Plant Science 141 (1999) 1 – 9

Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance N. Sreenivasulu a, S. Ramanjulu b, K. Ramachandra-Kini c, H.S. Prakash c, H. Shekar-Shetty c, H.S. Savithri d, C. Sudhakar a,* a

Department of Botany, Sri Krishnade6araya Uni6ersity, Anantapur-515 003, India Department of Plant Sciences, Weizman Institute of Science, Reho6ot 76100, Israel c Department of Studies in Applied Botany, Uni6ersity of Mysore, Mysore, India d Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

b

Received 25 May 1998; received in revised form 24 September 1998; accepted 20 October 1998

Abstract The effect of NaCl on total peroxidase activity, induction of isoperoxidases and lipid peroxidation in 5-day-old seedlings of two contrasting genotypes of Setaria italica L. (Prasad, a salt tolerant cultivar and Lepakshi, a salt susceptible cultivar), was studied. Total peroxidase activity increased under NaCl salinity and the degree of elevation in the activity was salt concentration dependent. Nevertheless, a greater activity was recorded in the tolerant cultivar (cv Prasad) compared to the susceptible (cv Lepakshi) one in all days of sampling. Further, the pattern of isoperoxidases was modified during stress conditions as evident from the electrophoregrams. Although, five acidic isoforms were detected in both cultivars, differences were found between the cultivars. Furthermore, it was observed that acidic isoperoxidases were strongly expressed and an acidic isoperoxidase, A3p (27 kDa) is specifically found in the tolerant cultivar (cv Prasad) under NaCl stress. This isoform was partially purified and found to be thermostable with pI 5.5 and the optimum pH 7.4. A close correlation exists between the rate of lipid peroxidation in terms of malonaldehyde (MDA) content and total peroxidase activity per gram fresh weight with salt tolerance of the two cultivars. The tolerant cultivar (cv Prasad) had low MDA content and high total peroxidase activity than the susceptible variety (cv Lepakshi) during salinity stress. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Isoenzymes; Peroxidase; Salinity; Salt tolerance; Setaria italica L.

1. Introduction Soil salinity is one of the important constraints and better understanding of the mechanisms that enable plants to adapt to salt stress is necessary for exploiting saline soils. The cellular mechanisms are especially important to glycophytes, in which physiological and biochemical processes contribute to the adaptation to salt stress. An increase in the peroxidase activity is a common response to oxidative and abiotic stresses * Corresponding author.

[1,2]. Enhanced production of oxygen free radicals are responsible for peroxidation of membrane lipids and the degree of peroxidative damage of cells was controlled by the potency of antioxidative peroxidase enzyme system. Increased total peroxidase activity in response to salinity were reported [3,4]. Sancho et al. [4] reported that the increased total peroxidase activity in the medium of the salt adapted cells reflect the changed mechanical properties of the cell wall, which in turn, could be related to the salt adaptation process since cell wall properties are known to be modified by salt stress and earlier reports [5,6] link total

0168-9452/99/$ - see front matter © 1990 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 2 0 4 - 0

2

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

peroxidase activity to changes in cell wall and cell membrane integrity properties under salt stress. To this end, we studied the activity of peroxidases and its isozymic pattern in order to understand the role of peroxidases in conferring stress resistance in two cultivars of fox-tail millet namely, cv Prasad and cv Lepakshi differing in salt tolerance. The present study showed high total peroxidase activity and low rate of lipid peroxidation (in terms of malonaldehyde content) in the tolerant variety (cv Prasad), compared to the susceptible one (cv Lepakshi). We also determined the molecular weight, isoelectric point, pH and thermostability of A3p isoform, which was specifically induced by NaCl salinity in tolerant cultivar (cv Prasad).

2. Materials and methods

2.1. Plant material and salt stress conditions Seeds of fox-tail millet cv Prasad (a salt tolerant) and cv Lepakshi (a salt susceptible) were procured from Andhra Pradesh Agricultural Experimental Station, Anantapur, India. Seeds were surface sterilised with 0.1% (w/v) sodium hypochlorite solution for 5 min and thoroughly rinsed with distilled water and allowed to germinate in petri plates lined with filter papers. Distilled water alone served as control, while for treatment 150 mM NaCl was used. The petri plates were maintained at 25°C under aseptic conditions for 5 days in dark. The total peroxidase activity and isoforms were studied in dried seed, 1, 3 and 5-day-old seedlings.

