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. 2015 Sep 8;169(3):2118–2128. doi: 10.1104/pp.15.00836

Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice1,[OPEN]

Xiangqian Zhang 1,2,3,2, Jing Sun 1,2,3,2, Xiaofeng Cao 1,2,3, Xianwei Song 1,2,3,*
PMCID: PMC4634063  PMID: 26351308

Epigenetic modification of a B3 transcription factor alters rice leaf angle via modulation of brassinosteroid homeostasis.

Abstract

Heritable epigenetic variants of genes, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Epialleles may also provide a new source of beneficial traits for crop breeding, but very few epialleles related to agricultural traits have been identified in crops. Here, we identified Epi-rav6, a gain-of-function epiallele of rice (Oryza sativa) RELATED TO ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) 6 (RAV6), which encodes a B3 DNA-binding domain-containing protein. The Epi-rav6 plants show larger lamina inclination and smaller grain size; these agronomically important phenotypes are inherited in a semidominant manner. We did not find nucleotide sequence variation of RAV6. Instead, we found hypomethylation in the promoter region of RAV6, which caused ectopic expression of RAV6 in Epi-rav6 plants. Bisulfite analysis revealed that cytosine methylation of four CG and two CNG loci within a continuous 96-bp region plays essential roles in regulating RAV6 expression; this region contains a conserved miniature inverted repeat transposable element transposon insertion in cultivated rice genomes. Overexpression of RAV6 in the wild type phenocopied the Epi-rav6 phenotype. The brassinosteroid (BR) receptor BR INSENSITIVE1 and BR biosynthetic genes EBISU DWARF, DWARF11, and BR-DEFICIENT DWARF1 were ectopically expressed in Epi-rav6 plants. Also, treatment with a BR biosynthesis inhibitor restored the leaf angle defects of Epi-rav6 plants. This indicates that RAV6 affects rice leaf angle by modulating BR homeostasis and demonstrates an essential regulatory role of epigenetic modification on a key gene controlling important agricultural traits. Thus, our work identifies a unique rice epiallele, which may represent a common phenomenon in complex crop genomes.

Epigenetic gene variants (epialleles) carry heritable changes in gene expression that do not result from alterations in the underlying DNA sequence (Kakutani, 2002). In eukaryotes, cytosine DNA methylation, a conserved epigenetic mark, plays essential roles in the silencing of transposable elements (TEs) and genes (Law and Jacobsen, 2010). In higher plants, the few known epialleles involve alterations in DNA methylation, indicating that this epigenetic marker makes a large contribution to epigenetic diversity. The Arabidopsis (Arabidopsis thaliana) clark kent epiallele, which has hypermethylated cytosines at the SUPERMAN locus, causes increased numbers of stamens and carpels (Jacobsen and Meyerowitz, 1997). Also, DNA hypomethylation at two direct repeats in the promoter region of FLOWERING WAGENINGEN (FWA; Soppe et al., 2000) causes the late-flowering phenotype in Arabidopsis plants carrying the fwa epiallele. Plants carrying the natural epiallele hypermethylated at Linaria cycloidea-like show altered floral symmetry, from bilateral to radial, in Linaria vulgaris (Cubas et al., 1999). In tomato (Solanum lycopersicum), the Colorless nonripening phenotype results from hypermethylation at the promoter of SQUAMOSA promoter-binding protein like (Manning et al., 2006). In melon (Cucumis melo), the transition from male to female flowers results from DNA hypermethylation in the promoter of CmWIP1, as mediated by a transposon insertion in gynoecious varieties (Martin et al., 2009).

Work in rice (Oryza sativa) has found only two epialleles, Epi-d1 and Epi-df, both of which show a dwarf phenotype (Miura et al., 2009; Zhang et al., 2012). Epi-d1 is a spontaneous epiallele that shows a metastable dwarf phenotype caused by DNA hypermethylation in the promoter region of DWARF1 (Miura et al., 2009). Epi-df is a gain-of-function epiallele caused by hypomethylation in the 5′ region of FERTILIZATION-INDEPENDENT ENDOSPERM1 (FIE1). The ectopic expression of FIE1 in Epi-df results in dwarf and various floral defects that are inherited in a dominant manner (Zhang et al., 2012).

