Plant Molecular Biology (2005) 59:289–307 DOI 10.1007/s11103-005-8881-1


Springer 2005

The methylation cycle and its possible functions in barley endosperm development Volodymyr V. Radchuk, Nese Sreenivasulu, Ruslana I. Radchuk, Ulrich Wobus and Winfriede Weschke* Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung (IPK), Molecular Genetics, Corrensstrasse 3, Gatersleben, 06466, Saxoinia-Anhalt, Germany (*author for correspondence; e-mail [email protected]) Received 8 March 2005; accepted in revised form 16 June 2005

Key words: Barley, DNA methylation, endosperm development, genes of methylation cycle enzymes, methyltransferases

Abstract Barley endosperm development can be subdivided into the pre-storage, intermediate, storage and desiccation phase. Nothing is known about DNA methylation events involved in different endosperm-specific developmental programmes. A complete set of methylation cycle enzyme genes was identified and investigated by mRNA expression analysis. During the pre-storage phase, methionine synthase and S-adenosylmethionine (AdoMet) synthase genes are expressed at high levels, mainly to produce AdoMet, which might be used for methylation processes as indicated by high expression of methyltransferases HvMET1, HvCMT1 and HvDnmt3-1 as well as AdoHcy hydrolase genes. The methyltransferases, core histones and DNA-unwinding ATPases are co-expressed at the mRNA level. On the contrary, storage protein (prolamin) gene expression is repressed due to CpG methylation. Expression of genes responsible for starch biosynthesis is also developmentally regulated but not methylation-dependent. Thus, during pre-storage phase, activity of HvMET1 and HvCMT1 possibly maintains DNA replication and suppresses specific pathways of maturation. Besides, HvDnmt3-1 might be responsible for differentiation-specific de novo methylation. Expression of methyltransferases HvDnmt3-2 and HvCMT2 peaks during the onset of massive starch accumulation. The enzymes are likely responsible for DNA methylation involved in determining plastid division and amyloplast differentiation as concluded from the patterns of co-expressed genes. Levels of AdoMet decarboxylase mRNA, but not methyltransferase- and AdoHcy mRNA, increase at the beginning of desiccation together with methionine synthase and AdoMet synthase levels. This increase may be indicative for utilization of AdoMet in polyamine production protecting aleuron and embryo cell membranes during desiccation. Abbreviations: ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; AdoMetDC, S-adenosylmethionine decarboxylase; AdoHcy, S-adenosylhomocystein; AdoMet, S-adenosylmethionine; AGPase, ADP-glucose pyrophosphorylase; CMT, chromomethylase; DAF, days after flowering; EST, expressed sequenced tag; GBSS, granule bound starch synthase; SBE, starch branching enzyme

Introduction Synthesis and utilisation of methyl groups in plant cells is a cyclic process (Figure 1). Four main

enzymes are responsible for the reactions of the methylation cycle: methionine synthase (3), S-adenosyl methionine (AdoMet) synthase (4), S-adenosyl methionine-dependent methyl transfer-

290

Figure 1. Scheme of the methylation cycle in plants. Metabolites: ACC, 1-aminocyclopropane-1-carboxylic acid; AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; Hcy, S-homocysteine; Met, methionine; SMM, S-methylmethionine. Enzymes: (1) cystathionine c-synthase; (2) cystathionine b-lyase; (3) cobalamin-independent methionine synthase; (4) AdoMet synthase; (5) AdoMet-dependent transmethylase; (6) AdoHcy hydrolase, (7) AdoMet:methionine S-methyltransferase; (8) SMM:Hcy S-methyltransferase; (9) ACC synthase; (10) AdoMet decarboxylase.

ases (5) and S-adenosyl homocysteine (AdoHcy) hydrolase (6). The terminal step of methionine biosynthesis is catalysed by methionine synthase (Eckermann et al., 2000). Besides being a building block for protein synthesis, methionine occupies a central position in plant cell metabolism. Via AdoMet synthase (EC 2.5.1.6), about 80% of methionine converts into AdoMet. In plants, a small gene family consisting of two (A. thaliana; Peleman et al., 1989) or three expressed members (C. roseus, Schro¨der et al., 1997; L. esculentum, Espartero et al., 1994) encodes AdoMet synthase. Both isoforms of AdoMet synthase in Arabidopsis show similar expression patterns and are preferentially expressed in xylem and phloem of the stem and in the root cortex as well as in leaves and in callus tissues. AdoMet is the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene (Ravanel et al., 1998). About 1% of AdoMet serves the syntheses of the polyamines spermine and spermidine via the

AdoMet decarboxylase reaction (Chiang et al., 1996). Another small part of AdoMet is converted into ethylene after transcriptional activation of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase). However, the majority of AdoMet (90%) serves as methyl group donor mostly for transmethylation of DNA (Ravanel et al., 1998). The transmethylation reactions release S-adenosyl-L-homocysteine (AdoHcy). Since most methyltransferases bind AdoHcy with a higher affinity than AdoMet, AdoHcy is a potent inhibitor of most AdoMet-dependent transmethylations (Hoffman et al., 1979; Hermes et al., 2004). Therefore, a fast removal of AdoHcy is required for efficient methyltransferase reactions. AdoHcy is utilized by AdoHcy hydrolase (EC 3.3.1.1), an enzyme catalysing the reversible hydrolysis of AdoHcy to adenosine and Hcy (Ueland, 1982). DNA methylation is a eukaryotic gene silencing mechanism protecting the genomes by inactivating selfish DNA elements and regulating selected endogenous genes (Chan et al., 2005). During DNA methylation, methyl groups are introduced into the C5 position of cytosine residues by different types of AdoMet-dependent methyltransferases. All known C5 DNA methyltransferases are characterized by several conserved motives present in catalytic regions (Cheng, 1995, Chan et al., 2005). Based on sequence similarity and function, the plant DNA methyltransferases are grouped into four distinct classes, the MET1 class, the chromomethylases (CMT), plant DNMT2, and the mammalian Dnmt3/plant DRM (domains rearranged methylation) class including the maize-specific homolog Zmet3 (Cao et al., 2000). Plant DNA methylation occurs at CG, CNG and CHH (where H is A, C or T) sequences (Chan et al., 2005). The Arabidopsis/maize MET1 class of methyltransferases maintains the majority of CpG methylations as implicated by mutational analysis (Finnegan et al., 1996; Ronemus et al., 1996; Kishimoto et al., 2001). Chromomethylases are plant specific (Papa et al., 2001) and characterized by a chromodomain embedded between the catalytic motives I and IV (Henikoff and Comai, 1998; Rose et al., 1998). Arabidopsis CMT3 lossof-function mutants were isolated and analysed in three independent studies (McCallum et al., 2000; Lindroth et al., 2001; Bartee et al., 2001). They

