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Plant Cell. Oct 2010; 22(10): 3232–3248.
Published online Oct 29, 2010. doi:  10.1105/tpc.110.079962
PMCID: PMC2990135

Arabidopsis SET DOMAIN GROUP2 Is Required for H3K4 Trimethylation and Is Crucial for Both Sporophyte and Gametophyte Development[C][W]


Histone H3 lysine 4 trimethylation (H3K4me3) is abundant in euchromatin and is in general associated with transcriptional activation in eukaryotes. Although some Arabidopsis thaliana SET DOMAIN GROUP (SDG) genes have been previously shown to be involved in H3K4 methylation, they are unlikely to be responsible for global genome-wide deposition of H3K4me3. Most strikingly, sparse knowledge is currently available about the role of histone methylation in gametophyte development. In this study, we show that the previously uncharacterized SDG2 is required for global H3K4me3 deposition and its loss of function causes wide-ranging defects in both sporophyte and gametophyte development. Transcriptome analyses of young flower buds have identified 452 genes downregulated by more than twofold in the sdg2-1 mutant; among them, 11 genes, including SPOROCYTELESS/NOZZLE (SPL/NZZ) and MALE STERILITY1 (MS1), have been previously shown to be essential for male and/or female gametophyte development. We show that both SPL/NZZ and MS1 contain bivalent chromatin domains enriched simultaneously with the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3 and that SDG2 is specifically required for the H3K4me3 deposition. Our data suggest that SDG2-mediated H3K4me3 deposition poises SPL/NZZ and MS1 for transcriptional activation, forming a key regulatory mechanism in the gene networks responsible for gametophyte development.


Histone methylation is one type of the epigenetic marks that play essential regulatory functions in the organization of chromatin structure and genome function (Yu et al., 2009; Liu et al., 2010). In general, active transcription depending on a permissive chromatin structure is associated with histone H3 lysine 4 (H3K4) and/or H3K36 methylation, whereas transcriptional repression is associated with H3K9 and/or H3K27 methylation. Enzymes catalyzing histone Lys methylation contain an evolutionarily conserved SET domain (Tschiersch et al., 1994), named after three proteins initially identified in Drosophila melanogaster: SuVar(3–9), E(z), and Trithorax. The Arabidopsis thaliana genome contains 47 SET DOMAIN GROUP (SDG) genes: SDG1 to SDG47 (http://www.chromdb.org), which could be classified into several distinct phylogenetic groups (Baumbusch et al., 2001; Springer et al., 2003; Zhao and Shen, 2004; Ng et al., 2007). So far, only some SDG genes have been investigated for their roles in plant growth and development (reviewed in Yu et al., 2009; Liu et al., 2010); the biological functions of a larger number of SDG genes remain unknown.

In Drosophila, Trithorax group (TrxG) SET domain proteins mediate H3K4 and/or H3K36 methylation and counteract transcriptional repression by Polycomb group (PcG)–mediated H3K27 methylation to sustain active expression of developmental regulatory genes (Schuettengruber et al., 2007). The TrxG family in Arabidopsis comprises 12 SDG genes, six of which have been assigned a biological function to date. Five of the six characterized genes, SDG8/ASHH2/EFS/CCR1, SDG25/ATXR7, SDG26/ASHH1, SDG27/ATX1, and SDG30/ATX2, are involved in flowering time regulation (Soppe et al., 1999; Kim et al., 2005; Zhao et al., 2005; Pien et al., 2008; Saleh et al., 2008; Xu et al., 2008; Berr et al., 2009; Tamada et al., 2009), whereas SDG4/ASHR3 is involved in late stages of pollen development (Cartagena et al., 2008; Thorstensen et al., 2008). In addition, ATX1 involved in H3K4 trimethylation (H3K4me3) is necessary for normal root, leaf, and floral organ growth (Alvarez-Venegas et al., 2003; Alvarez-Venegas and Avramova, 2005); and SDG8/ASHH2/EFS/CCR1, primarily involved in H3K36me2 and H3K36me3, is implicated in the regulation of organ size, shoot branching, fertility, and carotenoid composition (Soppe et al., 1999; Xu et al., 2008; Cazzonelli et al., 2009; Grini et al., 2009). In this study, we show that the previously uncharacterized TrxG family gene SDG2 (also named ATXR3) plays crucial roles in both sporophyte and gametophyte development.

As in other angiosperms, the Arabidopsis life cycle alternates between a prominent diploid sporophytic generation and a much-reduced haploid gametophytic generation. The gametophytic generation occurs late in development within sporophytic tissues of specialized floral organs. Female gametophytes, or megagametophytes, develop in ovules within the gynoecium of the flower (reviewed in Yang et al., 2010). A single megaspore mother cell (megasporocyte) differentiates from the subepidermal cell layer at the tip of each ovule primordium and undergoes meiosis to produce a tetrad of four haploid spores. Three of the spores degenerate, and one proceeds through three sequential rounds of mitotic division, forming the female gametophyte, the embryo sac, which at maturation consists of seven cells with four cell types (three antipodal cells, two synergid cells, one egg cell, and one two-haploid-fused diploid central cell). The male gametophytes, or microgametophytes, develop within the anthers of the flower (reviewed in Ma, 2005). Microsporocytes differentiate from the primary sporogenous tissue surrounded by the tapetum and undergo meiosis to form a tetrad of four haploid microspores. Each microspore undergoes one cycle of nuclear division, forming a generative cell and a vegetative cell. The generative cell undergoes one more round of mitosis to produce two sperm cells. At maturation, the male gametophyte, the pollen grain, is thus composed of a three-celled male germ unit. Therefore, both female and male gametophyte development consist of two phases: sporogenesis, which starts from reproductive organ differentiation and ends after meiosis by haploid spore formation; and gametogenesis, which consists of haploid cell activities leading to the formation of mature (functional) gametes. The highly coordinated processes of cell division, differentiation, and expansion that take place during female and male gametophyte development require precise fine-tuning of gene regulatory networks.

