Plant MolecularBiology11:203-214 (1988) © KluwerAcademicPublishers, Dordrecht - Printed in the Netherlands

203

DNA methylation and tissue-specific transcription of the storage protein genes of maize Michele W. Bianchi I and Angelo Viotti*

Istituto Biosintesi Vegetali, CNR, Via Bassini 15, 20133 Milano, Italy; 1present address: Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ UK (*author for correspondence) Received29 January 1988; acceptedin revisedform 26 April 1988

Key words: endosperm development, DNA methylation, storage protein genes, tissue-specific transcription, Zea mays

Abstract

We investigated the methylation state of a set of storage protein genes of maize, coding for zeins and glutelins, in different somatic tissues and in developing endosperms. These genes, present as multigene families in the maize genome and organized in clusters on different chromosomes, are coordinately and specifically transcribed only in endosperm cells. Southern blot analysis of DNA digested with methylation-sensitiverestriction enzymes shows a specific and extensive undermethylation of zein and glutelin sequences in the endosperm, while a common methylated pattern is detected in the different somatic tissues and in the embryo. However, a constant fraction of endosperm DNA ( - 35°7o) is methylated at all zein sequences, which are found to be heavily modified in pollen DNA as well. Undermethylation is extended along a zein duster and cannot be explained by reduced levels of 5-methylcytosine in endosperm DNA with respect to other tissues. The undermethylated state of storage protein genes is already established at an early stage of endosperm development, when transcripts levels for both genes are almost undetectable.

Introduction

The DNA of many organisms, from bacteria and unicellular eukaryotes to higher plants and vertebrates, contains modified bases produced postreplicatively by sequence-specific methylases [39]. Several functions of DNA modification, including restriction-modification systems, DNA repair and recombination have been clearly established in procaryotes [12, 34]. On the other hand, the selective pressures responsible for the presence of DNA methylation in eukaryotes are still not clear, especially since organisms such as Drosophila, nematodes or yeast are practically devoid of 5-methylcytosine

(5mC) [12, 40]. Transfection experiments in mammalian cells have shown that DNA methylation can inhibit transcription [12, 34], possibly through the assembling of methylated DNA in inactive chromatine structures [5, 23]. In vertebrates, transcription and methylation of tissue-specific genes are in general inversely correlated, but several exceptions do exist. DNA methylation has also recently been associated with parental genomic imprinting in mammals [35, 38, 45]. It is possible that the main functions of cytosine methylation in plants and animals are similar, but they are not necessarily identical, since animal methylases modify almost exclusively cytosine residues present at dinucleotide CpG, while

204 plant methylases also recognize the trinucleotide CpNpG [17] and are thus in part responsible for the high level of 5mC found in plants. For example, 26070 of all cytosines are methylated in maize, compared to 5-9070 in mammals [39]. At present, very few data are available on the distribution of 5mC in the genome of plants [9, 15], or on the methylation of plant genes in relation to their transcription [31, 48]. Studies on the T-DNA of crown gall tumors [20, 41] and on ribosomal genes [2, 49] indicate a correlation of transcription with undermethylation. DNA methylation is also associated with the inactive state of the maize transposable elements Ac [7] and Mu [6]. Information on the methylation of tissue-specific plant genes should contribute to the understanding of gene regulation in plants and the function of DNA methylation in higher eucaryotes. We studied the DNA modification of a set of storage protein genes in different tissues of maize, testing the methylation of cytosine residues present at the recognition sequences of methyl-sensitive restriction enzymes. These genes are organized in clusters on different chromosomes and constitute multigenic families whose transcription is tissue specifically activated during endosperm development. We report here evidence for an extensive and tissue-specific undermethylation in the endosperm tissue of zein genes belonging to two different subfamilies, and of a small family of glutelin genes. This work also provides the first conclusive evidence of a correlation between DNA undermethylation and transcription of tissue-specific genes in a higher plant.

Materials and methods

Plant material and sample collection All tissues were from the inbred line W64A. Plants were grown in the field or in the greenhouse during 1983, 1984 and 1985. Ears at 8 and 22 days after pollination (dap) were harvested, immediately frozen in liquid nitrogen and stored at -800C until use. Three-day etiolated seedlings were obtained by germinating mature kernels in sterile con-

ditions in moisted Petri dishes. Unfertilized ears (3-4 cm) harvested from field plants were frozen, lyophilized and then stored at - 20 ° C. Embryos and endosperms (22 dap) were obtained by hand dissection in a frozen state. Endosperms at 8 dap (2 mm long) were dissected by scalpel in a frozen state with the help of a bench lens. Mature pollen was harvested from field plants and stored at -80 °C.