291 show a genome-wide loss of CpNpG methylation and locus-specific effects on asymmetric methylation (Cao et al., 2003). By methylation profiling of Arabidopsis cmt3-2 and met mutants, Tompa et al. (2002) showed that CMT3 preferentially methylates transposons, a result confired and expanded to centromeric repeats by Cao and Jacobsen (2002a). Another class of plant methyltransferases contains catalytic domains similar to the mammalian Dnmt3 methyltransferases. Recombinant Dnmt3a and Dnmt3b enzymes from mouse displayed de novo activity when tested on unmethylated DNA templates in vitro (Okano et al., 1999). Therefore, DRM methylases are supposed to act as plant de novo methyltransferases. Experimentally, drm1 drm2 double mutants of Arabidopsis were shown to prevent the establishment but not the maintenance of transgene silencing (Cao and Jacobsen, 2002b). From a detailed analysis of methylation patterns found at the SUPERMAN locus in double (drm1 drm2) and triple (drm1 drm2 cmt3) mutants Cao and Jacobsen (2002a) concluded that DRM is responsible for de novo methylation and maintenance of asymmetric and CpNpG methylation. CMT3 has a function only in maintenance methylation of both, asymmetric and CpNpG sites, whereas MET1 has specificity for CpG maintenance methylation and influences CpNpG and asymmetric methylation indirectly (Cao et al., 2003). In transgenic plants, DNA methylation of promoter regions usually inhibits transcription, but methylation in coding sequences does not generally affect gene expression (Stam et al., 1998; Jones et al., 1999). In the Arabidopsis genes SUPERMAN and AGAMOUS, methylation of the coding parts causes transcriptional shut-down probably because of affecting important controlling elements in these regions (Sieburth and Meyerowitz, 1997; Ito et al., 2003). The FWA transcription factor gene, expressed only in the Arabidopsis endosperm, is silenced in all other tissues of the plant by methylation in the 5¢ end of the transcribed region (Chan et al., 2005). In the late barley endosperm, promoter regions of B-hordein genes are free of methylation, but completely methylated in leaves. On the contrary, the D-hordein promoter is unmethylated in both, endosperm and leaves (Sorensen et al., 1996). Barley seed development is divided into four stages (Wobus et al., 2004): the pre-storage phase

characterized mainly by cell division and elongation, the intermediate phase connecting cell division/elongation processes to the initiation of storage product accumulation (Sreenivasulu et al., 2004), the maturation phase associated with massive storage product accumulation and the desiccation phase. The intermediate phase is accompanied by massive transcriptional reprogramming. Stimulated by the high mRNA expression of methionine synthases during early grain development (Sreenivasulu et al., 2002), we started transcript analyses of the methylation cycle enzymes in developing barley grains. We have identified cDNAs of the methionine synthase, AdoMet synthase and AdoHcy hydrolase gene families as well as members of three classes of plant DNA methyltransferases expressed in barley seeds. Based on transcript analysis of the methylation cycle enzymes we assigned the expression of distinct isoforms of methyltransferases to the developmental stages of the barley endosperm. By expression analysis of the developing barley endosperm using a 12,000-unigene cDNA macro array filter, we were able to identify genes tightly co-regulated with methyltransferases and indicative of distinct developmental programmes. Genomic sequences associated with storage protein biosynthesis, but not those encoding enzymes of starch biosynthesis, show decreasing methylation and give rise to increasing mRNA amounts during endosperm development, indicative of transcriptional regulation via DNA methylation. Besides, we suggest different pathways for catabolizing AdoMet during early and late grain development.

Material and methods Plant material and tissue preparation Barley plants (Hordeum vulgare L. cv Barke, a two-rowed spring cultivar) were cultivated in a growth chamber at 19 C/16 C on a 16 h light/8 h dark cycle. Days after flowering (DAF) were determined and harvesting of seed material was done as described (Weschke et al., 2000). The developing seeds were harvested from the midregion of the ear at 2-day intervals from 0 till 26 DAF, and the endosperm was hand-dissected under a light microscope.

292 Sequence isolation and identification of methylation cycle genes

by Weschke et al. (2000) and given in relative units.

To identify genes encoding enzymes of the methylation cycle, sequence similarity searches were performed with publicly available sequences from monocot plants by BLASTN and ESTs with high percentages of identity were selected. To define gene family members, Arabidopsis and rice genome sequence annotations were used. The cDNA clones representing genes of the methylation cycle enzymes were selected, and the inserts were sequenced using the Megabase sequencer by using primers generated out of the flanking plasmid sequences as well as gene-specific primers (Metabion, Germany). Sequence data were processed using tools available at the Lasergene software (DNAstar, USA). The sequences were subjected to multiple alignments by using CLUSTAL W. The resulting alignments were visualized by using the BOXSHADE software.