2.2. Enzyme extraction The dried seeds, and seedlings of both cultivars were homogenized separately in 50 mM Tris–HCl buffer pH 7.4 at 4°C. The homogenate was centrifuged at 9000× g for 20 min in a refrigerated high speed centrifuge (Hitachi). The pellet was washed with same extraction buffer and centrifuged in the same way. The resultant supernatants were assayed for the peroxidase activity, isolation and purification of peroxidase enzyme.

2.3. Assay of peroxidase acti6ity Total peroxidase activity in the extracts was assayed as described by Hammerschmidt et al. [7].

The reaction mixture (3 ml) consisted of 0.25% (v/v) guaiacol in 10 mM sodium phosphate buffer, pH 6.0, containing 10 mM hydrogen peroxide. 25 ml of the crude enzyme extract was added to initiate the reaction which was measured spectrophotometrically at 470 nm (Shimadzu 1601). Total peroxidase activity was expressed as the increase in absorbance at 470 nm min − 1 g − 1 FW (0.01 OD=1 EU). Proteins in the extracts were quantified by the method of Bradford [8] using BSA as the standard.

2.4. Malonaldehyde (MDA) The levels of malonaldehyde content in 5-day-old seedlings was determined by the thiobarbituric (TBA) reaction as described by Heath and Packer [9]. One gram of tissue (FW) was homogenised in 5 ml of 0.1% (w/v) TCA. The homogenate was centrifuged at 10 000×g for 5 min and 4 ml of 20% TCA containing 0.5% (w/v) TBA was added to 1 ml of the supernatant. The mixture was heated at 95°C for 30 min and then quickly cooled on ice. The contents were centrifuged at 10 000×g for 15 min and the absorbance was measured at 532 nm in Shimadzu 1601 spectrophotometer. The concentration of MDA was calculated using a extinction coefficient of 155 mM − 1 cm − 1. MDA content expressed as mmol g FW − 1.

2.5. Electrophoresis Non-denaturing discontinuous poly acrylamide gel electrophoresis (PAGE) was done anodically in 10% separating and 5% stacking gels according to Davis [10]. Enzyme extracts (30 mg protein) were loaded onto the slots. After electrophoresis the gels were stained with benzidine for peroxidase isozymes as described by Schrauwen [11]. Isoelectric focusing was performed in 7.5% polyacrylamide gel containing 2% ampholyte (pH 3–10, Sigma, St. Louis, MO) using a multiphor-II (LKB) system according to the manufacturer’s protocol. The pI markers (Sigma), ranging from pI 3.6 to 9.3 were co-electrophoresed to determine the pI of acidic isoforms. Aliquots of 20 ml enzyme extracts were applied on the gel. The voltage was increased stepwise; 200 V for 20 min, 400 V for 40 min, 600 V for 60 min and 800 V for 80 min. The pI values were determined for the enzyme extract after the substrate staining [11] by comparing with standard pI marker stained with coomassie blue.

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

3

Fig. 1. Total peroxidase activity (a) and Malonaldehyde content (b) in 5-day-old seedlings of fox-tail millet under NaCl stress. (cp-control Prasad, sp-stressed Prasad, cl-control Lepakshi, sl-stressed Lepakshi).

Fig. 2. Peroxidase isoforms in the germination seedlings of susceptible (cv Lepakshi) and tolerant (cv Prasad) cultivars under control and NaCl stress from day-1 to day-5. (1 = day 1, 3S = day-3 stressed, 3C = day-3 control, 5S = day-5 stressed, 5C =day-5 control).

In order to determine the molecular weight of purified protein, IEF subjected gels were run for a 2nd dimension on SDS-PAGE under denaturing conditions on 12% polyacrylamide gel. Molecular weight markers ranging from 14.4 to 90 kDa (Pharmacia) were co-electrophoresed to estimate molecular weight of the purified protein. After running, the gel was stained with silver nitrate as described by Heukeshoven and Dernick [12].

2.6. Purification Ammonium sulphate precipitation: Solid ammonium sulphate was added to the crude enzyme to give a final 40–80% saturation. The resulting solution was centrifuged at 9000×g for 30 min and the pellet was redissolved in minimum amount of Tris–HCl buffer (pH 7.2) and dialysed against the same buffer over night with four changes.