Nucleic acid extraction and purification Maize DNA was extracted and purified from the various sources as previously described [43]. After the final purification step by equilibrium centrifugation in CsCI, DNA sizes ranged from 50 to 80 kb. DNA from mature pollen grains was extracted with a different procedure to avoid gelification of the homogenate in the presence of phenol or chloroform. Pollen grains were powdered in a mortar under liquid nitrogen for 40 minutes. In these conditions > 90°7o of the grains were disrupted as estimated under a light microscope. The powder was then gently homogenized in DNA extraction buffer (100 mM Tris, 150 mM EDTA, 0.5070 sodium laurosyl sarcosinate, pH 8.5). After two rounds of centrifugation at low speed, with re-extraction of the pellet in DNA extraction buffer, the combined supernatants were treated at 37°C with proteinase K for 1 h (0.2 mg/ml, final concentration). CsC1 and ethidium bromide were added to final concentrations of 0.95 and 0.025 mg/ml, respectively. After centrifugation at 10000 g for 30 min at 20°C in a swinging rotor, the upper pellicle and the amidaceous pellet were discarded. The DNA was then purified by equilibrium centrifugation in CsCI. Banded DNA was isopropanol-extracted to remove ethidium bromide, diluted 1:3 withTE (10 mM Tris, 1 mM EDTA, pH 7.6), ethanol-precipitated, and resuspended in TE. This solution was centrifuged at high speed to remove residual starch granules, extracted twice with saturated phenol-chloroform, once with saturated chloroform, reprecipitated with ethanol and resuspended in TE. DNA and RNA from 8 dap endosperms were purified from the same homogenate. The slurry was treated as previously described [46] for RNA preparations, with the following modifica-

205 tions: at the ethanol precipitation step DNA was spooled on a glass rod and more DNA was recovered by ethanol precipitation from the dialyzed supernatant. The pooled DNA solutions were further purified by equilibrium centrifugation in CsCI. Total RNA or poly(A) ÷ RNA were extracted and purified as previously described [46]. The purity and integrity o f the preparations were assessed by electrophoresis on 2°70 agarose gels. RNA was stored in 75°/0 ethanol or in TE at -80°C.

Restriction of DNA DNAs purified from the various sources were digested at 0.20/~g/t~l for 4 h, with a final 3-4-fold units excess of each restriction enzyme (5 for methylationsensitive enzymes), adding half of the units after 2 h. Glycerol concentration never exceeded 5°70 v/v and the other digestion conditions were those recommended by the suppliers (Bethesda Research Laboratories: Eco RII; New England Biolabs: Bst NI; Boehringer: all others). Each batch of purified DNA was tested for absence o f enzyme inhibitors by codigestion with lambda DNA. Completion o f digestion was confirmed by comparing hybridization patterns obtained with different reaction times and amounts of enzyme, or by rehybridizing the filters to probes which give already tested patterns.

Southern blot and hybridization Digested DNAs were fractionated by electrophoresis on agarose gels (0.8-1°70) in 50 mM Tris-acetate, 2 mM EDTA (pH 8) and then blotted onto nitrocellulose filters (Schleicher and Schuell). Filters were prehybridized for 3-6 h and hybridized at 42 °C in 50°/o formamide (v/v, deionized), 5 × SET (1 × SET is 150 mM NaCI, 2 mM EDTA, 30 mM Tris, pH 8), 5 × Denhardt's solution for 16-20 h. Washes were as follows: at room temperature for 15 min in 2 x SSC (1 × SSC is 150 mM NaCI, 150 mM sodium citrate), at 65 °C for 80 rain -2 h in 2 × SSC and then for 1 h in 1 × SSC for medium stringency or in 0.1 × SSC for high stringency conditions (only homologies > 95% are detected). Probes were labelled by nick

translation (spec. act. ca. 108 dpm/p.g) or by random priming (spec. act. ca. 7 x 108 dpm/tag) of DNA fragments purified from the appropriate plasmids by electrophoresis on low-melting agarose gel (Biorad) and phenol extraction of the agarose slice according to Higuchi et al. [21].