Evaluation of expression profiles from macro array analysis

Northern blot analysis Total RNA was extracted from tissue samples using the Gentra RNA isolation kit (Biozym, Germany). Isolated RNA (10 lg lane)1) was fractionated on a 1% agarose/formaldehyde gel. Filters were hybridised as described (Weschke et al., 2000) using gene-specific probes. As probe for HvCMT1, a 617 bp DNA fragment was isolated from the EST HZ65G10 (http://pgrc.ipkgatersleben.de/est/index.php) by SalI/NotI endonuclease digestion. A BamHI/HincII fragment (441 bp) from EST HF13M10 was used as probe for HvCMT2. HvMET1 was probed with a 579 bp EcoRI/HincII fragment from EST HA18G06. A mix of two DNA fragments (399 and 375 bp) isolated after BamHI digestion of EST HF06I13 was used as probe for HvDnmt3-1, and a 948 bp fragment, resulting after BamHI digestion of HT01M2, was used as probe for HvDnmt3-2. After enzyme digestion, all fragments were separated on 0.8% agarose gels, separated from agarose by using the Nucleospin Extract kit (Macherey-Nagel, Germany) and labelled with 32 P-dCTP using the Rediprime kit (Amersham Biosciences, USA). For quantification, filters were hybridized with a 26S rDNA fragment for estimation of RNA loading (data not shown). The hybridization signals were quantified as described

A custom made high-density 12,000 macro array of cDNA sequences expressed in developing grains was used to study the temporal mRNA expression profiles of members of gene families of methionine synthase, AdoMet synthase and AdoHcy hydrolase during endosperm development. The cDNA macro arrays were hybridised with 33P-labelled second strand cDNA probes generated from mRNA populations of developing endosperm tissue (0–26 DAF in two-day intervals). Synthesis of 33 P-labelled probes, hybridisation and processing of cDNA arrays has been carried out as previously described by Sreenivasulu et al. (2002). The expression values were quantified by the Array Vision spot detection software package. Local background was subtracted from spot intensities and signal intensity of the duplicated spots was averaged for each cDNA fragment. The resulting data were normalized using the median centring algorithm. Based on the results of functional annotation, gene family members were identified (see Section 2 of material and methods) and the corresponding expression values were extracted. The normalized expression values were plotted on the y-axis, and the corresponding developmental stage of the endosperm (0–26 DAF) was specified on the x-axis. Further, we have used J-express software to cluster gene expression patterns of query genes with expression profiles showing strong correlation during endosperm development. Especially, we used the approach to identify those expression profiles tightly correlated with the expression of methyltransferase genes. The analysis of the complete developmental process (0–26 DAF in two-day intervals) was repeated twice with independently grown biological material; the presented profiles result from the mean value of the two normalized signal intensities measured for each time point during development. Southern blot analysis Genomic DNA was isolated from endosperm tissue samples using the DNAeasy plant mini kit (Qiagen, Germany). 10 lg DNA from each sample

293 was digested with either HpaII or MspI (Fermentas, Lithuania), separated on a 1.0% agarose gel and transferred to nylon membranes (Hybond NX, Amersham, USA). Probe preparation, labelling and hybridisation procedures were performed in the same way as described for northern blot analyses. Each analysis was performed twice using independently grown plant material.

Results cDNAs of major enzymes of the methylation cycle (methionine synthase, AdoMet synthase, methyltransferases, AdoHcy hydrolase) were identified, and their transcript levels analysed during development of the barley endosperm. We focussed our interest on the different subtypes of methyltransferases and their role in regulation of distinct developmental programmes. Small gene families encode the enzymes of the methylation cycle. Genes included in the study were selected based on a set of more than 180,000 ESTs resulting from random sequencing of 24 cDNA libraries constructed from 18 different barley tissues. Among them, 47,066 ESTs were generated from cDNA libraries representing different tissues of the developing barley grain (Zhang et al., 2004). To search in the whole barley EST set for cDNA sequences encoding enzymes of the methionine cycle, published amino acid sequences from monocot plants were used. Because methionine synthases, AdoMet synthases and AdoHcy hydrolases are expressed in most of the tissues at relatively high levels (Table 1) and consensus sequences are generated by aligning overlapping ESTs (Zhang et al., 2004), we were able to identify full-length cDNA sequences for all members of the respective gene families. Phylogenetic trees were generated to group the distinct cDNAs to homeologous sequences available from the EMBL database. Two highly similar genes, HvMS1 and HvMS2 (92.7% similarity to each other on protein level) (Table 1) encode methionine synthase of barley. Both cDNAs show high similarity (between 86.4% and 93.9%) to other cloned methionine synthases from plants. The motif WVNPDCGLKTR, which is present at the C-terminal part, is strictly

conserved and present in all cloned sequences of cobalamin-independet methionine synthases (comparison not shown). For AdoMet synthase in barley, four distinct sequences were identified. Two of them (HvAMS1and HvAMS2) differ only at three amino acid positions. Thus, differences at the expression level could not be detected (see below). HvAMS3 and especially HvAMS4 show lower degrees of similarity (96.5% and 89.9%, respectively, compared to HvAMS1) (Table 1, Figure 2A). Clustal W groups HvAMS4 together with an O. sativa sequence and close to sequences of Arabidopsis, Pisum, Brassica and Solanum, whereas HvAMS1, 2 and 3 form a separate cluster (Figure 2A). Two genes, HvAHH1 and HvAHH2, encode AdoHcy hydrolases in barley. They are highly similar to each other (97.7% identity) and to AdoHcy hydrolases from other plant species (Figure 2B). Three sub-classes of methyltransferases were identified. As shown by in silico expression analysis (Table 1), methyltransferases are expressed at much lower level than the other methylation cycle enzymes. The chromomethylase HvCMT1 and the HvDnmt3-2 methyltransferase are represented as singletons (Table 1). For HvMET1, HvCMT2 and HvDnmt3-1, several ESTs were identified, each expressed in another tissue. No cDNA clone contained the complete coding region of the respective gene. Therefore, functional domains were identified and used to group the sequences to the three different methyltransferase classes (Figure 3). The MET1-class of methyltransferases is represented by only one gene, HvMET1 (Figure 3A). The partial sequence, composed of four ESTs (Table 1), is highly similar to that of the MET1 genes of Z. mays (92.0%) and O. sativa (80.7%). The two putative chromomethylase genes HvCMT1 and HvCMT2 show similarity to the maize chromomethylases Zmet5 and Zmet2, respectively, but at relatively low level (69.4% and 55.1%, see Table 1). However, the chromodomain present defines the sequence designated HvCMT1 clearly as being a chromomethylase. The second putative chromomethylase (HvCMT2) is represented only by two short ESTs. Nevertheless, homology searches define the sequence clearly as encoding a chromomethylase (score 213 to Zmet2). Two putative members, HvDnmt3-1 and HvDnmt3-2, represent the Dnmt3-type gene family in barley. The two partial cDNA sequences,