Transcriptome analyses of male and female gametophytes have provided lists of thousands of differentially expressed genes (Borges et al., 2008; Wuest et al., 2010). In comparison, fewer genes have been functionally characterized in male and/or female gametophyte development (reviewed in Wilson and Zhang, 2009; Yang et al., 2010). The MADS box transcription factor gene AGAMOUS (AG) specifies reproductive organ identity in flowers (Bowman et al., 1989) and acts in a negative feedback loop to terminate stem cell proliferation in the floral meristem (Lenhard et al., 2001; Lohmann et al., 2001). One of the earliest genes acting downstream of AG is SPOROCYTELESS/NOZZLE (SPL/NZZ), which encodes a MADS-like transcription factor (Schiefthaler et al., 1999; Yang et al., 1999). The AG protein binds the CArG-box–like sequence within the 3′-untranslated region of SPL/NZZ and activates SPL/NZZ expression (Ito et al., 2004). SPL/NZZ promotes differentiation of microsporocytes and anther wall cells in the stamens and is necessary for proximal-distal pattern formation, cell proliferation, and early sporogenesis in ovule development (Schiefthaler et al., 1999; Yang et al., 1999; Balasubramanian and Schneitz, 2000, 2002; Sieber et al., 2004). Ectopic expression of SPL/NZZ produces hyponastic leaves, defective shoot apical meristems, and abnormal floral organs (Li et al., 2008; Liu et al., 2009). Although direct targets of SPL/NZZ have not (yet) been identified, several genes are known to act temporally downstream during gametophyte development. The receptor-like protein kinase gene EXCESS MICROSPOROCYTES1 (EMS1)/EXTRASPOROGENOUS CELL and the small protein gene TAPETUM DETERMINANT1 (TPD1) are required for specifying tapetum and microsporocyte identity (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003). The bHLH transcription factor gene DYSFUNCTIONAL TAPETUM1 (DYT1) and the PHD domain transcription factor gene MALE STERILITY1 (MS1) are required for tapetum and microgametophyte development during and after meiosis, respectively (Zhang et al., 2006; Ito et al., 2007; Yang et al., 2007). Reverse genetic analysis revealed that BTB AND TAZ DOMAIN1 (BT1) to BT5 genes have functional redundancy and are essential for female and male gametophyte development (Robert et al., 2009). Large-scale screens of Ds transposon insertion lines have identified 67 male and 130 female gametophytic mutants (Pagnussat et al., 2005; Boavida et al., 2009). Among the identified mutant genes, the putative transcription factor genes EMBRYO SAC DEVELOPMENT ARREST31 (EDA31) and MATERNAL EFFECT EMBRYO ARREST65 (MEE65) are specifically involved in female gametogenesis and gametophyte function, whereas the exostosin-like gene EDA5 is required for early megagametogenesis as well as for pollen tube growth (Pagnussat et al., 2005; Boavida et al., 2009). Despite the above-described advances, the molecular mechanisms controlling gene transcription within these regulatory networks remain elusive, preventing a deeper understanding of gametophyte pattern formation.

Here, we demonstrate that sdg2 mutants exhibit both sporophytic and gametophytic development defects. SDG2 is required for activation of expression of at least 11 genes previously characterized as being essential for gametophyte development. We show that SDG2 is involved in H3K4me3 deposition at chromatin of some examined genes, including SPL/NZZ, MS1, and BT3. Both SPL/NZZ and MS1 contain bivalent chromatin domains enriched simultaneously with the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3. We propose that SDG2-mediated H3K4me3 deposition counteracts H3K27me3-mediated repression of SPL/NZZ and MS1, forming an important regulatory mechanism in the gene networks underlying gametophyte development.


Identification of Loss-of-Function Mutants of SDG2

The SDG2 gene is predicted to be >10 kb in length, containing 20 introns and 20 exons (Figure 1A). It encodes a putative 2335–amino acid protein containing a SET domain and is predicted at low confidence levels to contain a POST_SET, a GYF, and a NEBULIN domain (Figure 1A). To investigate the biological function of SDG2, we obtained six Arabidopsis lines, named hereinafter sdg2-1 to sdg2-6, each containing an independent transposon or T-DNA insertion within the SDG2 locus (Figure 1A). All mutations in these lines are recessive; homozygous, but not heterozygous, plants showed an obvious mutant phenotype, which was largely similar across these allelic mutants (Figure 1B). Homozygous mutant plants were smaller in size and were fully sterile (Figures 1B and 1C). RT-PCR analysis revealed that the transposon or T-DNA insertion effectively interrupted production of full-length SDG2 transcripts in these mutants (Figure 1D). Taken together, these observations establish that loss of function of SDG2 causes the phenotype in sdg2 mutants.

Figure 1.
SDG2 Gene Structure, Protein Domains, and Phenotype of Loss-of-Function Mutant Alleles.

Loss of Function of SDG2 Results in Smaller Plants

All six allelic sdg2 mutants have a similar phenotype; we hereafter concentrated on sdg2-1 for detailed characterization. At the vegetative stage, sdg2-1 showed a normal rate of rosette leaf initiation and has similar numbers of rosette leaves at bolting compared with wild-type Columbia (Col) plants (Figure 2A). Also, both sdg2-1 and Col plants bolted at roughly the same time after sowing. Therefore, differing from the previously characterized TrxG family Arabidopsis mutants (Soppe et al., 1999; Kim et al., 2005; Zhao et al., 2005; Pien et al., 2008; Saleh et al., 2008; Xu et al., 2008; Berr et al., 2009; Tamada et al., 2009), sdg2-1 exhibits a relatively normal flowering time under long-day photoperiod growth conditions. At later developmental stages (after bolting), sdg2-1 produced fewer secondary rosette leaves compared with Col (Figure 2A). The sdg2-1 rosette leaves are smaller in size compared with those of Col (Figure 2B). Fresh weight measurements of whole rosettes of 4-week-old plants further confirmed the smaller size of sdg2-1 (22.0 ± 8.7 mg, n = 6) compared with Col (60.0 ± 12.8 mg, n = 6). Light microscopy revealed smaller cell size in sdg2-1 compared with Col leaves (Figure 2C). The epidermal pavement cell surface is reduced to ~40% in sdg2-1 compared with Col leaves (Figure 2D). Taken together, these data indicate that cell expansion is drastically constrained, which might largely account for the reduced leaf size in sdg2-1. To investigate cell cycle progression, we compared the ploidy levels of sdg2-1 and Col leaves by measurement of the relative nuclear DNA content via flow cytometry analysis. The 2C and 4C DNA content corresponds to the G1 and G2 phases during mitotic division, respectively. The proportion of 2C cells is slightly lower in sdg2-1 compared with Col (Figure 2E), suggesting a relatively shorter duration of G1 in the mutant. Higher ploidy levels (≥8C) are the result of endoreduplication cycles in which nuclear DNA is replicated without a subsequent mitotic division. The relative proportion of cells with higher ploidy levels is slightly increased in sdg2-1 compared with Col (Figure 2E); moreover, cycle value, defined as the mean number of endoreduplication cycles per nucleus (Barow and Meister, 2003), is significantly (P < 0.001) higher in sdg2-1 (0.857 ± 0.083, n = 10,000) than in Col (0.614 ± 0.228, n = 10,000). These latter results indicate that mutant cells exit the mitotic cycle and undergo cell differentiation earlier.

