of grana and a significant appearance of lipidic vesicules. As pyruvate was utilised much more effectively than acetate as a substrate for fatty acid synthesis by the isolated linseed plastids we investigated the effect of light on pyruvate metabolism. Light enhanced the rate of fatty acid synthesis by a factor of 1.4 (Fig. 4), providing further support to the hypothesis that light energy can contribute to providing the necessary ATP / reducing power to drive fatty acid synthesis in the chlorophyllous embryos of linseed (2). The rate of fatty acid synthesis from pyruvate increased with increasing pyruvate concentration, whether incubations were illuminated or not (Fig. 4). This lack of saturation with respect to substrate concentration is similar to that seen for pyruvate utilisation by plastids isolated from castor endosperm (4), and unlike the saturatable activity seen for plastids from rapeseed embryos (3). Rate of fatty acid synthesis
nmoles of acetate.embryo-1.h-1
12 10 8 dark light
6 4 2 0 1 mM
2 mM
4 mM
concentration of substrate (pyruvate)
Figure 4: Influence of light on fatty acid synthesis from pyruvate by isolated plastids
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Conclusion We conclude that (i) starch synthesis and turnover may be important in linseed embryos, despite low levels of starch accumulation, (ii) that under the incubation conditions described pyruvate is the most effective substrate for fatty acid synthesis in comparison to a range of other well-characterised potential substrates, and (iii) that light energy can contribute to driving fatty acid synthesis although whether this occurs through the synthesis of ATP, NADPH or both is not clear. That the in vitro rate of fatty acid synthesis gave, at best, about 10% of the expected in vivo rate is not caused by too low a substrate concentration, by lack of light energy, nor by errors caused by choice of plastidial marker enzyme in making these calculations. It is possible that in our incubation conditions, NADPH and NADH are not produced endogenously in quantities that are required for fatty acid synthesis. Studying fatty acid synthesis from substrates such as pyruvate, in the presence of Glc -6-P, would address whether the plastidial oxidative pentose phosphate pathway could play a role in this provision (3,7).
References 1) Bradford , M.M. ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding. Anal Biochem 72, 248-54. 2) Browse, J., Slack, C.R. (1985) Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons. Planta 166, 74-80. 3) Eastmond, P.J., Rawsthorne, S. (2000) Coordinate change in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos. Plant Physiol. 122, 767-774. 4) Eastmond, P.J., Dennis, D.T., Rawsthorne, S. (1997) Evidence that a malate/inorganic phosphate exchange translocator imports carbon across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm. Plant Physiol. 114, 851-856. 5) Fox, S., Hill, L.M., Rawsthorne, S. (2000) Inhibition of the glucose-6-phosphate transport er in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis. Biochemical Journal 352, 525-532. 6) Kang, F., Rawsthorne, S, (1994) Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.). Plant J 6, 795-805. 7) Kang, F., Rawsthorne, S, (1996) Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos. Planta 199, 321-7.
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BREEDING HIGH-OLEIC SUNFLOWER LINES FOR COMPLEX DISEASE RESISTANCE R. NAGYNÉ KUTNI, R. SZALAY*, L. PÁLVÖLGYI Cereal Research Non-Profit Company, Department of Oil Crops Szeged 6726, Hungary *Corresponding author:
[email protected]
Abstract The most important oil crop in Hungary is sunflower. The specific sunflower oil with high oleic acid content may be gain the market for food and industrial purposes where high oxidative stability is required. Biodiesel fuel production may be the main promising industrial target for high-oleic sunflower varieties in Hungary. However, profitable production of high-oleic sunflower hybrids is limited by their disease sensibility under Hungarian growing conditions. The current opinion links the high oleic acid content with the highest field susceptibility to diseases. In contrast, our hypothesis is that high oleic acid content may be associated with good field disease-resistance. The first success with healthy high-oleic sunflower line selection is reported here. A large number of potential lines was developed from the crosses of a newly selected high-oleic female line and of a normal restorer line showing good field disease-resistance. In the case of 159 F4 entries, 57,9% of the analyzed plants expressed good field disease-resistance (score 5-8) and 57,6% of this material showed high oleic acid content ( = 75%). This study confirms no correlation between oleic acid content and poor sanitary state of sunflower lines. Our breeding model proposes an effective strategy to stabilize high oleic acid content and increases complex field disease-resistance simultaneously. Introduction Sunflower (Helianthus annuus L.) is the most important oil crop in Hungary and covers a total area of 500000 hectares. The major part of