1(1) 4(1), 5(1)

HvMET1 (partial)

DNA methyltransferase

2(1), 10(1), 17(1), 20(1)

HA18G06

HZ65G10 HF13M10

HY1DL08

HvAMS4 (full length)

HvCMT1 (partial) HvCMT2 (partial)

O. sativa (93.9%) HvAMS1 (89.9%)

HY05K15

HvAMS3 (full length)

Chromo methylase (CMT)

H. vulgare (96.5%) HvAMS1 (97.0%)

HZ47K19

HvAMS2 (full length)

Z. mays Zmet5 (69.4%) O. sativa (81.3%) Z. mays Zmet2 (55.1%) Z. mays Zmet1 (92.4%)

H. vulgare (99.0%) HvAMS1 (99.5%)

HZ46013

HvAMS1 (full length) H. vulgare (99.2%)

H. vulgare (92.7%) HvMSI (92.7%)

HY09B0S

HvMS2 (full length)

AdoMet synthase

H. vulgare (99.9%)

HY05K19

Highest similarity (%identity)

HvMS1 (full length)

Reference EST

Methionine synthase

EST abundance in barley tissues*

Members of gene family

Gene destination

AAK15805 XP476210 AAK11516 AACI6389

AAT94053

BAA09895

BAA09895

BAA09895

BAD34660

BAD34660

Acc. No

Table 1. Expression profiles of genes encoding methylation cycle enzymes in different barley tissues as estimated by in silico expression analysis.

Steward et al. (2000)

Papa et al. (2001) Papa et al. (2001)

Unpubl.

Unpubl.

Unpubl.

Reference

294

Unpubl. T. aestivum (96.7%) HvAHHI (97.7%) HY08B05

AAA34303

Unpubl. The Rice Full Length cDNA Consorium 2003 AAA34303 AK119539 T. aestivum (98.6%) O. sativa (95.7%) HY02112

AAF68437 AAF68437 Z. mays Zmet3 (76.0%) Z. mays Zmet3 (66.4%) HF06113 HT01M21

HvAHHI (full length) AdoHcy hydrolase

HvAHH2 (full length)

Dnmt3-1 (partial) Dnmt3-2 (partial) Dnmt3 class DNA methyltransferase

(Dnmt l/METI)

5(1), 1(1) 22(1)

O. sativa OsMETI (80.7%)

DAA01513

Teerawanichpan et al., 2004 Cao et al. (2000) Cao et al. (2000)

295

Figure 2. Phylogenetic trees of AdoMet synthase and AdoHcy hydrolase amino acid sequences of monocot and dicot plants. (A) Comparison of amino acid sequences of AdoMet synthases. The amino acid sequences of H. vulgare AdoMet synthases were aligned with the corresponding amino acid sequences of the following species: Arabidopsis thaliana I (Ac. nr. AT4g01850) and II (AT2g36880); Cataranthus roseus I (CAA958856), II (CAA958857), III (CAA958858); Medicago sativa (AAT40304); Oryza sativa (AAT94013); Pisum sativum I (BAC81655), II (CAA57580), III (S66352); Solanum brevidens (AAT47716). (B) Comparison of amino acid sequences of AdoHcy hydrolases. The amino acid sequences of H. vulgare AdoHcy hydrolases were aligned with the corresponding amino acid sequences of the following species: Arabidopsis thaliana (Ac. nr. AT3g23810), Cataranthus roseus (CAA81527), Lycopersicon esculentum (AAD50775), Medicago sativa (AAB41814), Medicago truncatula I (AAO89237), Medicago truncatula II (AAO89238), Nicotiana tabacum (P50248), Oryza sativa (AAO72664), Triticum aestivum (T06764).

defined as being Dnmt3-class DNA methyl transferases by their sub-domain structure (Figure 3B) show highest similarity to the Z. mays Dnmt3-type methyl transferase Zmet3 (76.0% and 66.4%, respectively).

296

Figure 3. (A) Comparison of the domain structure of maize Zmet1 (AAC16389) with the putative barley HvMET1, maize chromomethylase Zmet5 (AAK15805) with barley chromomethylases HvCMT1 and HvCMT2, and the maize Dnmt3-class methyltransferase Zmet3 (AAF68437) with barley HvDnmt3-1 and HvDnmt3-2. The bromo-adjacent homology domains (BAH), the chromodomains (CD), the conserved methyltransferase motives (I–X), and the ubiquitin-associated domains (UBA) are indicated by different shading. (B) Alignment of the conserved methyltransferase motives I–VI and IX–X of A. thaliana DRM2 (AAF66129) and Z. mays Zmet3 with those of HvDnmt3-1 and HvDnmt3-2. Dashes in the sequences represent gaps introduced by CLUSTALW to optimise the alignments. Identical residues are shown with a black background and similar residues with a grey background. Asterisks denote amino acids conserved between all assigned sequences.

297 In silico expression analysis of methionine synthases, AdoMet synthases and AdoHcy hydrolases The barley cDNA libraries used for EST generation are non-normalized. Therefore, the libraryspecific number of ESTs representing the same gene can be used as a measure for the expression level of this gene in the tissue the cDNA library was constructed from (in silico expression analysis). However, in silico analysis is possible only for highly expressed genes, i.e. in this study methionine synthases, AdoMet synthases and AdoHcy hydrolases. The different methlytransferase genes are excluded due to low abundance of their mRNA (see Table 1). Methionine synthase genes are preferentially expressed in seeds and roots. Whereas the mRNA levels of the methionine synthase genes are comparable in seeds, a remarkable difference exists in roots (Table 1). To check whether in silico analysis gives reliable results, abundance of the two isoforms in different barley tissues was analysed by Northern blotting. The analysis (data not shown) showed highest mRNA expression of the two genes in young internodes, a tissue not used for cDNA library production, but high expression also in roots with a 1:3 ratio of HvMS1 and HvMS2 expression as expected from the in silico analysis. A gene family consisting of four members encodes AdoMet synthases. Based on the total number of HvAMS-specific ESTs (1098), HvAMS3 is expressed about 30-fold higher than the other three AMS genes. In seeds, the overexpression level is reduced to about 14-fold. The highest transcript level of HvAMS3 was registered in germinating seeds. HvAMS1 and HvAMS2 are expressed to nearly the same level in roots and developing seeds, whereas a certain preference for developing seeds was found for HvAMS4. This isoform is expressed to nearly the same level in the aleuron/endosperm part of germinating seeds (see Table 1). Like methionine- and AdoMet synthases, AdoMet hydrolase HvAHH1 is expressed in nearly all tissues analysed and seems to fulfil house-keeping functions. HvAHH2 shows a certain preference of expression for seeds. Altogether, about 40% of all ESTs designated as AdoMet hydrolase-specific are detected in this organ.