4

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

Fig. 5. Ten percent native-PAGE following sephadex G-75 fractions, stained with benzidine for peroxidase isoformic activity. The arrow indicates the location of isoform A3p. Lane 1 relates to G75 fraction numbers 5 – 10, lane 2 to 11 – 16, lane 3 to 17 – 21 and lane 4 to 22 – 25. Fig. 3. Isoelectric focussing of total enzyme extract of tolerant cultivar of fox-tail millet. Separation was carried out in multiphor-II (LKB) system in pH 3–10 range carrier ampholytes. pI markers range between 3.6 and 9.3 (pI) is represented in IEF Mkr lane. Peroxidase isoforms were visualised in lane PS (peroxidase substrate staining) and CB (Coomassie Blue staining). Profile indicates acidic isoforms with pI of 3.68, 5.1, 5.3 and 5.5.

2.6.1. Gel filtration The dialyzed sample was subjected to Sephadex G-75 column (80×1.5 cm) equilibrated and eluted with Tris – HCl buffer (50 mM, pH 7.0). 2 ml fractions were collected, absorbance was read at 280 nm and total peroxidase activity was monitored for each fraction. The enzyme containing fractions were pooled, lyophilized and used for electrophoresis.

2.6.2. Ion-exchange chromatography G-75 activity showing fractions (10–16) were pooled, and subjected to ion-exchange chromatography on DEAE-Sepharose (Sigma) column (bed: 10×2.5 cm) which was pre-equilibrated with Tris–HCl buffer (50 mM pH 7.2). The sample was eluted with Tris–HCl buffer as flow-through and step-gradient NaCl (0.1, 0.5 and 1.0 M) as eluted fractions. All fractions were concentrated individually by lyophilization and used for electrophoresis as described by Govindaswamy and Balasubramanian [13]. 2.7. Enzyme kinetics The effect of pH on the activity of purified peroxidase was measured at 37°C over the pH range 3.0–9.0 in 50 mM buffers (citrate/Na2HPO4, pH 3.0–3.5; sodium acetate, pH 4.0–5.5; sodium

Fig. 4. Elution profile of proteins (monitored at 280 nm) and total peroxidase activity (D470 nm mg protein sephadex G-75 column.

−1

min − 1) from a

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

5

Fig. 6. DEAE Sepharose column chromatography profile of 0.5 M NaCl eluted fractions. NaCl elution profile of proteins (monitored at 280 nm) and total peroxidase activity (D470 nm mg protein − 1 min − 1) from DEAE sepharose column.

phosphate, pH 6.0–7.0; Tris–HCl, pH 7.1–9.0). Temperature optima of the enzyme was determined by measuring the activity remaining after pre-incubation of the enzyme at various temperatures (28 – 98°C) for 60 min in 50 mM Tris–HCl buffer (pH 7.4). 3. Results

3.1. Total peroxidase acti6ity Total peroxidase activity of the crude enzyme extracts by using guaiacol which reacts with all peroxidases, was measured in dried seed, 1, 3 and 5-day-old seedlings of susceptible (cv Lepakshi) and tolerant (cv Prasad) cultivars of fox-tail millet (Fig. 1). The time course of appearance of peroxidase was almost similar in both cultivars after 24 h of germination, but the tolerant cultivar (cv Prasad) had more activity compared to the susceptible one (cv Lepakshi). The same trend was continued in the tolerant cultivar, with a higher peroxidase activity, than susceptible cultivar on day-3 and day-5.

3.3. Differential expression of acidic peroxidase isoform Acidic peroxidase isoforms were detected in the extracts of both control and NaCl treated seedlings of both cultivars by using 10% native PAGE. Since the same amount of soluble proteins from each preparation was loaded on gel, the intensity of isoform bands reflects the induction patterns of individual peroxidase isoforms, in both cultivars. Five acidic isoperoxidases designated A1, A2, A3p, A4 and A5 were detected in crude extract (Fig. 2). However, the activity of isoperoxidases in the dried seeds of both cultivars was close to the detection limit (no activity). The activity of these

3.2. Malonaldehyde (MDA) content Salt stress(150 mM NaCl) caused a significant increase in the levels of MDA content in both the varieties. However, the degree of accumulation was more in the salt susceptible than in the salt tolerant variety indicating a high rate of lipid peroxidation in the susceptible variety due to salt stress (Fig. 1).