sunflower seed oil is traditionally processed to edible oil. Vegetable oils also can be converted by methyl esterification to biodiesel for transport uses. Potential of the high-oleic sunflower for biodiesel production is significant. High oleic acid content ensures low susceptibility of vegetable oils to oxidative changes during processing for human nutrition and for industrial application (Fuller et al., 1967). In connection with their oxidative stability, Baldini et al. (2003) found high-oleic sunflower hybrids ideal for biodiesel production. Up to now only one high-oleic sunflower variety was registered in the Hungarian National Catalogue. Hungarian sunflower growing environment creates harsh conditions for breeding competitive hybrids. Long periods of drought and very high temperatures are interspersed with rainfalls create ideal conditions for fungal pathogenesis. Pressure of fungal infection has a heavy influence on the yield performance of candidate hybrids. Relative importance of each fungal species is disputable. Several fungi including Plasmopara halstedii, Sclerotinia sclerotiorum, Diaporthe helianthi, Macrophomina phaseolina frequently attack sunflowers. Pacureanu-Joita et al.(2000) studied yield reducing capacity of a single fungus, exclusively that of Phomopsis / Diaporthe helianthi. In contrast to their principle, we propose an integrated breeding programme to investigate disease- resistance simultaneously with high oleic acid content. In Cereal Research Non-Profit Company (CRNPC) breeding programme for sunflower oil fatty acid composition was started in 1985. Further development (from 1996) on our programme is aimed at the selection of new healthy high-oleic female (HO) and restorer lines resistant to all important races of Plasmopara halstedii as well. Two restorer lines resistant to many downy mildew races having normal acid content (N) were involved into this programme. Objective of this paper was to present the first results of our work demonstrating the possibility of association of high oleic acid content with good field disease-resistance. Materials and Methods Plant Materials. Two restorer lines of normal oleic acid content (N) showing good field disease-resistance and three high-oleic (HO) cms female lines showing susceptibility to diseases were used in crossings aimed at creation of four initial source populations. These lines were previously developed by CRNPC. Crosses were carried out in 1999 and F1 plants were self-pollinated in 2000. Further selfing of selected individual plants was
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taking place in 2001, 2002, and 2003 in the pathological nursery (in monoculture) at Kiszombor, Hungary. Fungal natural infection was enhanced by irrigation. Artificial inoculation with Plasmopara halstedii by method of Gulya et al. (1991) was used to retest the presence of PL6 resistance gene. Evaluation of field disease resistance. Reaction to diseases was scored by bonification on a scale from 1 to 9 (1=worst, 9=best) three times during the vegetation period. Measurement of oil content. Ten to 15g of achenes were analyzed by nuclear magnetic resonance (Oxford 4000 NMR). Fatty Acid Composition Analysis. Ten to 20 achenes were ground, and total lipid was extracted with 3 ml 2M KOH dissolved in methanol. Afterwards, 3ml distillated H2 O was gently added to reaction mixture followed by extraction of nonsaponifiable materials with 3 ml petroleum ether. The clear upper layer was transferred to a glass tube and incubated for 30 minutes at 70 C°. Concentrated methyl -esters were eluted in 500 µl hexane. One µl of this sample was analyzed on a gas chromatograph (Hewlett-Packard model 5890 Series II) equipped with flame ionization detector (FID), on-column injector and 30 m long SUPELCOWAX™ 10 capillary column (Supelco INC, Bellefonte). As a carrier gas nitrogen was used. The oven temperature was programmed for 195 C°, the injector and the detector were heated to 220 C°. After 8 minutes running period fatty acids typically occurring in sunflower oil were determined: palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2). Results and Discussion In actual Hungarian climatic and agronomic conditions, the most promising energy crop for biodiesel production seems to be the high-oleic sunflower. Selection for resistance to an individual fungal pathogen is a difficult way to reduce the effect of fungal damages in high-oleic sunflower. It is also difficult to determine which fungal pathogens are important components of infection in various field conditions. However, selection for complex field resistance to diseases may be a possible way to be followed by the breeder. This study focuses on the better selection of healthy high-oleic restorer lines for these purposes. In our programme, the initial cross was made in 1999 between three newly developed high-oleic diseasesensitive cms female lines and two normal, relatively resistant restorer lines. Four populations were created and harvested in monoculture at our pathological nursery. Oil content and fatty acid composition of the selfpollinated plants was determined in every