Figure 2.
Comparison of Leaf Initiation and Phenotype between the sdg2-1 Mutant and Wild-Type Col.

Anther and Pollen Development Is Impaired in sdg2-1 Mutant Plants

The most remarkable phenotype of the sdg2 mutant plants is their sterility. Arabidopsis floral organs are arranged in four concentric whorls, from the outermost to the innermost: the sepals, petals, stamens, and pistil. sdg2 mutant flowers contain normal numbers of floral organs and a relatively normal morphology, except that the stamens remain short during later flower developmental stages (stage definition according to Smyth et al., 1990; Figure 1C). Homeotic conversion of floral organs, as had been previously reported for the atx1-1 (Alvarez-Venegas et al., 2003) and sdg8-1/ashh2-1 (Grini et al., 2009) mutants, was not observed in sdg2 mutants. In sdg2 mutants, the short stamens failed to reach the receptive stigmatic papillae of the pistil at anthesis for successful pollination. In addition, pollen production is also affected. Cytological analysis revealed aberrant anthers in sdg2-1 that lack one or more of the four locules (Figure 3A; see Supplemental Figure 1 online), indicating defects in initiation, specification, and/or development of primary sporogenous and tapetal cells in the mutant. In some locules, sporogenous cells developed and produced pollen grains; however, these pollen grains stuck to each other and anther dehiscence failed to occur efficiently. Over 40% of pollen grains showed collapsed morphology (Figures 3B and 3C), and Alexander staining revealed both viable and dead pollen grains in sdg2-1 locules (Figure 3D). Compared with Col pollen, viable sdg2-1 pollen was larger in size, and organization of the male germ unit showed irregular positioning of the two sperm and single vegetative cell nuclei (Figure 3E). To test whether viable sdg2-1 pollen is functional, we gently dissected pollen grains from mature anthers and used them to pollinate pistils of emasculated Col plants. From 24 pollinated pistils (containing a total of ~1200 ovules), we obtained 45 seeds, which were confirmed by PCR analysis to correspond to the expected heterozygous mutant genotype. The very low fertilization efficiency indicates that only a very small number of sdg2-1 pollen grains are fully functional.

Figure 3.
sdg2-1 Exhibits Wide-Ranging Defects in Anther and Male Gametophyte Development.

SDG2 Is Necessary for Male Gametogenesis

The sdg2-1 pollen phenotype and functional defects indicate that SDG2 is required for proper microgametogenesis. To gain further information, we examined tetrads dissected from sdg2-1 and Col immature anthers by 4’,6-diamino-2-phenylindole (DAPI) staining. Col tetrads contained the expected four haploid microspores, with four DAPI-stained nuclei visible (Figure 3F), whereas the sdg2-1 tetrads showed a variable reduced number of nuclei (Figure 3G). Quantitative analysis revealed that whereas >97% of Col tetrads contain the normal four DAPI-stained nuclei, only ~50% of sdg2-1 tetrads show such configuration and the remaining 50% of tetrads contain lower numbers of nuclei (Figure 3H). Abnormal sdg2-1 tetrads are also visible inside the pollen sac upon cytological examination (Figure 3I). Light microscopy images of sdg2-1 tetrads showed that microspores without DAPI staining are surrounded by a cell wall, suggesting that cytokinesis occurs relatively normally during cell division. The absence of DAPI staining and some aberrant DAPI-staining structures (Figure 3G) suggest that abnormal chromatin organization, nucleus degeneration, and DNA degradation had occurred during sdg2-1 microspore formation.

To examine gametophyte function under normal sporophytic growth, we investigated inheritance of sdg2 mutant alleles in heterozygous mutant plants. The sdg2-1 allele is associated with an insertion transgene expressing phosphinothricin resistance. Growth tests on seeds produced by self-pollination of heterozygous SDG2-1+/− plants revealed that phosphinothricin-resistant compared with phosphinothricin-sensitive plant numbers are significantly lower than the expected ratio of 3:1 (Table 1). The SDG2-2+/− and SDG2-3+/− lines behaved very similarly to SDG2-1+/− (Table 1). As no seed abortion could be observed, this suggested that male and/or female transmission of the sdg2 mutant alleles was decreased. To determine the inheritance of the sdg2 mutant alleles in the male and female gametes, reciprocal backcrosses of heterozygous mutant plants with the wild-type plants were performed. Genotyping by PCR analysis revealed that the inheritance of both the sdg2-1 and sdg2-3 alleles was reduced drastically through male and also slightly but significantly through female gametes (Table 1). Together, these genetic data establish a gametophytic function of SDG2, which is largely independent from its sporophytic function.

