H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice

Authors

  • Pengfei Sui,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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    • These authors contributed equally to this work.

  • Jing Jin,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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    • These authors contributed equally to this work.

  • Sheng Ye,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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  • Chen Mu,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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  • Juan Gao,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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  • Haiyang Feng,

    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
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  • Wen-Hui Shen,

    1. Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cédex, France
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  • Yu Yu,

    Corresponding author
    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
      (fax +86 21 55665673; e-mail aiwudong@fudan.edu.cn or yuy@fudan.edu.cn).
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  • Aiwu Dong

    Corresponding author
    1. State Key Laboratory of Genetic Engineering, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China
      (fax +86 21 55665673; e-mail aiwudong@fudan.edu.cn or yuy@fudan.edu.cn).
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(fax +86 21 55665673; e-mail aiwudong@fudan.edu.cn or yuy@fudan.edu.cn).

Summary

Methylation of histone lysine residues plays an essential role in epigenetic regulation of gene expression in eukaryotes. Enzymes involved in establishment of the repressive H3K9 and H3K27 methylation marks have been previously characterized, but the deposition and function of H3K4 and H3K36 methylation remain uncharacterized in rice. Here, we report that rice SDG725 encodes a H3K36 methyltransferase, and its down-regulation causes wide-ranging defects, including dwarfism, shortened internodes, erect leaves and small seeds. These defects resemble the phenotypes previously described for some brassinosteroid-knockdown mutants. Consistently, transcriptome analyses revealed that SDG725 depletion results in down-regulation by more than two-fold of over 1000 genes, including D11, BRI1 and BU1, which are known to be involved in brassinosteroid biosynthesis or signaling pathways. Chromatin immunoprecipitation analyses showed that levels of H3K36me2/3 are reduced in chromatin at some regions of these brassinosteroid-related genes in SDG725 knockdown plants, and that SDG725 protein is able to directly bind to these target genes. Taken together, our data indicate that SDG725-mediated H3K36 methylation modulates brassinosteroid-related gene expression, playing an important role in rice plant growth and development.

Introduction

The epigenetic regulation of gene expression depends largely on the status of chromatin structure in eukaryotes. As the basic building units of chromatin, nucleosomes consist of DNA and histones, which may be subjected to a number of covalent modifications, such as methylation, acetylation, phosphorylation and ubiquitination (Berr et al., 2011). Histone methylation plays a crucial and diverse regulatory function in chromatin organization and genome function. In general, methylation on histone H3 lysine 9 (H3K9), H3K27 and H4K20 results in transcription repression, whereas methylation on H3K4 and H3K36 correlates with gene activation. In addition, each lysine residue can be mono-, di- or tri-methylated, and the methyl number may have important functional implications (Yu et al., 2009). Most histone methyltransferases contain a conserved SET domain (named after three Drosophila genes: Su(var)3–9, E(z) and Trithorax), and are members of the SET domain group (SDG) proteins (Springer et al., 2003).

Rice (Oryza sativa) is one of the most important crops worldwide, and also serves as a model of monocot plants for basic research. In the past years, several rice SDG proteins involved in repressive H3K9 and H3K27 methylation have been reported. Analyses of SDG714 and other predicted H3K9 methyltransferases showed that these rice SDG proteins have either specific or redundant functions in regulating histone H3K9 methylation and retrotransposon repression (Ding et al., 2007a,b, 2010; Qin et al., 2010). Rice genes encoding potential H3K27 methyltransferases, including OsSET1 (also known as OsiEZ1) and OsCLF, have also been characterized (Liang et al., 2003; Thakur et al., 2003; Luo et al., 2009). High-resolution mapping of epigenetic modifications of the genome revealed the interplay between histone H3K4 methylation and gene expression in rice (Li et al., 2008). Although phylogenetic tree analysis suggests that nine SDGs may be involved in catalyzing H3K4 and/or H3K36 methylation in rice (Berr et al., 2009), none of them has been characterized so far. Interestingly, we found that SDG725-mediated H3K36 methylation is involved in regulation of aspects of plant growth and development in rice by the hormone brassinosteroid (BR).