The transcript levels of methyltransferases are high until the beginning of storage product accumulation, but low during later stages of endosperm development In the filial grain part, three different types of expression profiles of methyltransferases were found: (i) high expression during the pre-storage phase with a maximum at 2 DAF followed by very low expression during storage product accumulation (HvMET1; HvCMT1), (ii) high expression during both, early development and intermediate phase followed by decreasing mRNA expression during storage product accumulation (HvDnmt3-1) and (iii) high expression at the beginning of storage product accumulation (10 and 12 DAF; HvDnmt32 and HvCMT2, respectively), but low expression during pre-storage and later storage phase. Starting at 12 DAF (exponential phase of storage product accumulation), mRNA levels of all methyltransferase isoforms decline continuously (Figure 4B). mRNA abundances of methyltransferases and AdoMet-producing and -hydrolysing enzymes correlate during pre-storage and intermediate phase, but differ during storage product accumulation The mRNA expression profiles of HvMS and HvAMS genes were estimated by cDNA macro array analysis. Therefore, mRNA expression can be compared at the qualitative (mRNA profiles) as well as the quantitative level. Genes HvMS1 and HvMS2 are expressed in the endosperm with nearly identical profiles. However, the mRNA amount of HvMS1 is about 10-fold higher than that of HvMS2 (Figure 4B). Two maxima of expression are evident for both genes. The first peak correlates with the beginning of the intermediate phase (6 DAF; Sreenivasulu et al., 2004) and the second one with the end of grain filling (20 DAF; Weschke et al., 2000). The mRNA level of both genes is lowest 12 DAF. At least three members of the HvAMS gene family are expressed in the endosperm. Because of the high similarity between HvAMS1 and HvAMS2, they showed near identical profiles. Their summarized mRNA profile was designated HvAMS1/2 (Figure 4B). HvAMS3 shows the highest expression level in developing seeds (about 8-fold higher than HvAMS1/2 and 3-fold higher than HvAMS4). Similar to methionine synthase

298 b Figure 4. Transcript profiles of genes related to methylation processes in filial tissues of barley caryopses 0–26 DAF. The vertical grey shading indicates pre-storage (dark grey) and intermediate phase (light grey) of barley grain development, non-shading the storage/desiccation phase. (A) Transcript levels of the different subclasses of methyltransferases in barley seeds analysed by northern blot hybridisations. For quantification of the hybridisation signals see Material and methods section. (B) Normalized signal intensity representing mRNA expression levels of methionine synthase and AdoMet synthase genes as estimated by cDNA array expression analysis. Triangles in the HvAMS3 profile show mRNA levels of the AdoMet decarboxylase gene. For signal detection and quantification in expression analysis see Material and methods section. (C) Normalized signal intensities of the AdoHcy hydrolase genes estimated as described in (B).

genes, AdoMet synthase mRNA amounts are high during pre-storage and intermediate phase, low around 12 DAF and show a second peak 20 DAF. The mRNA profiles of the three gene family members show some similarity during grain filling, but differences are visible during early development. HvAMS1/2 mRNA level is highest during the pre-storage phase, that of HvAMS3 peaks during the intermediate phase. The level of HvAMS4 mRNA remains rather unchanged during early as well as later development. A remarkable similarity exists between the profiles of HvAMS and AdoMet decarboxylase mRNA (triangles in the HvAMS3 profile, Figure 4B) during the storage phase of grain development. In general, mRNA expression profiles of the HvMS and HvAMS genes correspond to those of HvMET1, HvCMT1 and HvDnmt3-1 during early development and intermediate phase (0–10 DAF) (cf. Figure 4A and B). However, the levels of HvCMT2 and HvDnmt3-2 mRNA are highest at 10/12 DAF when the amount of HvMS/HvAMS mRNA is lowest. Furthermore, the second increase of HvMS- and HvAMS mRNA abundance seen from 12 DAF onwards (see Figure 4B) is not found in any methyltransferase mRNA profile. The two isoforms of AdoHcy hydrolase show nearly identical mRNA levels in the filial part of developing grains as measured by expression analysis. Expression is highest 2 and 6 DAF and low at the beginning of storage product accumulation as well as during later development (Figure 4C). Gene expression of AdoHcy hydrolase corresponds to those of HvMET1, HvCMT1 and HvDnmt3-1 at 2 DAF and of HvDnmt3-1 at 6 DAF. However, no correlation exists between the

299 mRNA profiles of AdoHcy hydrolases and the mRNA expression of the methyltransferases HvCMT2 and HvDnmt3-2. During the pre-storage phase, methyltransferases HvMET1 and HvCMT1 are upregulated at the mRNA level together with chromatin-remodelling ATPases Using J-express, genes co-expressed with HvMET1/HvCMT1 during endosperm develop-

ment were identified. A tight correlation exists between the HvMET1/HvCMT1 profiles and those of 56 genes expressed during early development (Figure 5A and annex table 1). Forty genes are associated with the functional category DNA synthesis/chromatin structure (Figure 5A). More than half of these genes (21) encode the nucleosome core histones H2A, H2B, H3 and H4 (annex table 1). The cluster contains also genes involved in chromatin reassembly (see annex table 1), encoding for instance a histone deacetylase, a

Figure 5. Clusters of temporal expression profiles of genes co-expressed together with different methyltransferase genes. (A) Genes co-expressed together with the methyltransferases HvMET1 and HvCMT1. The continuous red line indicates the expression profile of the methyltransferase HvMET1, the dashed red line that of HvCMT2. Blue colour labels the expression profiles of the genes ISW1 (full line) and ISW2 (dashed line). (B) Genes co-expressed together with HvDnmt3-2. The red line indicates HvDnmt3-2 expression, blue lines label the expression profile of the plastid division genes FtsZ1 (full line) and FtsZ2 (dashed line). Expression profiles of genes related to starch metabolism are labelled in green (full line, sucrose synthase 2; dashed line, starch branching enzyme; dotted lines, two isoforms of the soluble starch synthase). Shadowing in yellow indicates the pre-storage phase, deep yellow the intermediate and light brown the storage phase of endosperm development. The grey-shadowed area includes expression profiles of all genes presented in annex table 1 [56 genes; grey shadowed area in (A)] and annex table 2 [42 genes; grey shadowed area in (B)]. The pie charts represent the percentages of genes out of the two clusters in (A) and (B) belonging to the functional classes indicated by the different colours.