Fig. 7. Ten percent native-PAGE following DEAE sepharose fractions stained with benzidine for peroxidase isoformic activity. Arrow indicates the location of isoform A3p. UBFflow-through fractions shows no activity.

6

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

Table 1 Purification and yields of an acidic isoperoxidase (A3p) Purification step

Total protein (mg)

Crude extract 39 Ammonium sulphate precipitation 29 Gel-filtration chromatography 6.3 Ion-exchange chromatography 0.53

Total activity (unit)

Specific activity (unit mg−1)

Yield (%)

463 448 361 113

12 15 57 213

100 97 78 24

isoperoxidases gradually increased from day-1 to day-5 under NaCl stress and was intensified especially during the 3rd and 5th day. The activity of these acidic isoperoxidases was relatively high in the tolerant (cv Prasad) than in the susceptible (cv Lepakshi) cultivar. The isoperoxidase representation of A1 could be detected during stressed conditions in both cultivars on day-1 and 5 and that was absent in control conditions. Further, an acidic isoperoxidase, A3p was specifically found to be expressed only in the tolerant (cv Prasad) cultivar. The pI values of these peroxidase isoforms were 3.6, 5.1, 5.3, and 5.5 (Fig. 3) as determined by isoelectric focussing. The expression of isoperoxidase, A3p in the stressed tolerant cultivar was also evident from SDS-PAGE profile. Further, the electrophoretic protein profile showed that this isoform, A3p with molecular weight 27 kDa, expressed in a tolerant (cv Prasad) cultivar under 150 mM NaCl stress (unpublished data).

with 0.1, 0.5 and 1.0 M NaCl and 0.5 M NaCl eluted fractions were exhibited highest total peroxidase activity with a single peak of A3p; while the flow-through fractions were eluted with Tris–HCl buffer (50 mM Tris–HCl buffer pH 7.0) and did not show any significant enzyme activity (Fig. 6). These fractions were lyophilized separately and subjected to non-denaturing discontinuous PAGE. The flow through fraction did not show any isoperoxidase activity while the 0.5 M NaCl eluted fractions showed a single major band (isoform A3p) in fraction 5 and 6 (Fig. 7). The activity decreased in later fractions. Further, purification steps and yields of an acidic isoperoxidase, A3p induced by NaCl salinity are summarised in Table 1. From 2-D electrophoresis, it was confirmed that the apparent molecular weight of A3p isoform is 27 kDa with pI 5.5 (Fig. 8).

3.4. Purification of acidic peroxidase isoform The following protocol allowed us to separate acidic isoperoxidases by three different steps. Ammonium sulphate precipitation at 40–80% saturation contained all the peroxidase isoforms. Gel-filtration chromatography fraction numbers 5–10, 11 – 16, 17 –21 and 22–25 exhibited high total peroxidase activity (Fig. 4). Protein peak containing fractions were pooled, lyophilized and subjected to non-denaturing discontinuous PAGE. In the Fig. 5, lane 1 relates to G75 fraction numbers 5 – 10, lane 2 to 11–16, lane 3 to 17–21 and lane 4 to 22–25, respectively. The profile showed predominantly the activity of isoform A1 in lane 2 and A2 and A3p activity in lane 3. Other lanes (4, 5, 6 and 7) did not show any significant activity (Fig. 5). Again, these fractions were pooled and subjected to step-gradient DEAE-Sepharose column. The bound proteins were eluted

Fig. 8. 2-Dimension pattern of purified peroxidase isoform (A3p). First dimension gel carry ampholytes (pH 3.5–10 range) following second dimension on 12% SDS-PAGE. Arrow indicates pI and molecular weight of A3p peroxidase isoform.

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

7

Fig. 9. Effect of pH and temperature on the purified peroxidase isoform (A3p).

3.5. Enzyme characterization The optimum pH of the purified isoperoxidase, A3p was 7.4 and the optimum temperature for the enzyme activity ranged between 37°C and 65°C and was found to be thermostable (Fig. 9).