Brassinosteroid is involved in a variety of plant physiological/developmental processes (Li, 2010). Compared to Arabidopsis, rice is less well studied with regard to both BR synthesis and signaling pathways. Nevertheless, a few evolutionarily conserved genes have been characterized in rice. D11 (OsDWARF11), D2, BRD1(BR-deficient dwarf1) and BRD2 encode cytochrome P450 family enzymes that are involved in several steps of BR biosynthesis, and their mutations cause typical BR knockdown phenotypes such as dwarfism, shortened internodes, erect leaves and small seeds (Hong et al., 2002, 2003, 2005; Tanabe et al., 2005). OsBRI1 and OsBZR1 have been identified based on their counterparts involved in BR signaling in Arabidopsis, and Osbri1 and Osbzr1 mutants showed dwarfism and erect leaves (Yamamuro et al., 2000; Bai et al., 2007). Recently, several factors were also found to act as positive or negative regulators in rice BR signaling pathways, including the α subunit of heterotrimeric G protein RGA1 (heterotrimeric G protein α1) (Oki et al., 2009), GRAS family protein DLT (DWARF AND LOW-TILLERING) (Tong et al., 2009), and the bHLH proteins BU1 (BRASSINOSTEROID UPREGULATED 1) (Tanaka et al., 2009), ILI1 (INCREASED LAMINA INCLINATION) and IBH1 (ILI1-binding bHLH) (Zhang et al., 2009). BR synthesis knockdown mutants could be rescued by application of bioactive BR compounds, but BR signaling defective mutants could not be rescued by exogenous BR treatment.

Our transcriptome analyses indicated that over 1000 genes were down-regulated more than twofold in the SDG725 knockdown mutant sdg725, including D11 as well as BRI1 and BU1. Consistently, the sdg725 mutants are less sensitive than the wild-type plants and can be partially rescued by exogenous BR treatment. Chromatin immunoprecipitation (ChIP) experiments showed that H3K36 di- and tri-methylation (H3K36me2 and H3K36me3) were decreased in some regions of BR-related genes in the sdg725 mutant. Using a specific monoclonal antibody, SDG725 protein was shown to directly associate with the D11, BRI1 and BU1 genes, supporting the view that they are target genes of SDG725, and that SDG725-dependent H3K36 methylation plays key roles in modulating BR-related gene expression and in regulating rice plant development.

Results

Rice SDG725 specifically methylates H3K36 in vitro

The recombinant SET domain of the SDG725 protein (named as SDG725s) was expressed in and purified from Escherichia coli in order to analyze its histone methyltransferase activity in vitro. Methyltransferase activity of GST–SDG725s for H3 was detected when mononucleosomes were used as the substrate (Figure 1a), using GST–SDG714 (Ding et al., 2007a), which shows H3K9 methyltransferase activity, as the positive control and GST as the negative control. We further examined the specificity of SDG725 for H3 methylation by using reassembled oligonucleosomes containing K→A mutations. Mutated oligonucleosomes containing H3K36A were not methylated by GST–SDG725s (Figure 1b). Therefore, we conclude that rice SDG725 is a robust and specific histone H3 lysine 36 methyltransferase.

Figure 1.

In vitro histone methyltransferase activity of SDG725.
(a) GST–SDG725s methylates histone H3 using mononucleosomes as substrate.
(b) GST–SDG725s specifically methylates H3K36. Reassembled oligonucleosomes containing K→A replacement mutations were used in the assay.
Upper panels show SDS–PAGE gels stained with Coomassie brilliant blue R-250 and lower panels show corresponding autoradiographs of histone methylation. GST–SDG714 and GST–SDG725s are indicated by black arrowheads and methylated histones are indicated by asterisks.

SDG725 knockdown plants display abnormal morphology

To uncover the function of SDG725 in rice, we generated SDG725 knockdown (sdg725) plants by RNAi to knockdown SDG725 expression. We obtained 24 independent transgenic lines containing the RNAi construct p35S:SDG725RNAi (subsequently referred to as 725Ri), and six showed a strong and stable dwarf phenotype after six generations. To confirm the phenotype of SDG725 knockdown plants, we also produced other SDG725 knockdown mutants using the artificial microRNA approach. We chose two artificial microRNA sequences from SDG725 and two microRNA backbones: MIR528 from rice and MIR319 from Arabidopsis. We obtained 20 independent transgenic lines containing the artificial microRNA construct p35S:MIR528-SDG725-1 (subsequently referred to as amiR725), and six showed a moderate and stable dwarf phenotype after three generations.