Table 1.
Segregation Analysis of sdg2 Mutant Alleles in Progeny Derived from Self-Pollination or Crosses

Ovule and Female Gametophyte Development Is Defective in sdg2-1 Mutant Plants

To investigate functionality of female gametophytes in homozygous sdg2-1 plants, we first examined their fecundity by pollination of mutant pistils with Col pollen grains. From a total of 90 pistils from 10 sdg2-1 plants examined in two independent experiments, we failed to obtain any seeds from pollination of sdg2-1 pistils, indicating that sdg2-1 is completely female sterile. We used light microscopy to examine ovule development. It is well known that in wild-type Arabidopsis plants, ovule development is synchronous and follows several distinct stages (Christensen et al., 1997). We observed that early-stage premeiotic ovules contain single megaspore mother cells in sdg2-1 as in Col (Figures 4A and 4B). After meiosis, the three spores closest to the micropyle of the ovule undergo programmed cell death and the chalazal megaspore undergoes mitosis to give rise to a two-nucleate embryo sac, as observed for Col (Figure 4C). However, most sdg2-1 ovules are defective, showing obvious abnormalities in megaspores and the development of the embryo sac (Figures 4D and 4E). Some sdg2-1 ovules show overproliferation of the nucellus at the tip (Figure 4D). We further analyzed embryo sac formation using confocal laser scanning microscopy. In Col ovules, the embryo sac at maturation is well surrounded by integument tissues and consists of one egg cell, one central cell, two synergid cells, and at the chalazal pole, three antipodal cells undergoing cell death (Figure 4F). sdg2-1 ovules showed abnormal phenotypes and could essentially be divided into three different classes. The first class, which accounted for ~56% of all ovules, showed relatively normal integument development and nucellus proliferation but did not contain an obvious embryo sac (Figure 4G). The second class represented ~28% of all ovules and showed inhibition of integument growth and nucellus overproliferation at the tip and also lacked an obvious embryo sac (Figure 4H). In these two classes, arrest of embryo sac development might occur before vacuole formation, which normally takes place at the two-nucleate stage. No clear nuclear morphology could be observed and some staining structures revealed cell death (Figures 4G and 4H), indicating that in both classes, megaspores are degenerated during early gametophyte development. Finally, the third class, accounting for ~16% of all ovules, contained a vacuolated embryo sac but displayed degeneration of nuclei and cell death prior to embryo sac maturation (Figure 4I). Taken together, our observations indicate that SDG2 plays crucial roles at various stages of ovule and female gametophyte development. The fact that SDG2 functions in megagametogenesis is also demonstrated by the reduced inheritance of mutant alleles in the heterozygous SDG2-3+/− and SDG2-1+/− plants (Table 1).

Figure 4.
sdg2-1 Shows Severe Defects in Ovule and Female Gametophyte Development.

SDG2 Transcripts Are Detected at High Levels in Sporogenous/Gametophytic Cells in Anthers and Ovules

SDG2 expression in Col plants was detected by RT-PCR in various tissue types, with the highest level observed in flower buds (Figure 1D). We further investigated SDG2 expression by in situ hybridization. We detected SDG2 transcripts at high levels in primordia and young floral organs (Figure 5A). At later stages, high levels of SDG2 transcripts were observed in sporogenous cells and microsporocytes in anther locules (Figure 5B). For comparison, AG transcripts were detected specifically in reproductive organ primordia and in anther cells prior to microsporocyte formation (Figure 5C), as previously reported (Bowman et al., 1991; Ito et al., 2004); and SPL/NZZ transcripts were detected in floral organ primordia, in tapetal and sporogenous cells as well as in microspores (Figure 5D), as previously reported (Schiefthaler et al., 1999; Yang et al., 1999). SDG2 transcripts were also detected in ovules within the pistil (Figure 5E) and in the embryo sac (Figure 5F). SDG2 transcripts were present at low levels in young embryos before the heart stage (Figure 5G) but were undetectable in mature embryos (Figure 5H). As expected, SDG2 transcripts were not detected in sdg2-1 flowers at all stages examined (shown for ovules in Figure 5I). As a negative control, hybridization with an SDG2 sense gene probe did not reveal detectable signals in Col or sdg2-1 in all tissues tested (data not shown). The observed SDG2 expression pattern in anthers and ovules is consistent with its proposed function during male and female gametophyte development.

Figure 5.
In Situ Hybridization Analysis of SDG2 Expression.

Genes Essential for Gametophyte Development Are Downregulated in sdg2-1 Flower Buds

To investigate the molecular mechanisms underlying the observed defects in sdg2-1 gametophyte development, we analyzed transcript profiles in the sdg2-1 mutant by microarray analysis (Agilent Technologies). We compared sdg2-1 and Col transcripts obtained from young flower buds around stage 8 of flower development (Smyth et al., 1990). At this stage, locules begin to appear in stamens, and ovule primordia are detectable as interdigitating finger-like protrusions in the pistil. This flower developmental stage was chosen to avoid the severe developmental defects that occur later during gametogenesis in the sdg2-1 mutant. We found 452 genes downregulated (see Supplemental Data Set 1A online) and 273 genes upregulated (see Supplemental Data Set 1B online) by more than twofold in sdg2-1 floral buds compared with Col.

Remarkably, 11 genes previously shown to be essential for gametophyte development were among the downregulated genes in sdg2-1 (Table 2). We further investigated the expression of several genes essential for gametophyte development by quantitative RT-PCR analyses. These included seven genes, SPL/NZZ, BT3, DYT1, MS1, MYB99, EDA31, and MEE65 (listed in Table 2), together with EMS1 and TPD1, which were not among the list of differentially expressed genes identified in sdg2-1 by microarray analysis but were previously shown to act early in the determination of tapetal cell identity (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003). Consistent with transcriptome analysis data, RT-PCR analysis showed that expression of SPL/NZZ, BT3, DYT1, MS1, MYB99, EDA31, and MEE65 was downregulated, whereas expression of EMS1 and TPD1 was unchanged in sdg2-1 flower buds compared with Col (Figure 6).

Table 2.
Downregulated Genes in sdg2-1 That Are Known to Be Functionally Essential for Gametophyte Development
Figure 6.
Quantitative RT-PCR Analysis of Gene Expression in Col and sdg2-1 Flower Buds at Developmental Stage 8.