4. Discussion Seeds of both tolerant (cv Prasad) and susceptible (cv Lepakshi) cultivars could germinate and grow in 150 mM NaCl. Nevertheless, the presence of NaCl limited their growth rate. The growth pattern (root and shoot lengths) diverged between genotypes, as the tolerant cultivar showed relatively better growth rate. The most striking efffect of NaCl stress was manifested by differences in the activity of isoperoxidases between the two genotypes. The qualitative and quantitative changes in the activity of several enzymes including peroxidase activity isolated from plants subjected to salinity stress were reported [14,8,9]. Peroxidase isozymes were reported to play a key role in salt tolerance [9]. A regulated balance between oxygen radical production and destruction is required, if metabolic efficiency and function are to be maintained either in normal or stress conditions. A constitutively high anti-oxidant capacity under stress conditions, can prevent damage and corre-

late with plants resistance to that particular stress. Hence, the mechanisms that reduce oxidative stress are expected to play an important role in imparting tolerance in plants under saline conditions. An increase in total peroxidase activity under saline conditions was reported [8,9]. In the present study, a significant elevation in the activitiy of peroxidase was recorded in both cultivars during NaCl stress conditions. Further, the degree of increase was found to be dependent on severity and duration of stress. Furthermore, the degree of elevation in enzyme activity was relatively high in the salt tolerant cultivar(cv Prasad) when compared to the susceptible one (cv Lepakshi). High peroxidase isozymic activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process [9]. Similar results from the present study indicated that there is a greater activity of acidic peroxidases in 5-dayold seedlings of tolerant variety under NaCl stress could be related to the salt adaptation of this variety. The peroxidase system of higher plants exists in multiple isoforms that are developmentally regulated and highly reactive in response to exogenous stimuli [15]. An increase in total peroxidase activity is a common response to various oxidative stress factors [16]. Enhanced production of oxygen free radicals are responsible for stress-dependent peroxidation of membrane lipids [17,18]. Increased

8

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

peroxidation of membrane lipids is known to occur during salinity stress [18]. It has been reported that salinity stress could modify the membrane structure, and may stimulate oxygen radical production which facilitate lipid peroxidation. MDA, which is one of the decomposition products of poly unsaturated fattyacids (PUFA) of biomembranes, showed greater accumulation under salinity treatment [19]. The greater increase in total peroxidase activity and low malonaldehyde content in NaCl stressed tolerant cultivar (cv Prasad), indicates involvement of peroxidases in cell membrane integrity. This was vice-versa in the susceptible cultivar (cv Lepakshi). Increased total peroxidase activity and/or induction of acidic isoperoxidases might be a useful adaptation under salt stress conditions. The extent of peroxidative damage of cells seems to be controlled by the potency of antioxidative systems like peroxidase and is an important defence system of plants against oxygen free radicals. In the present study, NaCl stress caused an induction of acidic isoperoxidases, namely A1,A2,A3p and A4 with pI 3.6, 5.1, 5.3 and 5.5, respectively. Similar results, with increase in two acidic isoperoxidases were reported in tobacco associated with resistance to blue mold and were assigned a role in catalyzing cross-linking of the cell wall extensins [20]. In the present study these acidic peroxidases might be involved in cell membrane integrity, regulation of early seedling growth under salt stress conditions as demonstrated earlier in some plant species [16]. The involvement of acidic peroxidases in plant growth during salinity stress was evident from the absence of isoperoxidases in seeds (on day zero germination) and their expression during germination and early seedling growth under salinity stress. Therefore, expression of such an isoperoxidases can be expected at least in part, to have some role in stress tolerance of fox-tail millet under NaCl stress. This can be further substantiated by the occurrence of a specific acidic isoperoxidase (A3p) only in the tolerant cultivar subjected to 150 mM NaCl stress. Though there are some reports of this kind [9], it is not clear to what extent isoperoxidases contribute to the overall tolerance to salinity in plant cells (particularly the acidic isoperoxidase confined to tolerant cultivar) and needs further study. Purified isoform A3p of tolerant cultivar (cv Prasad) was quite thermostable, and appear to

belong to the category of enzyme purified from the leaves of Haliminone portulacoides subjected to salinity [21]. In conclusion, exposure of cultivars to salinity resulted in changes in the induction of total peroxidase activity and its isozymes and such alterations in the induction and its isoform patterns vary between cultivars. The present data reveal that the relatively tolerant nature of cultivar Prasad could be due to induction of specific peroxidase isozyme (A3P), and that the two cultivars differed in their ability to respond to salinity by triggering these peroxidase gene expression.

Acknowledgements This research work was supported by Department of Science and Technology, New Delhi in the form of research grant (SP/SO/A-29/95-96) to C. Sudhakar.