In Arabidopsis, DNA methylation occurs in three sequence contexts: CG, CHG, and CHH (where H = A, C, or T) catalyzed by the de novo DNA methyltransferase DOMAINS REARRANGED METHYLASE2 (DRM2; Cao and Jacobsen, 2002). In the symmetrical contexts, DNA METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 maintain methylation in the CG and CHG contexts, respectively (Lindroth et al., 2001; Law and Jacobsen, 2010). Small interfering RNAs trigger de novo methylation in all sequence contexts and also trigger the maintenance of CHH methylation via RNA-directed DNA methylation predominately mediated by DRM2 (Cao and Jacobsen, 2002; Law and Jacobsen, 2010).

DNA methylation may be more prevalent and important in rice than in Arabidopsis, owing to the large numbers of TEs in the rice genome. In fact, Arabidopsis mutants of various DNA methyltransferases or DECREASE IN DNA METHYLATION1 show few or no developmental defects (Vongs et al., 1993; Lindroth et al., 2001; Cao and Jacobsen, 2002; Saze et al., 2003). By contrast, the rice met1a null mutant shows either viviparous germination or early embryonic lethality (Hu et al., 2014; Yamauchi et al., 2014). Moreover, impairment of the RNA-directed DNA methylation pathway proteins Dicer-like 3a and DRM2 causes drastic and pleiotropic developmental phenotypes (Moritoh et al., 2012; Wei et al., 2014).

Leaf angle is an important agronomic trait that directly affects crop architecture and grain yields (Sinclair and Sheehy, 1999). Crops with erect leaves capture more light for photosynthesis and are suitable for dense planting, all of which increase yields (Sakamoto et al., 2006). In rice, the brassinosteroid (BR) phytohormones participate in the determination of leaf angle (Tong and Chu, 2012; Zhang et al., 2014). BR-deficient or BR-insensitive mutants display erect leaves, while overexpression of BR biosynthesis genes or signaling components results in less erect leaves with large leaf inclination (Yamamuro et al., 2000; Hong et al., 2005; Bai et al., 2007). For example, loss-of-function mutants of rice BR INSENSITIVE1 (OsBRI1) and EBISU DWARF (D2), which encode the rice BR receptor kinase and a BR synthesis enzyme, respectively, show erect leaves (Yamamuro et al., 2000; Hong et al., 2003). In rice, RELATED TO ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1; RAV)-LIKE1 (RAVL1), a B3 DNA-binding domain-containing protein, maintains BR homeostasis via the coordinated activation of BRI1 and BR biosynthetic genes D2, DWARF11 (D11), and BR-DEFICIENT DWARF1 (BRD1; Je et al., 2010). The ravl1 mutant and RAVL1 overexpression lines showed erect leaves and large leaf angles, respectively (Je et al., 2010).

Here, we identified a natural epiallele of rice RAV6 and found that plants carrying this epiallele show increased leaf angle and small grains, as well as hypomethylation in the RAV6 promoter region and ectopic expression of RAV6. Our work revealed that epigenetic modification of RAV6 plays an essential role in the regulation of important agricultural traits in rice and found that RAV6 acts via BR homeostasis.

RESULTS

A Semidominant Mutant Shows Large Lamina Inclination and Small Seed Size

A spontaneously occurring rice mutant with large leaf angle and small seeds was isolated from the japonica rice ‘Zhonghua11’ and was named Epi-rav6 based on our subsequent characterization (see below). Compared with the wild type (cv Zhonghua11), almost all leaf angles throughout all developmental stages were larger in Epi-rav6 plants (Fig. 1A). The mutant had almost the same amount of rachis as the wild type but had obviously smaller grains (Fig. 1, B–D; Supplemental Fig. S1). Compared with the wild type, the Epi-rav6 seeds were markedly smaller, by 12.8% in length (average 7.8 mm in the wild type to 6.8 mm in Epi-rav6; Fig. 1, C and D), 25% in width (average 3.6 mm in the wild type to 2.7 mm in Epi-rav6; Fig. 1, B and D), and 20% in thickness (average 2.4 mm in the wild type to 1.9 mm in Epi-rav6; Fig. 1E). Thus, the 1,000-grain weight of Epi-rav6 seeds was only 57% of that of the wild type (Fig. 1E). Together, these findings indicate that Epi-rav6 influences leaf angle and grain size.