RNA transfer and hybridization Total RNAs or poly(A) + RNAs were ethanol precipitated, resuspended in TE, quantified spectrophotometrically, and the desired amounts brought to a volume of 25 #1 with sterile distilled water. 75/zl of formaldehyde and 2 0 x SSC (1:1, v/v) were added, and the solution was incubated at 65 °C for 15 minutes. The samples were applied in duplicate on nitrocellulose filters through a slotted template (for slot blots) following the supplier's instructions (Schleicher and Schuell). For quantitation, autoradiographs were scanned at 300 nm with a Camag TLC scanner II coupled to a Hitachi D-2000 chromator integrator.

HPLC analysis of the bases 10 #g samples of purified DNA were hydrolyzed in formic acid and prepared for analysis as described by Citti et al. [8]. Chromatography was performed isocratically on a Viosfer ods2 3/zm column (length 5 cm, internal diameter 4 mm) in 1 mM sodium hexane sulfonate (Fluka) at pH 3.8 [13]. Nucleobase standards were from Sigma.

Results

The approximately 100 zein sequences of maize can be subdivided into four to five subfamilies of high internal homology [19, 28, 36, 47] and are under the differential control o f several regulatory loci [I 1, 42]. A subfamily of 20 to 30 sequences, coding prevalently for light-chain zeins (23 kDa), is represented by gene E19, which is part of the genomic clone zE ([43]; Fig. 3e). In the characterization o f zE it was noticed that two restriction fragments generated by

206 methylation sensitive restriction enzymes were revealed by Southern hybridization in digests o f endosperm DNA, but not when the D N A was extracted from embryos or unfertilized ears. A different zein subfamily is represented by the c D N A clone pcM1 [44], which codes for a heavy chain zein (27 kDa), and is composed o f 15-20 sequences located on the long arm of chromosome 4 [46]. Mutations at the Opaque-2 locus severely reduce the levels o f M1 transcripts [25].

Zein and glutelin sequences are specifically undermethylated in the endosperm tissue Total D N A purified from different organs was digested with methyl-sensitive restriction enzymes and utilized in Southern blot hybridizations. Unless otherwise stated, embryos and endosperms were dissected from seeds collected 22 dap, a developmental stage at which storage proteins genes are actively transcribed in endosperm cells [3, 29]. The blot shown in Fig. la was hybridized to the 1059 bp fullsize insert of pcM1. The zein sequences homologous to M1 are clearly more susceptible to cleavage by the enzymes Sal I, H h a I and P v u II when the D N A is purified from the endosperm tissue (partial digestions are ruled out, see Materials and methods), irrespective of the presence of the opaque-2 mutation. H h a I, Sal I and P v u II do not cleave their recognition sequences G C G C , GTCGAC and CAGCTG, respectively, if the internal cytosines are methylated [30]. Barn H I recognize the sequence GGATCC and is inhibited by methylation of the internal cytosine as well, but this methylation is rare, since it is possible only when the restriction site is immediately followed by a G and only 50% of the external cytosines of C p C p G trinucleotides are methylated ([17]; see also Fig. 4a). In this work, no tissue-specific differences were detected in Barn H I hybridization patterns. Note, in Fig. la, the permanence of a high molecular weight smear in the H h a I and P v u II digests, and of a fraction of the B a m H I - B a m H I bands in the double digests Bam HI-Sai I, suggesting that not all endosperm D N A can be cleaved at M1 sequences by the methyl-sensitive enzymes. The same observation was made in digests of endosperm

Fig. 1. Undermethylation of glutelin and heavy-chain zein sequences in wild-type and opaque-2 endosperms. Southern blot of DNA purified from 22 days after pollination (dap) wild-type endosperms (es), 22 dap endosperms homozygous for the opaque-2 mutation (o2), 22 dap embryos (eb), 3 day shoots (sh) and unfertilized ears (ue). Restrictionenzymes:B, Barn HI; S, Sal I; P, Pvu II; H, Hha I. a: hybridization to the insert of the zein clone pcMl. b: same filter of panel a reprobed with the insert of the glutelin clone pcMY21. Fragment sizes are in kilobases, the positions of the MI homologousHha I fragmentsare also indicated in b.