References [1] E. Olmos, A. Piqueras, J.R. Martinez-Solano, E. Hellin, The subcellular localization of peroxidase and the implication of oxidative stress is hyperhydrated leaves of regenerated carnation plants, Plant Sci. 130 (1997) 97– 105. [2] M.A. Fieldes, K.E. Gerhardt, Flax guaiacol peroxidases can be used to illustrate the possibility of misinterpreting the effects of stress on the activity of developmentally regulated enzymes, Plant Sci. 132 (1998) 89 – 99. [3] B. Siegel, Plant peroxidases an organismic perspective, Plant Growth Regul.12 (1993) 303 – 312. [4] M.A. Sancho, S. Milrad de Forchetii, F. Pliego, V. Valpuesta, M.A. Quesada, Total peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells, Plant Cell Tiss. Org. Cult., 44 (1996) 161 – 167. [5] D.J. Bradley, P. Kjellbom, C.J. Lamb, Elicitor and wound induced oxidative cross-linking of a proline rich plant cell wall protein: A novel, rapid defense response, Cell, 70 (1992) 21 – 30. [6] Z. Chen, H. Silva, D.F. Klessig, Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid, Science 262, (1993) 1883 – 1886 [7] R. Hammerschmidt, E.M. Nuckles, J. Kuc, Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium, Physiol. Plant, 20 (1982) 73 – 82. [8] M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding, Anal. Biochem., 72 (1976) 248 – 254.

N. Sreeni6asulu et al. / Plant Science 141 (1999) 1–9

[9] R.L. Heath, L. Packer, Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fattyacid peroxidation, Arch. Biochem. Biophys. 125 (1968)189 – 198 [10] B.J. Davis, Disc electrophoresis-II method and application to human serum proteins, Ann. New York Acad. Sci., 121 (1964) 404–427. [11] J. Schrauwen, Nachweis Von Enzymen nach electrophoretischer Trennung an polyacrylamid sauren, J. Chromatogr. 23 (1966) 177–180. [12] J. Heukeshoven, R. Dernick, Simplified method for silver staining of proteins in polyacylamide gels and the mechanism of silver staining, Electrophoresis 6 (1985) 103 – 112. [13] V. Govindaswamy, R. Balasubramanian, Purification and properties of apoplastic chitenases from rust infected leaves of Arachis hypogea L., Bot. Helv. 104 (1994) 79–86. [14] S. Ramanjulu, K. Veeranjaneyulu, C. Sudhakar, Relative tolerance of certain mulberry (Morus alba L.) varieties to NaCl salinity, Serecolog. 34 (1994) 695–702. [15] T. Gaspar, C.L. Penel, T. Thorpe, H. Greppin, Peroxidases. A survey of their biochemical and physiological roles in higher plants, Universite de Geneve, Geneve, 1982, pp. 89–112.

.

9

[16] T. Gaspar, C. Penel, D. Hagage, H. Greppin, Peroxidases in plant growth, differentiation and developmental processes, in: J.H. Lobarzewsky, H. Greppin, C. Penel, T. Gaspar (eds.), Biochemical, Molecular, and Physiological aspects of Plant Peroxidases, University de Geneve, Geneve, 1991, pp. 249 – 280. [17] E.E. Elstner, Metabolism of activated oxygen species, in: D.D. Davies (eds.), The Biochemistry of Metabolism, Academic Press, San Diego, 1987, pp. 253 – 315. [18] R.S. Dhindsa, W. Matowe, Drought tolerance in two mosses correlated with enzymatic defense against lipid peroxidation, J. Exp. Bot. 32 (1981) 79 – 91. [19] K. Chaudhuri, M.A. Choudhuri, Effects of short-term NaCl salinity stress on free radical mediated membrane damage in two jute species, Indian J. Exp. Biol. 31 (1993) 327 – 331. [20] X.S. Ye, S.Q. Pan, J. Kuc, Activity, isozyme pattern, and cellular localization of peroxidase as related to systemic resistance to tobacco to blue mold (Peronospora tobaciana) and to tobacco mosaic virus, Phytopathology 80 (1990) 1295 – 1299. [21] A. Kalir, G. Omri, A. Poljakoff-Mayber, Peroxidase and catalase activity in leaves of Halimione portulacoides exposed to salinity, Physiol. Plant 62 (1984) 238–244.