300 SET-domain protein and two sequences showing similarity to ISW (Imitation SWitch) proteins (Figure 5A), described in S. cerevisiae as being the ATPase subunits ISW2 and ISW1, respectively, of two different chromatin-remodelling complexes (Tsukiyama et al., 1999; Mellor and Morillon, 2004). Besides of these two ATPase sequences, a third gene (cn9077, see annex table 1) was identified showing similarity (score 234) to Arabidopsis DECREASE IN METHYLATION 1 (DDM1), a gene encoding a protein related to a subunit of another chromatin remodelling complex of the SNF2 family (Lusser and Kadonaga, 2003). In vitro studies have shown that recombinant DDM1 has ATPase activity and is able to remodel nucleosomes (Brzeski and Jerzmanowski, 2003). During the intermediate phase, decreasing HvMET1 and HvCMT1 mRNA amounts correlate with decreasing CpG methylation and transcriptional activation of prolamin biosynthesis To examine whether expression profiles of methyltransferases coincide with changing patterns of DNA methylation, genomic DNA was isolated from barley endosperm at different developmental stages, digested by either one of the two isochizomers HpaII and MspI, sensitive or insensitive, respectively, against CpG methylation at the CCGG restriction site, and Southern blot analyses were performed using specific genes as labelled probes. Larger DNA fragments, indicating inhibition of HpaII digestion during the pre-storage phase were found for genes of the protein biosynthesis pathway, especially hordein B (Figure 6B) and the barley prolamin box-binding factor (BPBF) (Figure 6A), one of the two DOF transcription factors known to activate hordein B as well as trypsin-inhibitor BTI-CMe gene expression (Diaz et al., 2005). During the pre-storage phase, inhibition of HpaII digestion and no expression of the two genes (see profiles in Figure 6A and B) are inversely correlated with high expression of HvMET1 and HvCMT1 (Figure 4B). With proceeding endosperm development, methyltransferase expression decreases and correlates with changing HpaII restriction patterns and activation of hordein B and BPBF mRNA expression. Thus, the patterns presented in Figure 6A and B can be

explained by decreasing CpG methylation and, therefore, transcriptional activation of genes of the storage protein biosynthesis pathway during the intermediate phase. Unexpectedly, the genomic sequence encoding a putative member of the MYB family of transcription factors shows no changes of the HpaII patterns during endosperm development (Figure 6C). Members of the MYB family are known to be involved in the regulation of storage product accumulation in rice grains (Zhu et al., 2003). Furthermore, genes encoding key enzymes of the starch biosynthesis pathway as for instance the seed-specific small subunit of AGPase, granule bound starch synthase and soluble starch synthase 2 also show no changes in the HpaII pattern during endosperm development (data not shown), despite the fact that all those genes are transcriptionally upregulated during the intermediate phase (Sreenivasulu et al., 2004). Additionally, a-amylase inhibitor, an enzyme preventing accumulated starch from premature degradation is developmentally upregulated, but does not show any change in the CpG methylation pattern (Figure 6D). HvDnmt3-2 mRNA expression peaks at the beginning of storage product accumulation and correlates with the expression of plastid division proteins Expression profiles of HvDnmt3-2 during endosperm development cluster together with the profiles of 42 genes showing highest expression during the intermediate phase (Figure 5B and annex Table 1). 13 genes (31%) belong to the functional category ‘‘not assigned/unknown’’, followed by the category ‘‘starch metabolism’’ (8 genes, 19%), represented by genes encoding sucrose synthase 2, starch branching enzyme (SBE) as well as two isoforms of soluble starch synthase (Figure 5B). However, HvDnmt3-2 expression is most tightly correlated to those of two isoforms of the plastid division protein FtsZ. FtsZ proteins are described to be involved in plastid division processes of both, Arabidopsis (AtFtsZ1-1 and AtFtsZ2-1) and the moss Phycomitrella patens (Osteryoung et al., 1998; Strepp et al., 1998; McAndrew et al., 2001). Interestingly, a smaller peak was found within the HvDnmt3-2 cluster around 20 DAF, correlating with a small increase of expression of the two HvAHH isoforms (cf. Figures 5B and 4C).

301

Figure 6. Southern hybridisation analysis of the DNA methylation status during endosperm development, and mRNA expression profiles of the genes used for Southern blotting. Genomic DNA was digested with the restriction enzymes HpaII and MspI, sensitive and insensitive, respectively, to CpG methylation. The mRNA profiles result from cDNA macro array analysis. They are presented in relative units as percentages from their expression maximum. Restriction patterns and mRNA profiles originate from the following cDNA sequences: (A) DOF protein BPBF (Acc. Nr. BU976361); (B) B hordein (AL507502); (C) putative MYB factor (BU970890); (D) a-amylase inhibitor BDAI-1 (BU972477). DAF, days after flowering.

HvCMT2 expression peaks 48 h later than that of HvDnmt3-2 (Figure 4B). HvCMT2 is not part of a significant cluster of co-expressed genes.