D N A from a maize inbred line that has R F L P for M1 sequences with the line of this study (not shown). These results are also confirmed by the use of the isoschizomeres Eco RII and Bst NI, as shown in Fig. 4b. The H h a I and P v u II hybridization patterns of shoot, embryo and unfertilized ear DNAs (see also Fig. 5) are identical, suggesting that zein sequences bear the same modification pattern in the

207 different somatic tissues. A similar picture is revealed by reprobing the same blot with the insert of the cDNA clone pMY21 (Fig. lb), which is highly homologous (not shown) to a published 16 kDa glutelin-2 cDNA [33]. Interestingly, some of the H h a I sites located in the coding region are undermethylated in the endosperm and in the somatic tissues as well, giving rise to a band of ca. 300 bp (Fig. lb), while other restriction sites are undermethylated only in the endosperm tissue, giving rise to an additional intense H h a I fragment in the digests of endosperm DNA. (The hybridization signal corresponding to this intense band is present in the embryo and shoot digests as a high molecular weight smear hardly visible in the photograph.) To confirm these data and obtain more detailed information on the characteristics of the tissuespecific undermethylation of storage protein genes, we also analyzed the methylation state of the zein cluster zE/19.31, represented by the genomic clones

zE and 19.31. As shown in Fig. 3e, clone 19.31 comprises zE and extends in both directions, including portions of other two light-chain zein genes. There are four to six highly homologous copies of the 4.1 kb B a m H I - B a m HI fragment of zE per haploid genome [43], located on the short arm of chromosome 7 (A. Viotti and N. Pogna, unpublished), while, as we mentioned in the introduction, the zein genes present in cluster zE/19.31 have a much higher copy number. This makes identification of the methylation state of specific sites difficult, but it was possible to obtain considerable information by combining the use of the whole clone 19.31 as probe with the use of particular fragments which recognize the cluster more selectively. The result of the hybridization of the whole clone 19.31, is shown in Fig. 2. The enzyme Barn HI produces the same pattern irrespective of the source of the DNA. The four Bam HIBarn HI bands of clone 19.31 (arrows) are present together with several other bands containing homolo-

Fig. 2. Hybridization of clone 19.31 to DNAs from different tissues, after digestion with methyl-sensitive restriction enzymes. Filters were hybridized with an equimolar mixture of fragments spanning the whole cluster, excluding the Barn HI-Xba I fragment that contains a repeated sequence (scheme e, Fig. 3). Left panel: Hha I digests of endosperm DNA (es, 7 vg) and 19.31 DNA (~, 100 pg); the numbers assigned to the Hha I bands are those o f the sites generating the right end of each fragment according to scheme e of Fig. 3. One of the two expected external fragments of 19.31 is the unnumbered band below band 7; the other comigrates with band 10. Band 9 is visible with higher loads o f 19.31 DNA. Right panel: DNAs from different organs and from endosperms 22 days after pollination were restricted as indicated at the bottom. In the unfertilized ear series (ue), the Barn HI digest was not included in this blot. The patterns of ue digests differ only apparently from those of shoot or root DNA because o f higher loads of ue DNA; in a shorter exposure no qualitative differences are detectable. On the right, arrows mark the positions o f the Barn HI-Barn HI fragments of cluster zE/19.31. Abbreviations of restriction enzymes as in Fig. 1.

208 gous zein genes. The enzymes Hha I and Sal I cleave much more frequently the sequences homologous to 19.31 when the DNA is extracted from endosperms; this qualitative difference remains also when the comparison is made with overloaded digests of unfertilized ear DNA. As for M1 sequences, also in this case the same pattern is present in the somatic tissues (see figure legend). Several bands generated by Sal I and/or Hha I in endosperm DNA correspond in size to fragments predictable from the restriction map of 19.31. This is particularly clear when the genomic digests are directly compared with digests of 19.31 DNA (Fig. 2, shown only for Hha I digests), which has lost methylation at Hha I and Sal I sites during propagation in Escherichia coli. To obtain selective information on the methylation of the zE/19.31 cluster, we utilized the fragments defined in Fig. 3e as probes 1, 2, 3 and 4. The results, considered together with the data from the blot shown in Fig. 2, are summarized in Fig. 3e. In this analysis we did not use the isoschizomeres Eco RII/Bst NI because of the paucity of restriction sites for these enzymes in clone 19.31. Therefore, in some cases it is not possible to distinguish with certainty between restriction site polymorphism among copies of the zE/19.31 cluser and heterogeneity of methylation, since methyl-insensitive isoschizomeres of Hha I and Sal I are not available. Probe 1 also recognizes sequences located outside the cluster, since it also hybridizes to other Barn HIBam HI fragments besides the 3.15 kb fragment of zE/19.31 (Fig. 3a), while probes 3 and 4, and probe 2 in particular, are more specific for the expected Bam HI-Barn HI fragments of 4.5 and 4.15 kb, respectively (panels c, d and b). The 4.5 kb Barn HIBam HI fragment also has a copy number of 4-6, as estimated from reconstruction hybridizations (Xba I digests of panels c and d). Probes 2 and 3 also recognize uniquely an expected Eco RI-Eco RI fragment of approximately 9 kb (not shown, see panel e). Blots a, b, c and d show that: i) sites H2-Hll, $2 and $3 are completely methylated in somatic organs, with the possible exception of site $2, which could be partially undermethylated; ii) these sites, with the exception of H9, are, to different extents, undermethylated in the endosperm tissue; iii) a constant fraction of endosperm DNA is totally methylated at