Both 725Ri and amiR725 transgenic plants displayed a dwarf phenotype, and their overall phenotypes were similar, except that amiR725 plants showed less severe phenotypes than 725Ri plants (Figure 2a), correlating well with their levels of SDG725 expression (Figure 2b). Therefore, the amiR725 plant could be considered as a weak sdg725 line. The sdg725 plants had short internodes (Figure 2c), erect leaves (Figure 2d), reduced spikelets and rachis branches (Figure 2e) and small grains (Figure 2f), compared with wild-type (WT) rice (Table 1). Thus knockdown of SDG725 causes pleiotropic defects in rice plant growth and development.

Figure 2.

 Phenotypes of the SDG725 knockdown plants.
(a) Morphological analyses of a wild-type (WT) plant (left), amiR725-1 mutant (center) and 725Ri-1 mutant (right). Scale bar = 25 cm.
(b) Relative expression level of SDG725 in WT, amiR725-1 and 725Ri-1.
(c) Three uppermost internodes (1–3) of WT (left), amiR725-1 (center) and 725Ri-1 (right). Scale bar = 10 cm.
(d) Second leaf of WT (left), amiR725-1 (center) and 725Ri-1 (right). Lamina joints are indicated by white arrows. Scale bar = 2 cm.
(e) First panicle of WT (left), amiR725-1 (center) and 725Ri-1 (right). Scale bar = 5 cm.
(f) Unhulled grains of WT (left), amiR725-1 (center) and 725Ri-1 (right). Scale bar = 0.5 cm.

Table 1.   Measurements of mature wild-type (WT) and SDG725 knockdown plants
 WTamiR725-1725Ri-1
  1. Values are means ± SD of 20 plants in each line, except for the weight of 1000 unhulled grains.

Plant height (cm)76.1 ± 2.1852.7 ± 2.8346.1 ± 1.67
Flag leaf (cm)27.1 ± 2.1322.7 ± 1.8218.9 ± 1.34
Panicle (cm)19.5 ± 1.3416.2 ± 0.9615.1 ± 0.92
First internode (cm)31.7 ± 1.0721.7 ± 1.2720.1 ± 1.2
Second internode (cm)12.4 ± 0.686.7 ± 0.565.7 ± 0.46
Third internode (cm)6.5 ± 0.583.4 ± 0.382.7 ± 0.23
Lamina joint angle (°)12.9 ± 2.910.3 ± 2.16.6 ± 1.5
Weight of 1000 unhulled grains (g)25.7 ± 1.122.8 ± 1.017.48 ± 0.87

SDG725 is required for normal expression of a large number of genes

Because methylation on H3K36 correlates with gene activation in eukaryotes, we analyzed the transcriptome of sdg725 plants using the Agilent Rice Oligo Microarray, which contains more than 40 000 probes for rice transcripts. Two sdg725 mutants (725Ri-1 and 725Ri-3), with similar expression levels of SDG725, were selected for microarray analysis. Compared with the wild-type, 1738 and 1907 genes were down-regulated more than twofold in 725Ri-1 and 725Ri-3, respectively, and 1052 and 1164 genes were up-regulated more than twofold in 725Ri-1 and 725Ri-3, respectively. Of these, 1060 genes are down-regulated and 717 genes are up-regulated in both 725Ri-1 and 725Ri-3, and these genes are involved in many biological processes such as transcription, phytohormone, metabolism, transport and so on (Table S1). These data suggest that SDG725 is required for normal expression of a large number of genes throughout plant development.

BR-related genes are mis-regulated in SDG725 knockdown plants

SDG725 knockdown plants display phenotypes that are very similar to those of BR-deficient mutants, prompting us to examine the expression of BR-related genes within the microarray data for 725Ri mutants. We found that the BR biosynthesis-related gene D11 (Os04g0469800) is among the genes that are down-regulated in 725Ri mutants (Table S1). Quantitative real-time RT-PCR validated the reduction of D11 expression in 725Ri-1 (Figure 3a). We further investigated the expression levels of several rice BR-related genes, including two other genes involved in BR biosynthesis (D2 and BRD1) and three genes involved in BR signaling pathways (BZR1, BRI1 and BU1). Quantitative RT-PCR analyses showed that expression of BRI1 and BU1 was down-regulated, but expression of D2, BRD2 and BZR1 was not obviously changed in the 725Ri-1 mutant compared with wild-type rice plants (Figure 3a).