year. In these four combinations, 159 F4 candidate lines were analyzed to determine influence of the high oleic acid content on the complex field disease-resistance. In the case of all the four populations, 27% of analyzed entries were grouped in normal oleic content class, 23,3% of the material showed intermediate values and 49,7% of F4 lines selected in similar environmental conditions pooled in high oleic class (data not presented). According to the model of Fernandez-Martinez et al. (1989), the number of selected high-oleic F4 lines indicates a high degree of dominance of stable genetic factors controlling the high oleic acid content. Table 1. shows the average of the parameters of four high-oleic populations in comparison with the population mean, and with that of parental lines. Significant differences between oil content, oleic acid content and field disease-resistance were observed. High-oleic F4 line from the F931210 population had the highest oil content and oleic acid content, too. These higher values might be attributable mainly to better combining ability of normal line with high –oleic line. However the high oleic parent may also positively influence these parameters studied. Significant differences were found to be due to high oleic parent in the case of F741185 population. Disease symptoms on all the four populations were noted by visual observations of plants during the 2003 cultivation period in Kiszombor. Differences in complex responses of entries to fungal pathogens were observed in the field. The F741185 and F931210 population showed the least diseased plants, consequently, the highest number of plants was selected with the best potential of field disease-resistance. Present results indicate that in every four combinations it was possible to obtain better field resistant pools than in the parental high-oleic lines. An important modification of field disease-resistance was observed in these four populations. Retesting incorporation of the PL6 resistance gene against Plasmopara halstedii was made by artificial inoculation published by Gulya et al (1991) and confirmed the presence of gene PL6.
199
Table 1.
Mean performance of 159 F4 lines, of line with highest oleic acid content and that of each parent of four populations.
Oil content %
Character Oleic acid content %
Population mean
55,9
62,9
4,94
Line with highest oleic acid content High oleic female parent (1)
59,7 53,7
90,9 89,2
7 5
Normal male parent (I) LSD 0,05*
49,8 2,1
23,4 6,5
7 0,61
55,1 58,4 53,7 52,5 2,4
58,4 91,3 89,2 28,3 26,0
3,83 5 5 6 1,33
53,1 54,5 54,9 52,5 4,1
75,6 91,9 92,1 28,3 8,8
3,56 4 3 6 0,78
Population identification
Sanitary state (bonification 1-9) 1=worst; 9=best
F741185 population
F861189 population Population mean Line with highest oleic acid content High oleic female parent (1) Normal male parent (II) LSD 0,05* F901192 population Population mean Line with highest oleic acid content High oleic female parent (2) Normal male parent (II) LSD 0,05* F931210 population Population mean 55,4 66,9 5,17 Line with highest oleic acid content 62,1 93,3 6 High oleic female parent (3) 59,5 92,3 5 Normal male parent (II) 52,5 28,3 6 LSD 0,05* 2,2 12,0 0,51 * = Indicates the least significant difference between the line with highest oleic acid content and the population mean at the 0.05 significance level.
Correlation analysis was conducted to demonstrate the association of disease symptom data with good field disease resistance of normal – and high-oleic line selections (Figure 1.). Linear correlation coefficient between measured oleic acid content per plant and observed complex field disease-resistance was 0,041 , indicating that field disease-resistance has not influence on the seed oleic acid content. Figure 1. Correlation analysis between oleic acid content and complex field disease-resistance.
Field resistance
9 7 y = -0,0018x + 4,8776 r=0,041
5 3 1 10
20
30
40
50
60
70
80
90
100
Oleic acid content %
Data of the Figure 1. confirm our hypothesis that high oleic acid content may be associated with good field disease-resistance. This study demonstrates no correlation between high oleic acid content and the poor sanitary state of candidate lines. This kind of selection would affect performance, mainly disease resistance of the candidate lines, since no correlation of oleic acid content with field disease-resistance was detected.
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Conclusions These results may appear in contrast to current opinion that associates high oleic acid content with highest field susceptibility to disease. The present study revealed an important variability for oleic acid content and field disease-resistance in four F4 sunflower populations. It was demonstrated that it was possible to develop an effective breeding strategy to stabilize high-oleic acid content and to increase complex disease-resistance at the same time. Our results indicate a reasonable effective selection model for high oleic acid content under field pathogenic pressure. Further efforts to define relationships between high oleic acid content and complex field disease-resistance are in progress.