Discussion The methylation cycle in plants provides methionine for protein synthesis and S-adenosyl-methionine for a variety of methylation reactions, which play key roles in the regulation of cellular and developmental processes. Methylation in developing seeds have been poorly analysed, and nothing is known about the function of distinct subtypes of methyltransferases for regulation of endosperm development. Here, we used a large barley EST collection (Zhang et al., 2004) to identify a complete set of methylation cycle enzyme genes and a 12k cDNA array of seed-expressed genes to assign

the expression of distinct isoforms of methyltransferases to the developmental stages of the barley endosperm. Genes co-expressed with distinct subtypes of methyltransferases were identified influencing chromatin assembly as well as cellular differentiation processes. Decreasing methyltransferase expression correlates with increasing expression of genes of the storage protein biosynthesis pathway indicating a regulatory function of methyltransferases in endosperm development. Expression of HvMET1, HvCMT1and HvDnmt3-1 peaks during cell division/differentiation and may serve endosperm-specific DNA methylation MET1 methyltransferases maintain the majority of CpG methylation during DNA replication (Finnegan et al., 1996; Ronemus et al., 1996; Kishimoto et al., 2001) whereas CpNpG and

302 asymmetric methylation is only indirectly influenced (Cao et al., 2003). Furthermore, the Arabidopsis chromomethylase CMT3 was shown to function in only maintenance methylation of both, asymmetric and CpNpG sites (Cao et al., 2003). HvMET1 is expressed during very early endosperm development with a maximum 2 DAF. A nearly identical profile of mRNA expression shows chromomethylase HvCMT1 (see Figures 4A and 5A). High mRNA levels at 0 and 2 DAF were also found for HvDnmt3-1, one of the two barley homologes of the Arabidopsis DRM genes. DRM genes in Arabidopsis were shown to be required for de novo methylation (Cao and Jacobsen, 2002b), but a role in maintenance of asymmetric and CpNpG methylation was also discussed (Cao et al., 2003). Cell division in the barley endosperm starts 2 DAF, presumably in the embryo-surrounding micropylar region of the endosperm (Engell, 1989). Under the growing conditions used for barley plants in this study, the endosperm coenocyt is visible 3 DAF. Differentiation processes determining the cells of the first alveolic endosperm cell row to become either transfer cells, aleuron or starchy endosperm (Olsen, 2001), start around 3 DAF. Endosperm cellularisation is completed 4 DAF (Weschke et al., 2003). Hence, in addition to intensive DNA replication and cell division, cellular differentiation processes characterize the early phase of endosperm development. We suggest that expression of HvMET1, HvCMT1 and HvDnmt31 serves CpG and CpNpG maintenance methylation as well as maintenance methylation of asymmetric loci, to guarantee intensive DNA replication and cell division and prevent premature differentiation. In addition, HvDnmt3-1 may function by de novo methylation to establish specific DNA methylation patterns, which allow differentiation into distinct endosperm tissues. Co-expression of methyltransferases and DNAunwinding ATPases suggests concerted action for DNA replication during early endosperm development Fifty-six genes are co-expressed with HvMET1 and HvCMT1 (Figure 5A and annex table 1). The cluster is characterized by a high number of sequences encoding histones H2A, H2B, H3 and H4B, which build up the octameric protein core of

the nucleosome (Tyler, 2002; Robinson and Schultz, 2003). Nucleosomes as the fundamental repetitive elements of chromatin are assembled in a DNA replication-coupled manner (Krude and Keller, 2001; Mello and Almouzni, 2001), but can also be assembled or reassembled independently of DNA replication. Figure 5A shows the expression profile of genes (HZ50L06 and HZ62K02) similar (score 234 and 177, respectively; see annex table 1) to two putative ATPases, ISW1 and ISW2, which are part of the Imitation SWitch subfamily of chromatin remodelling factors in S. cerevisiae. ISW1 is part of a foursubunit complex with nucleosome stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activity. ISW2 is a member of a two-subunit complex, also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activity but no detectable nucleosome disruption activity (Tsukiyama et al., 1999). Thus, the complexes weaken the interaction between DNA and histones to allow regulated access of protein factors to DNA sequences (Mellor and Morillon, 2004) but may also directly influence transcriptional regulation (Tsukiyama et al., 1999). The HvMET1/HvCMT1 cluster (Figure 5A) contains the sequence cn9077 (annex table 1) similar (score 249) to the Arabidopsis decrease in DNA methylation 1 (DDM1) gene. The gene encodes a protein related to the ATPase-subunit of a second type (SWI2/ SNF2-like) of chromatin remodelling complexes (Lusser and Kadonaga, 2003). Analysis of Arabidospis mutants showed that both MET1 and DDM1 are essential for CpG methylation (Saze et al., 2003; Kankel et al., 2003). Furthermore, a gene (cn1897) similar to histone deacetylase 6 (hda6) was identified. Like MET1 and DDM1, HDA6 is required for CpG methylation (Aufsatz et al., 2002). The presented data indicate, that HvMET1 and HvCMT1, chromatin remodelling complexes and histone deacetylases act together to establish and/ or maintain the silenced status of genomic loci during early endosperm development. Methyltransferase expression and CpG methylation correlate with suppressed gene expression of the prolamin-, but not the starch biosynthesis pathway Expression of the methyltransferase genes declines continuously from 2 DAF onwards (Figures 4A

303 and 5). Decline of methyltransferase mRNA amount is accompanied with decreasind CpG methylation of the genomic sequences encoding the barley prolamin box-binding factor BPBF and hordein B. In good accordance to the decreasing methylation, expression of the two genes increases (Figure 6A and B). However, DNA sequences of genes encoding key regulators of the starch biosynthesis pathway (endosperm specific small subunit of AGPase, GBSS and soluble starch synthase) do not undergo CpG methylation over all periods of endosperm development (data not shown). No changes in the CpG methylation patterns are visible also for a MYB factor and for a gene encoding one subunit of a dimeric a-amylase complex, despite of their increasing expression (see Figure 6C and D). From the presented data, we conclude that during early development, high methyltransferase expression leads to heavy CpG methylation of genes associated to prolamin biosynthesis preventing storage protein accumulation in non-differentiated tissues. Down-regulation of methyltransferase expression during later development is indicative for decreasing methylation and beginning expression of the regulatory gene BPBF followed by expression of the target gene hordein B. Regulation of expression of key genes of the starch biosynthesis pathway is largely independent of CpG methylation. HvDnmt3-2 and HvCMT2 may be responsible for DNA methylation processes, which parallel beginning starch accumulation HvDnmt3-2 and HvCMT2 mRNA expression peaks 10 and 12 DAF, respectively (Figure 4A), i.e. at the onset of massive starch accumulation in the starchy endosperm. This correlation is reflected by parallel mRNA expression of key enzymes involved in starch synthesis, as AGPase, GBSS and SBE. Initiation of storage product accumulation is accompanied by endoreduplication processes in storage organs of plant seeds, as shown for maize endosperm (Knowles and Phillips, 1985; Knowles et al., 1990; Engelen-Eigles et al., 2000), barley aleuron (Keown et al., 1977) and V. faba cotelydons (Borisjuk et al., 1995). During endoreduplicaton, cells amplify their genome without chromatin condensation, segregation or cytokinesis, i.e. endoreduplication can be described as an