Among the identified genes, BT3 is involved in both male and female gametophyte development (Robert et al., 2009). Nevertheless, because the bt3 mutant displays a wild-type phenotype and only the double mutant bt2 bt3 shows defects in gametophyte development (Robert et al., 2009), we believe that downregulation of BT3 alone, as identified in the sdg2-1 mutant, has little effect on the sdg2-1 mutant phenotype. SPL/NZZ is unique in that it is required early in both male and female gametophyte development (Schiefthaler et al., 1999; Yang et al., 1999). The other nine genes are known to be involved downstream of SPL/NZZ and at later developmental stages in either male or female gametophyte development (Table 2). Some of the early gametophyte developmental defects observed in sdg2-1 partially phenocopy the spl/nzz mutant. In addition, both SPL/NZZ and SDG2 expression can be found in sporogenous cells and microsporocytes in anthers and in megasporocytes in ovules (Schiefthaler et al., 1999; Yang et al., 1999; Figure 5). To examine expression differences in a tissue-specific manner, we compared SPL/NZZ expression in sdg2-1 and in Col by in situ hybridization. As shown in Figure 7, SPL/NZZ expression is clearly reduced in ovule primordia and in sporogenous cells and microsporocytes within anthers in sdg2-1 compared with Col. This is consistent with transcriptome (Table 2) and RT-PCR (Figure 6) analysis data.

Figure 7.
In Situ Hybridization Analysis of SPL/NZZ Expression in Col and sdg2-1 Floral Organs.

SDG2 Activates Gene Transcription through H3K4 Trimethylation

To gain insight into the molecular mechanism of SDG2-mediated activation of gene expression, we investigated histone methylation levels in sdg2-1. Protein immunoblot analysis (Figure 8A) showed that compared with Col, sdg2-1 plants contain a dramatically reduced level of H3K4me3, a slightly reduced level of H3K4me2, and an enhanced level of H3K4me1. By contrast, levels of H3K36me1, H3K36me3, and H3K27me3 were unchanged in sdg2-1 compared with Col (Figure 8A). This indicates that SDG2 is required primarily for H3K4me3 deposition and, to a lesser degree, H3K4me2 deposition in Arabidopsis. H3K4me1 deposition likely involves a different enzyme, and defects in converting monomethyl to di-/trimethyl by sdg2-1 might have elevated H3K4me1 levels as observed in the sdg2-1 mutant plants.

Figure 8.
Comparison of Histone Methylation in sdg2-1 and Col.

We further investigated H3K4me3 and H3K27me3 at specific genes by chromatin immunoprecipitation (ChIP) assays (Figures 8B and 8C). H3K4me3 levels were drastically reduced at BT3 and SPL/NZZ in sdg2-1 compared with Col. Compared with BT3 or SPL/NZZ, MS1 contains lower levels of H3K4me3 in Col. Nevertheless, significant (P < 0.01) reductions in H3K4me3 were also observed at MS1 in the sdg2-1 mutant compared with Col. By contrast, EDA31 and MEE65 contain low levels of H3K4me3 that are barely affected in sdg2-1. At all examined genes, levels of H3K27me3 were not significantly different in sdg2-1 compared with Col.

Interestingly, relatively high levels of both H3K4me3 and H3K27me3 were detected at SPL/NZZ and MS1 in Col plants. To investigate whether H3K4me3 and H3K27me3 simultaneously mark SPL/NZZ and MS1 chromatin or if they are derived from subpopulations of cells exhibiting different chromatin configurations, we performed sequential double ChIP analysis. Chromatin was immunoprecipitated first with anti-H3K27me3 and then with anti-H3K4me3 antibodies. The results obtained are shown in Figure 8D. Consistent with previously reported data (Jiang et al., 2008), we found that chromatin at both FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC) concomitantly carries both H3K27me3 and H3K4me3, whereas ACTIN2 (ACT2) chromatin does not. Like FT and FLC, SPL/NZZ and MS1 chromatin also simultaneously carry both H3K27me3 and H3K4me3 marks. Reduced levels in sdg2-1 were observed specifically at SPL/NZZ and MS1 loci but not at FT and FLC (Figure 8D).

Taken together, our ChIP data show that SDG2 mediates H3K4me3 deposition selectively at BT3, SPL/NZZ, and MS1, which is consistent with the transcriptional repression of these genes in sdg2-1. H3K4me3 levels in sdg2-1 were unchanged at FLC and FT, which is in agreement with unchanged expression of these genes and the unchanged flowering time phenotype of the mutant. EDA31 and MEE65 do not show detectable changes in H3K4me3, suggesting that their reduced expression could be a secondary effect in the sdg2-1 mutant. Our results also reveal that SPL/NZZ and MS1 are embedded in bivalent chromatin domains, which simultaneously contain the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3.


Over two-thirds of all Arabidopsis nuclear genes contain chromatin marked by H3K4 methylation (Zhang et al., 2009). Among previously characterized TrxG family mutants, only atx1 showed a mild reduction in the global level of H3K4me3 (Alvarez-Venegas and Avramova, 2005). Gene locus-specific reduction of H3K4 methylation was observed in atx1 (Alvarez-Venegas and Avramova, 2005; Pien et al., 2008), and also in atx2 (Saleh et al., 2008) and sdg25/atxr7 (Tamada et al., 2009). Our study establishes that SDG2 is a major factor for H3K4me3 deposition in Arabidopsis. sdg2-1 showed a global reduction of H3K4me3 in total histone extracts (Figure 8), which is more pronounced than that observed in atx1 (see Supplemental Figure 2 online). Consistent with this, SDG2 has a broad function, and sdg2 mutants show pleiotropic phenotypes.

SDG2 in Regulation of Sporophyte Development

The sdg2 mutant plants are small in size, which is visible across a variety of organs, including leaves, stems, and flowers. The previously characterized atx1-1 and sdg8/efs/ccr1 mutants also exhibit reduced plant and organ sizes (Soppe et al., 1999; Alvarez-Venegas et al., 2003; Xu et al., 2008). These data thus reveal that global levels of H3K4me3 and H3K36me2/3 have an overall positive role in plant growth. Plant size is intrinsically determined by cell division and cell expansion activities. The initiation of a leaf begins with the periclinal division of a cell in the L2 layer of the shoot apical meristem, which grows out into the leaf primordium and then forms the mature leaf. In contrast with the indeterminate growth of apical meristems, leaves show determinate growth with a fixed period of development. Our investigation shows that leaf initiation is relatively normal during vegetative growth in sdg2-1; however, final leaf size is drastically reduced in sdg2-1 compared with Col. The reduced leaf size is largely associated with a major reduction of cell expansion. Moreover, cell division and differentiation in sdg2-1 is also affected; the G1 phase is relatively shorter, and polyploidy levels are slightly enhanced in the sdg2-1 mutant leaves.