References 1
Directive 2003/30/EC of the European parliament and of the council of 8 may 2003 on the promotion of the use of biofuels or other renewable fuels for transport. Off. J. of the European Union L123/42. Ahloowalia, B.S., Maluszynski, M., Nichterlein, K. (2004) Global impact of mutation-derived varieties. Euphytica 135:187-204. Baldini, M., Vischi, M., Di Bernardo, N., Turi, M., Vannozzi, GP., Olivieri, A.M. (2003) High oleic sunflower varieties for biodiesel: a new perspective for sunflower crop. In Proc. XLVII1 Italian Society of Agricultural Genetics Annual Congress, Verona, Italy Fuller, M.J., Diamond, J., Applewhite, T. (1967) High oleic safflower oil. Stability and chemical modification. J. Am. Oil Chem. Soc. 44:264-267. Fernandez-Martinez, J., Jimenez, A., Domingez, J., Garcia, J.M., Garces, R., Mancha, M. (1989) Genetic analysis of the high oleic acid content in cultivated sunflower (Helianthus annuus L.). Euphytica 41:39-51. Fernandez-Martinez, J., Munoz, J., Gomez-Arnau, J. (1993) Performance of near-isogenic high and low oleic acid hybrids of sunflower. Crop Sci. 33:1158-1163. Gulya, T., Miller, J., Virányi, F., Sackston, W.E. (1991) Proposal internationally standardized methods for race identification of Plasmopara halstedii. Helia 14:11-20. Pacureanu-Joita, M., Vranceanu, A.V., Stanciu, D., Raranciuc, C. (2000) High oleic acid content in sunflower genotypes in relation with resistance to disease. pp. J49-J56.In Proc. 15h Int.. Sunflower Conf., Toulouse, France
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Effect of heavy metals upon lipid metabolism in P. perfoliatus Rozentsvet O.A., Bosenko E.S., Guschina I.A. Institute of Ecology of the Volga River Basin, Russian Academy of Sciences,
10 Komzin St., Togliatti, Russia, Fax 007 (8482) 489405 1.
Introduction
Heavy metals brought in the natural water by industrial waste and washouts from soil cause serious pollution of the environment [1]. The most dangerous toxic elements in water ecosystems are metals belonging to Group II of the Mendeleyev’s Table, as they can cause many chronic and episodic diseases. Reacting to the toxic effect, water inhabitants develop certain mechanisms helping them adapt to the new ambient conditions, which appear under anthropogenic influence [2, 3]. Despite remarkable success in the study of heavy metal effects, the picture of formation and integration of physiological and biochemical processes underlying plants’ adaptation to metal-induced stress has not been made sufficiently clear. The present work examines the influence of metal nitrates (Cd +2 , Cu +2 , Zn+2 ) in the concentration range 1 to 1,000 ìMole/l on a water plant Potamogeton perfoliatus L., growing in a natural habitat. This species is widespread in freshwater pools, resistant to excessive content of biogenic and industrial pollutants present in the water and bottom sediments. Accumulation of metals, content of general lipids and ratio of individual lipid components, responsible for the structure and function of biological membranes, were monitored in the plant’s leaves during a 3-day period of incubation 2. Materials and Methods In the shallow water area of the Saratov water reservoir with abundant thicket of pondweed (Potamogeton perfoliatus L.), tanks were placed on the bottom to encapsulate some 10 to 12 plants with roots and isolate them from surrounding water. The volume of natural water in the tanks was maintained at 135 to 140 l level. During three days and after each 24 hours salts of metals were introduced into the tanks to obtain concentrations 1, 10, 100 and 1,000 ìMole/l. Plants inside the tanks, where no metal nitrates were added, were used as Control samples (C). Leaves from the 4th to 7th shoot layers were used for analysis. The collected leaves were thoroughly washed in running water, dried, weighed and analyzed for lipid content. Following de-fermentation with hot isopropanol, plant samples of 1 to 2-g fresh weight were homogenized and extracted with a chloroform-methanol mixture. Polar lipids were separated using twodimensional thin-layer chromatography (6x6 cm glass plates) with a fixed silica gel layer. Lipids in the extracts were analyzed in aliquots of the extract previously dried in vacuum to constant weight. Total and individual phospholipids were quantified by phosphorus, while a densitometric method was used for glycolipid analysis.