Figure 1.

Figure 1.

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Characterization of a semidominant rice mutant with larger leaf angle and smaller seeds. A, The wild-type (WT), heterozygous Epi-rav6 (+/–), and homozygous Epi-rav6 (−/−) plants at tilling stage. B and C, Comparison of grain and seed width (B) and length (C) between the wild type and Epi-rav6 (+/–). D, Characterization of seed weight, seed length, seed width, and seed thickness in the wild type and the Epi-rav6 mutant. Values are means ± sd. In each graph, statistically significant differences are indicated by double asterisks (**P < 0.01). Bars = 20 cm (A) and 1 cm (B and C).

To determine the inheritance of Epi-rav6, we examined the phenotypes of 260 progeny of self-pollinated Epi-rav6 plants. Three phenotypes segregated in these progeny: 62 were like the wild type, 143 were like Epi-rav6, and 55 had severe defects, including fewer tillers, very large leaf angles, and sterility (Fig. 1A). The ratio of the wild type to Epi-rav6 like to severe phenotypes was 1:2.3:0.89 (χ2 = 2.98, P > 0.05). In addition, 27 F1 plants derived from the cross between Epi-rav6 and cv Zhonghua11 showed nearly a 1:1 ratio of wild-type progeny and Epi-rav6-like progeny (Supplemental Fig. S2). This genetic evidence demonstrated that the mutant is controlled by a single, semidominant locus and the derived allele is heterozygous. Thus, we regarded Epi-rav6-like progeny as heterozygotes and refer to them as Epi-rav6 (+/–); we also regarded the progeny with severe defects as homozygotes and refer to them as Epi-rav6 (–/–).

Cloning and Characterization of RAV6

To explore the molecular mechanism responsible for the Epi-rav6 phenotype, we used map-based cloning to isolate the causal gene. Using an F2 population derived from a cross between the Epi-rav6 mutant and indica var Huajingxian74, we mapped Epi-rav6 to an 18.5-kb region on chromosome 2. Based on the MSU7.0 annotation (http://rice.plantbiology.msu.edu/), this region contains only the promoter region of a putative gene, Os02g45850 (Fig. 2A). However, we found no nucleotide sequence difference between the mutant and the wild type in this region. We next investigated the expression level of Os02g45850, the only annotated gene in the mapped region. Compared with its low expression level in wild-type leaves, Os02g45850 showed much higher expression in Epi-rav6 (+/–) and Epi-rav6 (–/–) leaves (Fig. 2, B and C). Moreover, the degree of up-regulation was much higher in homozygous than heterozygous lines (Fig. 2, B and C).

Figure 2.

Figure 2.

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Molecular cloning and expression analysis of RAV6. A, Map-based cloning of RAV6. The RAV6 locus was mapped to the long arm of rice chromosome 2 (Chr. 2) between markers RM3512 and RM318. The gene was further delimited to an 18-kb genomic region between the markers ID8-26 and ID5-3 within the bacterial artificial chromosome (BAC) clones AP004178 and AP004255. The number of recombinants is marked corresponding to the molecular markers. This 18-kb interval contains the candidate gene Os02g45850. Gray and black boxes indicate the untranslated regions and the coding sequence, respectively. B and C, Expression analysis of RAV6 by reverse transcription-PCR (B) and real-time reverse transcription-PCR (C) in the wild type (WT), Epi-rav6 (+/–), and Epi-rav6 (–/–). Rice elongation factor 1a (OsEF1a) was used as a control. Values are means ± sd of three biological replicates.

To further confirm that the ectopic expression of Os02g45850 is responsible for the developmental defects in Epi-rav6, we overexpressed Os02g45850 in the wild type under the control of the maize (Zea mays) Ubiquitin promoter. All the positive transformants with high expression of Os02g45850 displayed large leaf inclination and small grains (Fig. 3). Thus, we concluded that the abnormal phenotype in this mutant results from high expression of Os02g45850. Os02g45850 encodes a B3 DNA-binding domain-containing protein, which belongs to the RAV family and was named RAV6 (Romanel et al., 2009). Therefore, we named the allele Epi-rav6.