Figure 3.

 Expression levels of BR-related genes in SDG725 knockdown plants, and lamina joint bending assay.
(a) Transcription levels of the BR-related genes relative to OsACTIN1. Error bars show SD from three replicates.
(b) Alterations in the second leaf lamina joint of wild-type and 725Ri-1 plants in response to BL treatment.
(c) Measurements of angles at the second leaf lamina joint. Values are means ± SD (= 20).

To further assess BR-related physiological defects in sdg725 plants, we performed a lamina joint bending assay by treatment of the 725Ri-1 and wild-type rice plants with brassinolide (BL, a type of bioactive BR). When the wild-type plants were treated with increasing concentrations of BL, the angles of the lamina joint increased. For the 725Ri-1 mutant plants, the angles also increased with BL treatment, but the extent was less than that for wild-type plants under the same conditions (Figure 3b,c), indicating that sdg725 plants can respond to exogenous BL but with reduced sensitivity. All these data suggest that the BR biosynthesis and signaling pathways are disturbed in SDG725 knockdown plants.

Levels of H3K36me2 and H3K36me3 in BR-related genes are decreased in SDG725 knockdown plants

To obtain insight into the molecular mechanism of SDG725-mediated gene expression, we firstly investigated histone methylation levels in D11 chromatin (Figure 4a) by ChIP assay. Compared with wild-type plants, the 725Ri-1 mutant showed an obvious reduction in H3K36me2 and H3K36me3 in most regions of D11 chromatin, and an increased level of mono-methylated H3K36 and tri-methylated H3K4 (Figure 4b). Notably, H3K36me2 and H3K36me3 accumulated predominantly in 3′ coding regions of D11 (Figure 4b). With regard to the repressive marks, there was an increased level of tri-methylated H3K9 but no obvious change in H3K27 tri-methylation in the 725Ri-1 mutant compared to wild-type plants (Figure 4b). ChIP experiments were again performed to test the enrichment of H3K36 methylation in 3′ coding regions of BR-related genes. As shown in Figure 4(c), H3K36me2 and H3K36me3 levels were decreased to variable extents in some regions of tested genes, with decreases at BRI1 and BU1 loci among the more obvious. These results indicate that the down-regulation of genes involved in BR biosynthesis and signaling pathways in SDG725 knockdown plants is due to the altered methylation status on histone H3 at the chromatin regions of these genes.

Figure 4.

 ChIP analyses at BR-related gene loci.
(a) Schematic representation of the D11 gene structure. Black boxes represent exons, lines represent promoter and introns, and bars labeled 1–8 represent regions amplified by quantitative real-time PCR.
(b) ChIP analysis at the D11 locus using various antibodies against modified histone H3, as indicated at the top of each panel. The fold enrichment is normalized by the value of the input, and is referenced to that of OsACTIN1. Error bars show SD from three replicates.
(c) Levels of H3K36 methylation at BR-related gene loci. The results show the enrichment of mono-, di-, and tri-methylation of H3K36, respectively, relative to OsACTIN1. Error bars show SD from three replicates.
(d) The relative binding activity of SDG725 at BR-related gene loci by ChIP analysis using an antibody against SDG725. The values are normalized by input, and is expressed relative to that of OsACTIN1. Error bars show SD from three replicates.

SDG725 protein directly binds to certain chromatin regions of target genes in the nucleus

To further investigate the role of SDG725 in transcription regulation, we performed analysis at the SDG725 protein level. Consistent with the SDG725 function in the nucleus, we found that the EYFP–SDG725 fusion protein was localized in the nucleus in transgenic tobacco (Nicotiana tabacum) BY2 cells. Although EYFP-SDG725C (amino acids 1240–2150) was also localized in the nucleus, EYFP–SDG725N (amino acids 1–1255) was mainly located in the cytoplasm (Figure S1), indicating that the NLS (nuclear localization site) is probably located in the C-terminal region of the SDG725 protein. To analyze the localization of endogenous SDG725 protein in rice, we produced specific antibodies against SDG725. The antibody specificity was confirmed by Western blot analysis using recombinant GST–SDG725N containing amino acids 1–528 of SDG725 (Figure S2). Using this antibody, we investigated the chromatin-binding activity of SDG725 by ChIP assay. ChIP analysis showed that the chromatin-binding ability of SDG725 with target genes was reduced at 3′ coding regions of the D11, BRI1 and BU1 genes in the 725Ri-1 mutant compared to wild-type plants (Figure 4d).