zE/19.31 sequences, as already established for M1 sequences (in all cases approximately 35% of total endosperm DNA; a possible interpretation will be discussed). Undermethylation of sites H1 and S1 in the endosperm is suggested by the blot reported in Fig. 2. We report only some main observations concerning these blots. In the blot shown in a, all the bands expected from cleavage of zE/19.31 at site H2, $2, $3 and H3 and (panel e) are detectable in the appropriate digests of endosperm DNA. The fragments expected from cleavage at site H2 have lower intensities than those expected from cleavage at site $2 (1.5 and 1.8 kb bands compared with 0.8 and 1.9 kb bands). These differences can be explained by polymorphism among the zein sequences hybridizing to probe 1, but weaker undermethylation at the H2 site compared with site $2 cannot be excluded. Site H2 is completely methylated in root DNA (compare Bam HI and Bam H I + H h a I digests), while a fraction of the Bam HI-Sal I band generated by cleavage at $2 is also present in root DNA. Identical results are obtained with embryo DNA (not shown). Blot b concerns sites $3 and H4, H5 and H6. These sites are completely undermethylated in the endosperm, excluding the constantly methylated fraction of endosperm DNA, since only bands corresponding to total digestion are detected. Blot c and d refer to sites H7-Hll (note that the distance from H7 to H8 and H10 to Hll is the same: 1150 basepairs). Each of these sites is completely methylated in roots (and in embryos as well, not shown). Methylation at these sites is certainly heterogeneous in the endosperm tissue, since polymorphism alone among the four to six copies of the cluster cannot account for all the bands detected in the Hha I and Hha I+Bam HI digests of endosperm DNA.

Endosperm DATA is not undermethylated compared with DNAs from other tissues

The undermethylation of zein and glutelin genes in the endosperm would be simply explained by a general lack of 5mC in the DNA of this tissue. This possibility is ruled out by the following considerations: i) there are no differences among the ethidium

209

Fig. 3. Undermethylation of the Hha I and Sal I sites of cluster zE/19.31 in endosperm tissue, a, b, c, d: Southern blots of DNA purified from 22 dap (days after pollination) endosperms, 22 dap embryos and 3 day roots (rt), restricted as indicated and hybridized respectively to probes 1, 2, 3 and 4, as defined in scheme e. X, Xba I; R, Eco RI; h, 19.31 DNA; other abbreviations as in Fig. 1. In panels c and d, the Xba I digestions refer to endosperm DNA (6 tzg) and to DNA of clone 19.31 (24 pg, corresponding to 5 copies/haploid genome). Scheme e: map of relevant restriction sites of clones zE/19.31 and general representation of the modification state of the Hha I and Sal I sites in different tissues. Circles: methylation states deduced from the hybridization to probes 1-4, as in blots a-d and in other Southerns not shown. Diamonds: methylation states deduced only from hybridization to the whole cluster as reported in Fig. 2. Empty and closed simbols indicate undermethylated and totally methylated sites respectively; partially filled symbols indicate heterogeneity of methylation and/or restriction site polymorphism. Dark bars: probes, open boxes: transcribed sequences; wavy line: region containing a repetitive sequence. Arrows in the open boxes indicate direction of transcription. The El9 zein gene is indicated.

210 bromide-stained digests of DNA from different tissues, when digested with methylation-sensitive restriction enzymes; ii) the transcribed region of the large rDNA unit is apparently methylated to a very similar extent in root and endosperm DNAs (Fig. 4a); iii) we determined, by HPLC analysis of the DNA bases, that the level of total 5mC in endosperm DNA does not differ significantly from that of unfertilized ear DNA (data not shown).