Endoreduplication occurs after cells have ceased mitotic cycles, and endoreduplicated cells do not reenter the mitotic cell cycle. Endoreduplication is thus characteristic of a switch between cell proliferation and differentiation. It is also believed to be essential for enhancing metabolic capacity and supporting cell growth and for maintaining an optimal balance between cell volume and nuclear DNA content (reviewed in Kondorosi et al., 2000; Inzé and De Veylder, 2006). Curiously, sdg2-1 shows slightly elevated polyploidy levels but reduced cell size. Ploidy-dependent epigenetic regulation has been reported to be involved in differential reprogramming of orthologous gene expression and in stable silencing of epialleles (Lee and Chen, 2001; Baubec et al., 2010). Based on its global effect on H3K4me3 deposition, it is reasonable to speculate that SDG2 is involved in regulation of chromatin structure and gene expression in diploid and polyploid cells, playing important roles in the coordination of cell division, differentiation, and expansion to determinate organ size.

Although SDG2 transcripts were detectable in the inflorescence meristem and in floral organ primordia, sdg2 mutant flowers showed the normal order of the four whorls and normal numbers of floral organs. Flower organ identity is determined by the interplay between homeotic transcription factor genes, including AG, PI, AP3, AP2, and AP1, which are subjected to chromatin-remodeling regulation (reviewed in Shen and Xu, 2009). Consistent with its phenotype, sdg2-1 did not show any detectable alteration of expression of these floral homeotic transcription factor genes in our microarray analysis. By contrast, downregulation of AG, PI, AP2, and AP1 was shown in atx1-1, with flowers exhibiting homeotic conversions and variable aberrations (Alvarez-Venegas et al., 2003). sdg8-1/ashh2-1 has also been reported to display downregulation of PI, AP2, and AP1, with a low proportion of flowers exhibiting homeotic conversions (Grini et al., 2009). Furthermore, the sdg2 mutants differ in the flowering time phenotype from previously studied TrxG family gene mutants. Both activation and repression of FLC depend on chromatin remodeling activities (for recent reviews, see He, 2009; Berr and Shen, 2010), and sdg8/efs/ashh2, sdg25/atxr7, atx1, and atx2 mutants exhibit reduced FLC expression associated with a decrease of H3K4me2/me3 and/or H3K36me2/me3 at FLC chromatin (Kim et al., 2005; Zhao et al., 2005; Pien et al., 2008; Saleh et al., 2008; Xu et al., 2008; Berr et al., 2009; Tamada et al., 2009). Despite its broad effects, sdg2-1 did not alter H3K4me3 levels at FLC, and the mutant plants showed a wild-type flowering time under long-day photoperiod growth conditions, revealing the selectivity of SDG2-dependent H3K4me3 deposition and transcription activation. Overlapping and specific roles of different members within the TrxG family might allow for more flexibility in functions associated with plant growth and developmental plasticity.

SDG2 in Regulation of Gametophyte Development

The sdg2 mutant plants show complete sterility. At least three defects contribute to sdg2-1 sterility: first, stamen filaments are too short to allow effective pollination of the stigma; second, anther dehiscence and production of functional pollen is drastically impaired; and third, ovules lack a fully developed, functional embryo sac. In sdg2-1, anther and pollen development show a variety of defects from early to late stages, including sporophytic locule initiation, microsporogenesis, tapetum development, and microgametogenesis. Late function of viable pollen grains also seems to be affected as indicated by the very low efficiency of seed production obtained using sdg2-1 pollen in pollination of wild-type pistils. In addition, for >80% of sdg2-1 ovules, megagametogenesis is arrested before the completion of the mitotic haploid divisions. For <20% of sdg2-1 ovules, a vacuolated embryo sac is visible, but the megaspore nucleus degenerates before embryo sac maturation. Defects were also observed in sporophytic tissues within sdg2-1 ovules. High levels of SDG2 expression were detected in reproductive organs with specific patterns that are consistent with the important role of SDG2 in gametophyte development. The pleiotropy and variable expressivity of the sdg2-1 phenotype makes it distinct from transcription factor mutants that display a specific defect in one or a few stages of male or female gametophyte development. The main requirement for SDG2 is likely in the sporophyte for proper development or function of anthers and ovules and sporogenesis. Nevertheless, SDG2 also has important roles in gametogenesis as evidenced by late stage defective gametophytes observed in sdg2-1 and more importantly by the reduced transmission efficiency of mutant alleles in heterozygous mutant plants (Table 1).

Nuclear degeneration and genomic DNA degradation likely occur in both microspore and megaspore cells in sdg2-1. We hypothesize that SDG2-mediated H3K4me3 deposition plays an important role in chromosomal organization and reprogramming during meiosis. In yeast, H3K4me3 serves as a prominent mark of active meiotic recombination initiation sites, and loss of function of the sole H3K4 methyltransferase SET1 causes reduced formation of DNA double-strand breaks, which are essential for proper chromosome segregation and fertility (Borde et al., 2009). In mammals, germ cell–specific PRDM9 (also known as Meisetz), which is involved in H3K4me3 but not H3K4me2 or H3K4me1 deposition, is necessary to guide recombination hotspots of homologous chromosomes during meiotic prophase (Hayashi et al., 2005; Hochwagen and Marais, 2010). Nevertheless, we currently do not know if meiotic recombination is affected in the sdg2 mutants. Alternatively, SDG2 might regulate chromatin structure and function during gamete formation after meiosis. Distinct from yeast and mammals, plant gametogenesis occurs within highly structured sporophytic tissues. Coordination with sporophytic tissue development is essential for proper plant gametogenesis, and SDG2 plays an important role in the establishment of expression patterns of sporophytic and gametophytic genes.