Figure 3.

Figure 3.

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Overexpression of RAV6 phenocopies the Epi-rav6 mutant phenotype. A, The morphologies of wild-type (WT), Epi-rav6 (+/–), and transgenic plants overexpressing RAV6 at seedling stages. B, Relative expression analysis of RAV6 in wild-type, Epi-rav6 (+/–), and the RAV6-overexpressing plants at seedling stage. C and D, The grain morphologies of the transformed wild-type plants. OV-1 and OV-2 indicate transgenic plants overexpressing RAV6. Bars = 1 cm (A) and 2 cm (C and D).

RAV6 Regulates Leaf Angle via Mediating BR Homeostasis

The plant-specific RAV family belongs to the B3 transcription factor superfamily and consists of 12 members in rice, including RAV6 (Romanel et al., 2009). Phylogenetic analysis showed that the RAV6 (encoded by Os02g45850) is more closely related to RAVL1 (encoded by Os04g49230) than to other members of the RAV family (Fig. 4A). Comparison of deduced amino acid sequences suggested that RAV6 exhibits a high degree of sequence identity with RAVL1, especially in the B3 DNA-binding domain (Fig. 4B). These observations indicate that RAV6 and RAVL1 may share similar functions. RAVL1 maintains BR homeostasis via the coordinated activation of BR synthetic and receptor genes in rice (Je et al., 2010). In addition, plants overexpressing RAVL1 showed increased lamina inclination, like the Epi-rav6 mutants (Je et al., 2010), suggesting that RAV6 may also be involved in the BR pathway via activation of BR biosynthetic and signaling genes. To confirm this, we measured expression of representative genes encoding BR biosynthesis enzymes and the BR receptor, known RAVL1 targets, in Epi-rav6 mutants. As expected, expression of BR synthetic genes, including D2, D11, and BRD1, was dramatically up-regulated in the Epi-rav6 mutant (Fig. 5A). Similarly, we also observed higher levels of induction for the BR receptor gene BRI1 in Epi-rav6 mutants. These data suggest that RAV6 affects the expression of genes involved in BR signaling and BR biosynthesis.

Figure 4.

Figure 4.

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Evolutionary relationship between RAV6 and RAVL1 in rice. A, Phylogenetic relationship of the RAV subfamily of B3 DNA-binding domain-containing proteins in rice. The bar indicates substitutions per site. B, Amino acid sequence alignment of rice RAV6 and RAVL1. Identical and similar amino acids are boxed in black and gray, respectively. The signature B3-binding domain is underlined.

Figure 5.

Figure 5.

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RAV6 regulates leaf angle by controlling genes affecting BR homeostasis. A, Real-time reverse transcription-PCR analyses of BRD1, D2, D11, and BRI1 in wild-type (WT), Epi-rav6 (+/–), and Epi-rav6 (–/–) plants. 25S ribosomal RNA was used as a control. Values are means ± sd of three biological replicates. B, Lamina inclination of 45-d-old seedlings in wild-type, Epi-rav6 (+/–), and Epi-rav6 (–/–) plants subjected to Pcz treatment for 15 d. Bar = 20 cm.

To further confirm that the large lamina inclination of rav6 mutant results from BR overdose, we postulated that treatment with a BR inhibitor would restore, at least partially, the mutant phenotype. As expected, in the presence of propiconazole (Pcz), a BR biosynthesis inhibitor (Hartwig et al., 2012), the lamina inclination of Epi-rav6 mutant was restored (Fig. 5B). Taken together, these results suggest that RAV6 is involved in BR-mediated developmental processes.