Undermethylation of zein and glutelinn sequences correlates with tissue-specific transcription

Zein transcripts account for 15-20°70 of the mRNA population of developing (20 to 30 dap) endosperm cells [29], are undetectable in other maize tissues (Fig. 5 and 6b; [3]), and are present to some extent in embryo RNA preparations. Experiments of runoff transcription with nuclei isolated from three-day shoots and roots demonstrated that zein tissuespecificity is regulated at the transcriptional level [3]. My21 transcripts are likely to have the same tissue specificity of zein genes, as indicated by dot blot analysis of total RNAs (Fig. 5). The levels of zein and glutelin transcripts found in embryos RNA preparations are probably due to contamination from surrounding endosperm tissue (difficult to avoid), since zein mRNAs are not detected in embryos by in situ hybridization [37] and zein peptides are not found in embryos by cyto-immunological methods [10].

Early developmental establishment of the undermethylated state At eight days after pollination, endosperm cells are apparently still undifferentiated and rapidly divid-

Fig. 4. Modification state of the transcribed region of the large rDNA unit and of zein sequences in endosperm and root DNA. DNA purified from 22 dap (days after pollination) endosperms and 3 day roots (es and rt, in alternate loads) was digested as indicated. Ms: Msp I, does not cut its recognition sequence CCGG when the first cytosine is methylated; Hp: Hpa II; isoschizomere of Msp I, cuts only if both cytosines are demethylated; Bs: Bstn I, recognizes CCAGG and CCTGG and is not sensitive to methylation; Ec: Eco RII, isoschizomere of Bstn I, does not cut when the internal cytosine is methylated; Hh: Hha I; Pv: Pvu II, a: hybridization to a fragment of the transcribed region of the maize 17s rDNA unit. b: same filter of a, reprobed to the insert of pcM1.

Fig. 5. Dot blot analysis of total RNAs from different tissues. The filter was probed with the insert of clone pcMY21. Numbers indicate/zg of poly(A) ÷ RNA purified from 3 days shoots (sh) and roots (rt), or of total RNA from 22 dap (days after pollination) endosperms (es) and embryos (eb). The insert of pcM1 and the Sal I-Sal I fragment of cluster zE containing gene El9 (see Fig. 3e) gave identical results.

211

Fig. 6. Modification state and relative transcript levels of zein genes at an early stage of endosperm development A: restricted DNAs

from endosperms at 8 (e8) and 22 (e22) days after pollination (dap), and from unfertilized ears (ue), probed with the insert of pcMl. Bands sizes as in Fig. 1. R, Eco RI; H, Hha I; P, Pvu II. B: slot blot analysis of total RNAs isolated from endosperms at 8 and 22 dap. r: total RNA from rat liver; rt: poly(A)+ RNA from 3 day roots. Panel a' represents a longer exposure of a. Panel b: control of RNA loading by hybridization with the flax rDNA insert of pBG35 [16]. Hybridization with the Sal I-Sal I fragment of cluster zE containing gene El9 (Fig. 3e) and with the insert of pcMI'21 gave similar results.

ing [24]. At this early stage o f development, the levels o f zein a n d glutelin transcripts are at least 900-1100 less t h a n those f o u n d in 22 d a p e n d o s p e r m s R N A (Fig. 6b). Nevertheless, the m e t h y l a t i o n p a t t e r n s o f zein a n d glutelin sequences in 8 d a p e n d o s p e r m s are n o t distinguishable from those f o u n d at 22 d a p (Fig. 6a). M1 sequences also a p p e a r to be heavily m o d i f i e d in the male g e r m i n a l line, since the H h a I sites present in these sequences are extensively methylated in m a t u r e pollen D N A (Fig. 7).

Fig. 7. Hybridization of the insert of pcM1 to Hha I digests of

DNA isolated from mature pollen cells (pl, 0.8/~g). Double loads (1 and 6 t~g) of Itha I digested DNA from unfertilized ears (ue) or 22 dap (days after pollination) endosperms (es) are included for comparison and control of detectability.