Combined with the decreased H3K36me2 and H3K36me3 levels in BR-related genes, we conclude that SDG725 can directly bind its target genes and regulate gene expression via H3K36 methylation.

Discussion

In this study, we have demonstrated that the rice SDG725 gene encodes a specific H3K36 methyltransferase that plays key roles in rice plant development. Native mononucleosomes or reassembled oligonucleosomes but not recombinant H3 or core histones (Figure S3) were suitable substrates for SDG725 in our in vitro activity assay, suggesting that the higher-order structure of nucleosomes is essential for SDG725 activity. Knockdown of SDG725 leads to a significant decrease in planta of H3K36me2 and H3K36me3 for several examined genes. By contrast, the level of H3K36me1 was increased in most of the analyzed chromatin regions, implying that SDG725 may be responsible for H3K36 di- and tri- but not mono-methylation. Although their substrate specificity has not been examined in vitro, Arabidopsis SDG4, SDG8 and SDG25 have been previous reported to be involved in H3K36 methylation in planta (Kim et al., 2005; Zhao et al., 2005; Cartagena et al., 2008; Berr et al., 2009). Similar to the rice sdg725 mutant, the Arabidopsis sdg8 mutant also exhibited increased levels of H3K36me2/3 but reduced levels of H3K36me1 (Xu et al., 2008). This is consistent with sequence comparison showing that the SDG725 and SDG8 proteins are closely related (Berr et al., 2009) and share great similarity in terms of evolutionarily conserved functional domain organization (Figure S4).

The abnormal morphologies of the SDG725 knockdown plants, such as dwarfism and erect leaves, are common features of rice BR-related mutants. Similar to many known BR-related mutants, the dwarf phenotype of SDG725 knockdown plants is probably caused by reduced cell elongation, because the cell length was obviously decreased in the elongated zone of the second internode in the 725Ri-1 mutant compared with wild-type plants (Figure S5). By molecular analyses, we found that three previously identified rice BR-related genes were obviously down-regulated in 725Ri mutants, i.e. D11, which is involved in BR biosynthesis, and BRI1 and BU1, which are involved in BR signaling pathways. The level of H3K36me2/3 and the chromatin binding activity of SDG725 in these gene loci were also significantly decreased in SDG725 knockdown plants. By contrast, expression of some other BR-related genes, including D2, BRD2 and BZR1, and the binding of SDG725 to these three genes were not obviously altered in SDG725 knockdown plants, indicating that only specific BR-related genes are targeted by SDG725. Similar BR-related functions were not previously reported in the Arabidopsis sdg8, sdg4 or sdg25 mutants. Therefore, it remains to be examined whether H3K36 methylation plays critical BR-related functions in plants other than rice. The sdg8 mutant plants exhibited pleiotropic phenotypes, including early flowering, increased shoot branching, altered carotenoid composition, mis-formed reproductive organs and reduced fertility, and impaired plant defense against fungal pathogens (Kim et al., 2005; Zhao et al., 2005; Dong et al., 2008; Xu et al., 2008; Cazzonelli et al., 2009; Grini et al., 2009; Berr et al., 2010). The distinct phenotypes of plant growth and development between the rice sdg725 and Arabidopsis sdg8 mutants are probably due to the divergence of the two orthologs in regulating organism-specific targets in different plant species. In support of this, the sdg8 early-flowering phenotype is associated with down-regulation of FLC (Kim et al., 2005; Zhao et al., 2005), a key floral repressor that is present in Arabidopsis but absent in rice (Tadege et al., 2003).