‘alternative cell cycle’ (Larkins et al., 2001). Chromosomes of dividing cells normally replicate only once per cell cycle. Thus, different types of cell cycle regulation characterize dividing and endoreduplicating cells. Typically, cells that have undergone endoreduplication are bigger and have a larger number of organelles (Larkins et al., 2001). Both, endoreduplication and plastid division taking place in parallel in storage organs, require reprogramming of gene expression. Therefore, DNA de novo methylation can be expected. The tight correlation between expression of the de novo methyltransferase HvDnmt3-2 and the two genes similar to the key plastid division proteins FtsZ1 and FtsZ2 (Figure 5B) backs this conclusion. The plastid division proteins are homologs of the bacterial cell division protein FtsZ (Strepp et al., 1998), a tubulin-homolog (Erickson, 1997). Two distinct protein families, FtsZ1 and FtsZ2, are encoded in the nuclear genome of Arabidopsis. Both have been shown to be essential for chloroplast division (Stokes et al., 2000; McAndrew et al., 2001). Osteryoung and Nunnari (2003) developed a model for chloroplast division, which includes besides FtsZ proteins plant genomederived factors as ARC6 and ARTEMIS, and the MIN proteins derived from the bacterial ancestor like FtsZ. However, nothing is known about the gene products encoded in the nuclei to serve plastid division in differentiating storage organs. High expression of the two FtsZ-like proteins (Figure 5B) is a first hint to amyloplastspecific division processes. Methionine- and AdoMet synthase expression during late endosperm development is not related to methylation processes but may serve polyamine synthesis The levels of methionine and AdoMet synthase mRNA are high during pre-storage and intermediate phase of barley grain development and correlate during these developmental stages with the high expression of methyltransferase as well as AdoHcy hydrolase genes. Thus, AdoMet produced during early grain development is mainly consumed by DNA methylation processes. High amounts of methionine and AdoMet synthase protein seem to be typical for mitotically active tissues as concluded from a proteomics study of

304 germinating Arabidopsis seeds. Methionine and AdoMet synthase were identified as fundamental compounds controlling metabolism in the transition from a quiescent to a highly active state during seed germination (Gallardo et al., 2002). However, expression of methionine synthase and AdoMet synthase genes in barley grains peaks for a second time around 20 DAF (see Figure 4B) when expression of methyltransferase genes is low and desiccation becomes evident because of the decreasing grain fresh-weight (Weschke et al., 2000). Besides being the source for methylation in plant cells, AdoMet serves additional processes like ethylene and polyamine synthesis. Key enzymes for ethylene and polyamine production are ACC synthase and AdoMet decarboxylase (AdoMetDC), respectively. The AdoMetDC mRNA profile (triangles in the HvAMS3-profile of Figure 4B) correlates to those of methionine synthases as well as AdoMet synthases during storage phase and beginning desiccation, but not during early development (Figure 4B). Weak correlation was found also with the ACC mRNA profile (data not shown). Seeds accumulate polyamines like spermin and spermidine at relatively high levels (Lepri et al., 2001). Generally, polyamines are recognized to be important for an orderly pattern of growth and development in most organisms (Tabor and Tabor, 1984; Cohen, 1998). Polyamines interact with various macromolecules and membranes. They show specific and differential binding to phospholipids and affect membrane rigidity (Schuber, 1989). Spermine and spermidine are involved in a variety of stress responses in plants, especially in the defence against drought as shown recently by AdoMetDC over-expression in rice (Capell et al., 2004). Over-expression of an AdoMetDC cDNA of Datura stramonium in rice plants alone is sufficient to increase the level of spermine and spermidine in transgenic seeds without any manipulation of the two additional synthases also involved in their biosynthesis (Thu-Hang et al., 2002). Thus, the increased level of AdoMetDC mRNA at the beginning of desiccation in the mature barley grain might be indicative of an increase of spermine and spermidine production protecting membranes of the surviving aleuron as well as embryonic cells during desiccation. Another part of AdoMet is

possibly used for biosynthesis of ethylene, which is known to be a major regulator of maturation processes. Methionine and AdoMet synthesis in the barley endosperm is regulated at both, transcriptional and post-transcriptional levels Our conclusions are largely based on an analysis of mRNA levels as compared to developmental processes and assume that mRNA amounts roughly mirror enzyme activities. Some evidence exists that enzymes associated with methionine biosynthesis are transcriptionally regulated but additionally influenced at the protein level (Mathur et al., 1991, 1992; Schro¨der et al., 1997). However, no data are available for developing seeds where transcriptional regulation generally plays a major role (Thomas, 1993). Interestingly, the level of methionine synthase mRNA is reduced in barley suspension cultures co-cultivated with AdoMet (V. Radchuk, A. Tewes, unpublished results). However, a combined analysis of AdoMet synthase mRNA and protein levels as well as enzyme activity in Cantharanthus roseus suspension cultures under changing NaCl and sucrose concentrations showed that no simple correlation exists between transcript amounts, protein detected in immunoblots and enzyme activity in this system (Schro¨der et al., 1997). The authors state that ‘…transcriptional changes are only one aspect in the regulation of the enzyme activity.’

Acknowledgements We are grateful to Angela Stegmann, Gabi Einert and Elsa Fessel for excellent technical assistance. W. W. thanks Hans Weber for the idea to correlate methyltransferase expression to endopolyploidisation. This work was supported by a BMBF grant (GABI-SEED, FKZ 0312282).

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