SDG2 Positively Regulates H3K4me3 Deposition and Expression of Some Gametophyte Development Genes

At least 11 genes previously shown to play essential functions in gametophyte development were identified as downregulated in sdg2-1 flower buds (Table 2). Among them, the MADS-like transcription factor gene SPL/NZZ is of particular interest. It acts upstream of many stamen- and ovule-expressed genes and is required for initiation of both microsporogenesis and megasporogenesis (Schiefthaler et al., 1999; Yang et al., 1999; Yu et al., 2005; Zhang et al., 2006; Alves-Ferreira et al., 2007; Wijeratne et al., 2007). Although knowledge is currently scarce about how SPL/NZZ regulates downstream processes, a gain-of-function study suggested that SPL/NZZ might be involved in auxin homeostasis by repression of YUCCA genes in lateral organ development (Li et al., 2008). The phytohormone auxin was recently shown to have an important role in gamete cell-type specification during embryo sac development (Pagnussat et al., 2009). We believe that SDG2 activates SPL/NZZ expression, forming a key step in the regulatory network of male and female gametophyte development. Nevertheless, sdg2-1 reduces but does not fully silence SPL/NZZ expression, and residual levels of H3K4me3 are detectable at SPL/NZZ chromatin in sdg2-1, suggesting that additional TrxG family members could be involved in SPL/NZZ activation. Moreover, SDG2 is also required for H3K4me3 deposition and activation of other genes (e.g., BT3 and MS1) which are known to regulate gametophyte development (Ito et al., 2007; Yang et al., 2007; Robert et al., 2009). The fact that SDG2 regulates several different genes and affects them to varying degrees might account for the variable sporophytic and gametophytic defective phenotypes observed in sdg2-1 anthers and ovules.

Our study demonstrates that both SPL/NZZ and MS1 are imbedded within a bivalent chromatin domain consisting of transcriptionally active H3K4me3 and transcriptionally repressive H3K27me3 marks. The simultaneous presence of H3K4me3 marked by TrxG and H3K27me3 marked by PcG was first described in mammalian stem cells and was proposed to represent a pluripotent chromatin state that poises genes for activation upon appropriate developmental cues (Azuara et al., 2006; Bernstein et al., 2006). In Arabidopsis, both TrxG and PcG factors regulate stem cell maintenance genes and floral homeotic genes (reviewed in Shen and Xu, 2009), and the antagonistic function between ATX1/SDG27 and the PcG gene CLF was shown to regulate AG expression (Saleh et al., 2007). Currently, however, the most extensively studied TrxG and PcG function is in flowering time regulation (for recent reviews, see He, 2009; Berr and Shen, 2010), and the simultaneous presence of H3K4me3 and H3K27me3 at FLC and FT has been demonstrated using sequential ChIP analysis (Jiang et al., 2008). SDG2 is specifically required for marking H3K4me3 for active transcription of SPL/NZZ and MS1 but not FLC nor FT. The ATX1/SDG27 and ATXR7/SDG25 proteins bind FLC chromatin (Pien et al., 2008; Tamada et al., 2009). Whether or not the SDG2 protein directly binds chromatin at SPL/NZZ and MS1 requires future investigation. The PcG factors involved in H3K27me3 deposition at SPL/NZZ and MS1 chromatin are currently unknown. Future identification of new factors involved in marking H3K27me3 and H3K4me3 at SPL/NZZ and MS1 will be of great interest for understanding regulation of gene expression and the molecular mechanisms underlying male and female gametophyte development.


Plant Growth and Mutant Genotyping

Mutant seeds were obtained from the ABRC (http://www.Arabidopsis.org): sdg2-1 (WISCDSLOX361D10), sdg2-2 (GK-480F05), sdg2-3 (SALK_021008), sdg2-4 (SALK_120450), sdg2-5 (SALK_138889), sdg2-6 (SALK_055991), sdg25-1 (SALK_149692), and atx1-2 (SALK_149002). Plants were grown on soil at 21°C under a 16-h-light/8-h-dark photoperiod in a glasshouse. Genotyping was performed by PCR analysis using specific primers (sequences available in Supplemental Table 1 online).

Histochemical Assays and Microscopy

Anther transverse sections were stained with toluidine blue as previously described (Sanders et al., 1999) and visualized by light microscopy. Pollen viability was examined using Alexander's staining solution (Alexander, 1969). DAPI staining was performed by incubation of dissected anthers in a solution containing 0.1% Nonidet P-40, 10% DMSO, 50 mM PIPES, pH 6.9, 5 mM EGTA, and 5 μg/mL DAPI for at least 30 min prior to examination under fluorescence microscopy. For ovule images, pistils were fixed and mounted as previously described (Christensen et al., 1997). Plant organs were examined using a Leica MZ12 dissecting microscope. Higher magnification light and fluorescence images were acquired using a Nikon Eclipse 800 microscope equipped with a CDD camera DXM1200. Confocal plan images were acquired using a Zeiss LSM510 Meta inverted confocal laser microscope (Carl Zeiss). Stacked images were deconvolved to reassign blurred images and subsequently flattened into a single image using ImageJ software and the plug-in DeconvolutionLab (NIH Image). Scanning electron microscopy images were taken using a Hitachi S-3400N (Hitachi High-Technologies Europe). Images were processed with Adobe Photoshop 6.0 (Adobe Systems).

Flow Cytometry

Nuclei were prepared from leaves of 2-week-old plants and stained with propidium iodide as previously described (Galbraith et al., 1983) and analyzed on a FACStarPLUS flow cytometer (BD Biosciences) equipped with an argon laser INNOVA 90-C (Coherent). Propidium iodide fluorescence was excited with 500 mW at 514 nm and measured in the FL1 channel using a 630-nm band-pass filter. A total of 10 to 16 plants were used for each sample, and typically 10,000 nuclei per sample were analyzed. Two replicates were performed for each sample.

In Situ Hybridization

Digoxigenin labeling of RNA probes, tissue preparation, and in situ hybridization was performed as previously described (Zhao et al., 2005). Tissue sections were 8 μm thick. A fragment containing the 396 bp 3′-untranslated region of SDG2 was obtained by PCR amplification using specific primers (see Supplemental Table 1 online), cloned into the pGEM-T Easy vector (Promega), and used for preparation of the SDG2 sense (negative control) and antisense probes. Similarly, a fragment containing 359 bp of the 5′ region of SPL/NZZ was amplified (for primer sequences, see Supplemental Table 1 online), cloned, and used for in situ hybridization.