Like SDG8, SDG725 contains a CW domain (Figure S4), which was recently shown to bind methylated H3K4 (Hoppmann et al., 2011). In addition to the changes in H3K36 methylation, D11 chromatin also contains an increased level of tri-methylated H3K4 in sdg725 mutant plants. Both increases and decreases of H3K4 methylation have been observed previously in the sdg8 mutant (Kim et al., 2005; Zhao et al., 2005; Xu et al., 2008). It appears that an interplay between H3K4 and H3K36 methylation exists in Arabidopsis as well as in rice. We also detected an increase in tri-methylated H3K9 at D11 chromatin in sdg725 mutant plants. The increased level of the repressive mark tri-methylated H3K9 appears to be correlated with down-regulation of D11 expression. It was reported that ELF6 (Early Flowering 6) and its homolog REF6 (Relative of Early Flowering 6), which are JmjN/C domain-containing histone demethylases in Arabidopsis, interact with BES1 (also known as BZR2), a transcription factor that is homologous to rice BZR1, and probably modify BR-related gene expression by affecting H3K9 methylation (Yu et al., 2008). More recently, AtIWS1 (Interact With Spt6) was also reported to interact with BES1 and to be required for BR-induced gene expression in Arabidopsis (Li et al., 2010). In yeast and humans, IWS1 is implicated in transcription elongation after RNA polymerase II recruitment by interacting directly with elongation factor Spt6 (Krogan et al., 2002; Liu et al., 2007). Human IWS1 is able to recruit the human H3K36 methyltransferase HYPB (also known as Setd2) to the RNA polymerase II elongation complex (Yoh et al., 2008). Gowever, in Arabidopsis, loss-of-function of AtIWS1 caused inhibition of H3K27me3 but not H3K36me3 at the nitrate transporter gene NRT2.1 in response to induction by high nitrate supply (Widiez et al., 2011). In addition, REF6 was recently shown to be an H3K27-specific demethylase (Lu et al., 2011). Therefore, more studies are necessary to unravel the complex layers of active and repressive histone methylation marks involved in BR-related gene expression and physiological function in Arabidopsis and rice.

In conclusion, our study demonstrates that SDG725 binds directly to chromatin, methylates H3K36, and activates expression of the BR-related genes D11, BRI1 and BU1. The study highlights the importance of an epigenetic mark in the regulation of biosynthesis and signaling gene expression of the important phytohormone BR. The SDG725-mediated regulation of BR-related genes modulates several traits, e.g. rice plant architecture and height, seed size and seed number, that are of great interest for rice breeding.

Experimental Procedures

Plant materials and growth conditions

The plants used in this work are in the Oryza sativa spp. japonica background. Plants for phenotype analysis were cultured in the field. Seedlings for RT-PCR and ChIP experiments were grown on agar-solidified half-strength MS medium M0222 (Duchefa, http://www.duchefa.com), at 30°C under 12 h light/12 h dark conditions.

Isolation of SDG725 cDNA and protein subcellular localization

Full-length cDNA of SDG725 amplified by RT-PCR and verified by sequencing was fused in-frame with EYFP and inserted into an estradiol-inducible vector pER8 (Zuo et al., 2000) to produce pER8:EYFP-SDG725. The inducible vector pER8 is often used to avoid gene silencing or harmful effects of the expressed protein on cells/plants. Vector pER8:EYFP-SDG725N, encoding amino acids 1–1255 of SDG725, and vector pER8:EYFP-SDG725C, encoding amino acids 1240–2150, which are also induced by estradiol were constructed in a similar way. The primers used to produce the constructs are listed in Table S2. The plant expression vectors were introduced into Agrobacterium tumefaciens LBA4404, and the resulting strains were used to transform tobacco BY2 cells as described previously (Ding et al., 2007a). The subcellular localizations of EYFP-fused proteins were imaged using an LSM 510 confocal microscope (Zeiss, http://www.zeiss.com/).

Recombinant protein production and histone methyltransferase assay

To generate the SET domain of SDG725 protein (SDG725s) for the in vitro histone methyltransferase assay, the fragment of SDG725 encoding amino acids 1216–1552 of SDG725 protein was amplified by PCR using the primers in Table S2, and was then inserted into pGEX-4T1 (GE Healthcare, http://www.gehealthcare.com) to produce GST fusion proteins in E. coli. The pGEX-4T1:SDG725N vector encoding amino acids 1–528 of SDG725 was constructed similarly for SDG725 antibody test. Expression and purification of GST-fused proteins from bacteria were performed as described previously (Dong et al., 2005). Histone methyltransferase assays were performed as previously described (Ding et al., 2007a). A 30μl reaction mixture containing substrate, enzyme and 250 nCi S-adenosyl-[methyl-14C]-l-methionine (Amersham, http://www.amersham.com) in buffer containing 50 mm Tris/HCl, pH 8.5, 20 mm KCl, 10 mm MgCl2, 100 μm ZnCl2, 10 mmβ-mercaptoethanol and 250 mm sucrose was incubated for 2 h at 37°C. Reactions were stopped by boiling in SDS loading buffer, and proteins were separated by 15% SDS–PAGE, then visualized using Coomassie brilliant blue R-250 and autoradiography. The mononucleosomes prepared from HeLa cells and the in vitro reconstituted oligonucleosomes were provided by Dr Yi Zhang (University of North Carolina, USA) and Dr Bing Zhu (NIBS, Beijing, China), respectively.