3. Results and Discussion As we have previously demonstrated [4], over a 2-day period the pondweed’s leaves can actively accumulate metals; the greater concentration of metals in the water, the higher accumulated amount (Rozentsvet et al., 2003à, 2003b). On the third day, however, the concentration-to-accumulation relationship held no longer, possibly as a result of changes in metabolic processes and/or initiation of protection mechanisms in the leaves under by the prolonged effect of toxic concentration of the metal. Using different concentrations of metal ions allowed observe different sensitivity of individual components involved in the lipid metabolism. 1 to 10 ì M concentrations of metals did not cause any notable changes in phospholipids, compared to 100 and 1,000 ì M concentrations. The glycolipid ratio changed at all tested concentrations (Rozentsvet et al., 2004). The present work shows the results of comparative investigation into the effect of metal salts at the concentration 100 ìM. It has been demonstrated that prolonged time of influence of the metal on the plants caused a decrease of the total lipid content (Fig. 1) for all examined metals. The higher toxicity of the metal, the greater decrease of the total lipid level was observed.
mg/g wet weigh
30 25 20 15 10 5 0 6
24
48
72
Time, h
Fig. 1. Dynamics of total lipid changes in the leaves of P. perfoliatus under the influence of metal salts. ♦-Cîntrol sample, - Zn+2, - Cu +2, - Cd+2
Under the influence of metals (Cu +2 , Cd +2 , Zn+2 ) at the given concentration the share of phosphatidylethanolamine (PE) in phospholipids went down with the notable increase of phosphatidylglycerol (PG) (Fig. 2). The content of (phosphatidylcholine) PC went down under the influence of Cd +2 . II
70 60 50 40 30 20 10 0 6
24
48
72
Relative phospholipid content %
Relative phospholipid content %
I 60 50 40 30 20 10 0
Time, h
Time, h
IV
60
Relative phospholipid content %
Relative phospholipid content %
III
50 40 30 20 10 0 6
24
48 Time, h
72
60 50 40 30 20 10 0 6
24
48
72
Time, h
Fig. 2. Dynamics of individual phospholipid changes in the leaves of P. perfoliatus under the influence of metal salts: I- Control sample, II- Zn(No 3)2, III- Cu(No 3)2, IV- Cd(NO 3)2. Concentration of salts in the water is 100 µÌ. ♦phosphatidylcholine, – phosphatidylethanolamine, - phosphatidylglycerol, – phosphatidylinositol, – phosphatidic acid, -diphosphatidylglycerol
The glycolipids showed a lower content of monogalactosyldiacylglycerol (MGDG) but increased sulphoquinovosyldiacylglycerol (SQDG) (Fig. 3).
II
60
Relative glycolipid content,%
Relative glycolipid content %
I
50 40 30 20 10 0 6
24
48
50 40 30 20 10 0
72
6
24
Time, h
Relative glycolipid content %
Relative glycolipid content %
60 50 40 30 20 10 0 24
72
48
72
IV
III
6
48 Time, h
48
72
Time, h
60 50 40 30 20 10 0 6
24 Time, h
Fig. 3. Dynamics of individual glycolipid changes in the leaves of P. perfoliatus under the influence of metal salts: IControl sample, II- Zn(No 3)2, III- Cu(No 3)2, IV- Cd(NO 3)2. Concentration of salts in the water is 100 µ Ì. ♦ - monogalactosyldiacylglycerol, – digalactosyldiacylglycerol, - sulphoquinovosyldiacylglycerol
It has been demonstrated that the plants growing under metal-induced stress show an active re-structuring of the cell metabolism directed to support cell structures, which provide for the pigment apparatus’s functioning and photosynthesis, and the lipid changes correlate with the levels of metal toxicity. The changes in the quantitative characteristics of lipid metabolism testify to their ecological importance in the development of plants’ adaptive reactions to changes in the ambient conditions in their habitats. This work has been by RFBR (grant No. 04-04-96506). Reference: 1. Rama Deli S., Prasad M.N.V. Membrane lipid alteration in exposed plants. In Heavy metal stress in plants. From molecules to ecosystems. Berlin: Springer, 1999. P. 99-117. 2. GummingJ.R., Taylor C.J. Mechnisms of metal tolerance in plants: physiological adaptation for exclution of metal ions from the cytoplasm.In: Allen N.S. , ed. Stres responses in plants: adaptation and accumulation. 1990. New York: Wiley – Liss,. P. 328-356. 3. Van der Werff Madelijn, Pruyt Margreet J. Long-term effects of heavy metals on aquatic plants. // Chemosphere 1982. V.11. N.8. 727-739. 4. Rozentsvet O.A.,. Mursaeva S.V. , Guschina I.A., Bosenko E.S. Accumulation of a cadmium and physiologo biochemical state Potamogeton perfoliatus L. Depending on concentration of nitrate cadmium in aquatic environment. // Izvestiya Akademii Nauk (Samara Herald). 2003. No.1. P. 180- 188.
SPONSORS of 16 PLANT LIPID SYMPOSIUM th
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