RT-PCR was performed using Improm-II reverse transcriptase (Promega) on total RNA extracted from seedlings and various plant organs using the TRIzol kit (Invitrogen) according to the manufacturer’s instructions. Gene-specific primers used for PCR amplifications are listed in Supplemental Table 1 online.

For quantitative real-time RT-PCR, gene-specific primers were designed using the LightCycler Probe Design 2 program (Roche) and are listed in Supplemental Table 1 online. Total RNA was isolated from the sdg2-1 mutant and wild-type flower buds corresponding to developmental stage 8 (Smyth et al., 1990). cDNA was synthesized from 2 μg of total RNA treated with 2 units of DNase I in a total volume of 40 μL using 2 μM oligo(dT)20, 0.5 mM deoxynucleotide triphosphates, 5 mM DTT, and 200 units of SuperScript III reverse transcriptase in 1× first strand buffer(Invitrogen). A total of 25 to 50 ng cDNA in a total reaction volume of 10 μL SYBR Green Master mix was analyzed using a LightCycler 480 II instrument according to the manufacturer's instructions (Roche). Melting curve analysis was performed to verify amplification of a single PCR product. PCR amplification was performed on three technical replicates on each of the two biological repeat samples. Several conventionally used reference genes were evaluated for their respective stability in our experimental conditions using geNorm (Vandesompele et al., 2002) and Norm Finder (Andersen et al., 2004), and the housekeeping genes GAPDH, TIP41-like, and At4g26410 (unknown function) were selected for use as internal references. After normalization with each of the three reference genes, the relative expression level of each gene in the sdg2-1 mutant was compared with that of the wild type.


Total RNA was isolated from sdg2-1 mutant and wild-type young flower buds corresponding to developmental stage 8 (Smyth et al., 1990), which were pooled from at least 12 individual plants. The microarray analyses were performed using Aligent’s Whole Arabidopsis Gene Expression Microarray (G2519F, V4, 4x44K) via custom service of the Shanghai Huaguan Biochip Co. All microarray procedures and data analyses were performed according to Aligent’s manual (Agilent Technologies). Quantile normalization was performed to make the distribution of probe intensities for each array in a set of arrays the same. Microarray data were deposited at the Gene Expression Omnibus according to “Minimum Information About a Microarray Experiment” standards. Experiments were repeated with independently grown plants, pool of flower buds, RNA preparation, and microarray analysis. Significant genes regulated with a factor of ≥2.0 and a P value of ≤0.05 in two independent experiments were selected.

Histone Extraction and Immunoblot Analysis

Arabidopsis thaliana histones were extracted from 20-d-old seedlings as previously described (Yu et al., 2004) separated by electrophoresis on 15% SDS-PAGE, and transferred to an Immobilon-P polyvinylidene difluoride transfer membrane (Millipore). Immunoblots were performed using specific antibodies: anti-monomethyl-H3K4 (Upstate Catalog No. 07-436; Millipore), anti-dimethyl H3K4 (Upstate Catalog No. 07-030; Millipore), anti-trimethyl-H3K4 (Upstate Catalog No. 07-473; Millipore), anti-monomethyl-H3K36 (Abcam; ab9048), anti-trimethyl-H3K36 (Abcam; ab9050), anti-trimethyl-H3K27 (Upstate Catalog No. 07-449; Millipore), and anti-H3 (Upstate Catalog No. 05-499; Millipore).

ChIP Analysis

ChIP was performed according to Xu et al. (2008) with the following modifications. Flower buds at developmental stage 8 were used. Chromatin was sheared with a Bioruptor sonicator (Cosmo Bio) twice for 15 min with a 50% duty cycle and high power output to obtain 200- to 1000-bp DNA fragments. Immunoprecipitation was performed using anti-trimethyl-H3K4 or anti-trimethyl-H3K27 antibody together with Protein A-magnetic beads (Millipore). Negative controls were performed without antibody. DNA was recovered using Magna ChIP spin filters according to the manufacturer’s instructions (Millipore). ChIP DNA was analyzed by quantitative real-time PCR using gene-specific primers (see Supplemental Table 1 online). Enrichment of H3K4 and H3K27 trimethylation in sdg2-1 mutant and wild-type flower buds, relative to ACT2/7 for H3K4me3 and FUSCA3 for H3K27me3, was calculated using the Pfaffl equation (Pfaffl, 2001).

Sequential ChIP

Sequential ChIP was performed essentially as previously described (Bernstein et al., 2006; Jiang et al., 2008). Briefly, chromatin prepared from flower buds at developmental stage 8 was first immunoprecipitated with anti-trimethyl-H3K27 antibody and washed, eluted for 30 min at 37°C in 10 mM DTT, and further diluted 1:50 in lysis buffer (Xu et al., 2008). Half of the eluted chromatin was used as a mock control in a second immunoprecipitation without antibody, and the other half was subsequently immunoprecipitated using anti-trimethyl-H3K4 antibody. DNA fragments were recovered and purified for quantitative real-time PCR analysis. Enrichment of bivalent chromatin (with both H3K4me3 and H3K27me3 marks) in sdg2-1 mutant and wild-type flower buds, relative to the FT locus known to be bivalent (Jiang et al., 2008), was calculated using the Pfaffl equation (Pfaffl, 2001). FLC, another bivalent locus, and ACT2/7, a constitutively expressed locus lacking H3K27 trimethylation, served as positive and negative controls, respectively (Jiang et al., 2008).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database under accession number At4g15180 (SDG2). Microarray data can be found at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18513.

Supplemental Data

The following materials are available in the online version of the article.

Supplementary Material

Supplemental Data:


We thank L. Xu and M.F. Mbengue for assistance in early stages of sdg2 mutant identification, M. Erhardt for assistance in microscopy analysis, and A. Alioua for assistance in quantitative PCR analysis. This work was supported in part by the Centre National de la Recherche Scientifique and Agence Nationale de la Recherche (ANR 06-BLAN-0054-01).


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