Discussion We utilized restriction enzymes that are i n h i b i t e d by the presence o f 5 m C residues in their r e c o g n i t i o n se-

212 quences and interpreted differences in the products of their digestion in terms of the presence or absence of methylated cytosines. Our confidence in these conclusions is based on the following considerations: i) completeness and reproducibility of digestions were assured as described in Materials and methods; ii) no differences were ever observed among the hybridization patterns of the DNAs of the various sources after restriction with methylinsensitive enzymes; iii) when possible, results were confirmed using isoschizomeres differing in their sensitivity to 5-methyl cytosine; iv) the only stable modified bases detected up to now in plant DNA are 5mC and, in much lower amounts, 4mC and 6mA. Moreover, treatment of maize seedling with the inhibitor of cytosine methylation 5' -azacytidine leads to demethylation of total seedling DNA and to a shift in the hybridization patterns of zein sequences after restriction with methyl-sensitive enzymes, which then become similar to those normally found in endosperm DNA (M. W. Bianchi, unpublished data). In general, zein and glutelin sequences were found to have two possible methylation states: a heavily methylated one, detected in all somatic tissues investigated, and an extensively undermethylated one, specific to endosperm cells. We tried to minimize the intrinsic limitations of the use of restriction enzymes by analyzing several storage protein genes: this allowed us to complement detailed information concerning the zE/19.31 zein cluster with more general data on relatively large groups of sequences of different chromosomal position. Therefore, chromosomal location effects for the extensive undermethylation of cluster zE/19.31 are unlikely. Undermethylation does not seem to be restricted to particular gene regions, e.g. transcribed regions, canonical regulatory regions [19], or binding sites of trans-acting factors [26], since Hha I and Sal I sites scattered along the cluster are undermethylated in endosperm DNA. On the other hand, some degree of site specificity is suggested especially by the data on glutelin sequences, which are undermethylated at several Hha I and Pvu II sites in all the tissues investigated, but show demethylation at additional sites in the endosperm (Fig lb). Since zein sequences are heavily methylated in mature pollen cells and in all sporophytic tissues inves-

tigated, it is most likely that the undermethylated state of storage protein genes is acquired during endosperm development. Two questions become evident at this point: how is the loss of the methyl groups temporally related to the developmentally regulated transcription of these genes? How are both transcription and undermethylation related to the massive DNA amplification which, from 10-12 to 14-18 days after pollination [24] accompanies, in the bulk of endosperm, the end of the cell divisions and differentiation of the tissue? In endosperms dissected at 8 days after pollination, the transcript levels of these genes are barely detectable, while the undermethylated state is already established. Therefore, undermethylation of zein and glutelin sequences is likely to be a very early step in their activation, perhaps reflecting changes in chromatine conformation [1, 3, 23], rather than being a consequence of active transcription, which could also depend on the binding of trans-acting factors [26]. The lack of effect of the opaque-2 mutation on the methylation of heavy-chain chain zein genes is in accordance with this idea. It should be possible to determine if the methylation of storage protein genes is responsible for their total transcriptional repression in tissues other than the endosperm, utilizinng transgenic plants transformed with in vitro methylated constructs. The undermethylated state of these genes is also a potentially useful molecular marker of very early steps of endosperm development, which could, for example, be used to screen defective kernel mutants in search of mutation that interfere with early differentiation events. There is also an alternative hypothesis to that of a post-fertilization undermethylation of storage protein genes: undermethylation could occur before fertilization in the two maternal polar nuclei, while the pollen genome would remain methylated and therefore account for most of the fraction of endosperm DNA ( - 35%) which is methylated at zein and glutelin sequences. This hypothesis, which has striking analogies with what has been inferred from genetic data on the maternal derepression of the R locus of maize [22], is being tested. The RFLPs that these sequences show among different maize inbred lines permits to easily distinguish between the mater-

213 nal and paternal nuclear genomes in hybrid endosperms. This work shows that in a higher plant, as in vertebrates, undermethylation of tissue-specific genes correlates with their transcription. It remains to be established whether this fact reflects a convergent evolution of similar mechanisms which help control gene expression, or is a consequence of the maintenance of DNA methylating functions under other selective pressures, such as post-replicative mismatch repair [18], parental genomic imprinting [38, 45] and, for plants only, transposon inactivation.

Acknowledgements We are grateful to Professor B. A. Larkins and to Dr N. Di Fonzo for providing us with the X-19.31 clone. This work was supported in part by grants from Consiglio Nazionale delle Ricerche, Progetto Strategico Agrotecnologie.

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