Transgene constructs and plant transformation

The DNA fragment containing nucleotides 132–663 of the SDG725 coding region was used as inverted repeats to obtain the hairpin structure, using the primers listed in Table S2. The full hairpin structure was inserted into plant expression vector pHB containing a double CaMV 35S promoter, creating the RNAi vector (p35S:SDG725RNAi) for plant transformation. The artificial microRNA725-1 (5′-taaatagaaatgcttctgggc-3′, mismatched bases are italic) and microRNA725-2 (5′-tacacttttagacgtgcccgcc-3′), corresponding to nucleotides 4323–4344 and 5286–5307, respectively, of SDG725, were inserted into the rice MIR528 or Arabidopsis MIR319 backbones, and then sub-cloned into pHB vector to form four artificial microRNA vectors (p35S:MIR528-SDG725-1, p35S:MIR528-SDG725-2, p35S:MIR319-SDG725-1 and p35S:MIR319-SDG725-2) as described previously (Schwab et al., 2006). Agrobacterium tumefaciens (strain EHA105)-mediated plant transformation was performed as described previously (Sun and Zhou, 2008).

Microarray and gene expression analysis

Two independently derived sets of 14-day-old seedlings, including two independent transgenic lines of SDG725 knockdown plants 725Ri-1 and 725Ri-3, as well as the wild-type (50–60 plants per set), were harvested for microarray analysis using the Agilent Rice Oligo Microarray, which was performed by Shanghai Biotechnology Co.,Ltd. (http://www.ebioservice.com/eng/index.asp). Total RNA was prepared using a TRIzol kit according to the manufacturer’s instructions (Invitrogen, http://www.invitrogen.com). The RNA was then used for microarray analysis or reverse transcription using standard procedures with Improm-II reverse transcriptase (Promega, http://www.promega.com). Quantitative real-time PCR was performed using a kit from Takara (http://www.takara-bio.com). Gene-specific primers used in PCR are listed in Table S2.

Lamina joint bending assay

Leaves containing the second lamina of 7-day-old seedlings were first placed in distilled water for 24 h, and then in solutions with various concentrations of BL for another 24 h. The angles of the lamina joints were measured as described previously (Hong et al., 2005).

Chromatin immunoprecipitation assays

Leaves of 14-day-old rice seedlings were used for ChIP assays, using previously described methods (Ding et al., 2010). Anti-trimethyl-H3K4 (07-473), anti-trimethyl-H3K9 (07-442), anti-trimethyl-H3K27 (07-449) and anti-dimethyl-H3K36 (07-369) antibodies were purchased from Millipore (http://www.millipore.com), and anti-monomethyl-H3K36 (ab9048) and anti-trimethyl-H3K36 (ab9050) were purchased from Abcam (http://www.abcam.com). The monoclonal antibody against SDG725 was produced by Abmart (http://www.ab-mart.com.cn), using the protein fragment comprising amino acids 326–516 of SDG725 as the antigen. Quantitative real-time PCR was performed to determine the enrichment of DNA immunoprecipitated in the ChIP experiments, using gene-specific primers listed in Table S2.

Acknowledgements

We thank Dr Xuelu Wang and Dr Hong Ma for critical reading of the manuscript. We are grateful to Dr Yi Zhang (UNC Chapel Hill, USA) and Dr Bing Zhu (NIBS, Beijing, China) for providing nucleosomes. The vector pHB was a gift from Dr Hongquan Yang (SIBS, Shanghai, China). This work was supported in part by grants from the National Basic Research Program of China (2012CB910500) and the Chinese National Scientific Foundation (30800629) to Y.Y., and from the National Basic Research Program of China (2009CB825601 and 2011CB944600) to A.D.

Accession numbers:

The GenBank/EMBL accession number for the SDG725 sequence is NM_106379.

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