Hypomethylation of the Promoter Region of RAV6 in Epi-rav6 Plants

We found no nucleotide sequence difference between the wild type and Epi-rav6, but we did observe alteration of RAV6 expression levels in the Epi-rav6 plants, suggesting that the mutation may result from an epigenetic modification. We therefore investigated the DNA methylation status of the RAV6 locus. The promoter region of RAV6 contains many TEs, including an adh5-like miniature inverted repeat transposable element (MITE), a short interspersed nuclear element (SINE), two unclassified retrotransposons, and a Snabo-like MITE (Fig. 6A; Supplemental Fig. S3A). TEs, especially those proximal to genes, can act as epigenetic mediators to influence nearby gene expression (Hollister and Gaut, 2009; Hollister et al., 2011; Wei et al., 2014). So, we performed bisulfite sequencing to analyze methylation in a 2,358-bp genomic region consisting of 573 bp of gene body, 1,321 bp of upstream region, and 464 bp of 5′ distal retrotransposon sequence (Supplemental Fig. S3A). In the wild type and homozygous Epi- rav6 mutants, we found no DNA methylation in all three sequence contexts in the 573-bp gene body of RAV6. However, we observed higher CG and CHG but not CHH DNA methylation in proximal and distal upstream regions of RAV6 in the wild type compared with the homozygous Epi-rav6 mutant. All the changed methylation sites occurred in a contiguous 96-bp region at –600 to –504 bp relative to the transcriptional start site, including the closest MITE (adh5-like MITE). This region is hypermethylated in the wild type but hypomethylated in Epi-rav6, containing four CG sites and two CHG sites (Fig. 6B; Supplemental Fig. S3B). To further confirm that the ectopic expression of RAV6 in Epi-rav6 results in hypomethylation of the promoter region, we treated the wild-type seeds with 5-aza-2′ deoxycytidine (5-aza-dC), an inhibitor of DNA methylation (Chang and Pikaard, 2005). The expression levels of RAV6 were measured in 7-d-old seedlings with or without 5-aza-dC treatment. Treatment with 5-aza-dC up-regulated RAV6 expression to varying degrees in three cultivated rice strains, including two japonica (cv Zhonghua11 and Nipponbare) and one indica (cv Kasalath) accessions (Fig. 6C). These three cultivated accessions also contain the MITE (see below); therefore, these results indicate that the DNA methylation mediated by the TE in the 5′ upstream sequence of RAV6 plays an essential role in the regulation of RAV6 expression.

Figure 6.

Figure 6.

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DNA methylation analysis of the RAV6 locus. A, Schematic representation of RAV6 with putative MITE, MITE-adh5-like DNA transposon. Boxes indicate exon (black), untranslated regions (white), and MITE (green). The red line shows the region used for analysis of DNA methylation by bisulfite sequencing (B). B, DNA methylation status of bisulfite-sequenced region (as indicated in A) in wild-type (WT) and Epi-rav6 (–/–) plants. Histograms represent the percentage of CG (red), CNG (blue), and CHH (green). C, Real-time reverse transcription-PCR analyses of RAV6 expression in 7-d-old seedlings treated with (+) or without (–) 5-aza-dC, an inhibitor of DNA methylation. Rice elongation factor 1a (OsEF1a) was used as a control. Values are means ± sd of three biological replicates.

The Effect of MITE-Mediated DNA Methylation on RAV6 Expression Is Conserved in Cultivated Rice

MITEs are short (less than 600 bp), nonautonomous DNA transposons. As the TEs with the highest copy numbers in the rice genome, MITEs mainly occur in the chromosomal arms, especially in the vicinity of genes (Jiang et al., 2004). According to the Plant Repeat Database, the japonica rice ‘Nipponbare’ genome contains 2,984 copies of the adh5-like MITE (Ouyang and Buell, 2004). The adh5-like MITE in the RAV6 promoter region is 100 bp long and located 198 bp upstream of the RAV6 gene body.

To further explore the evolutionary significance of the epigenetic regulation of RAV6, we investigated the natural variation of the MITE in the promoter region of the RAV6 locus in 40 accessions of cultivated rice, which represent all of the major groups of Asian cultivated rice (Xu et al., 2012). The results showed that the TE insertion in the promoter region of RAV6 is conserved in all cultivated rice accessions (data not shown). To further confirm this, we aligned the sequences of the MITEs, as well as the 5′ region of RAV6, in four known assembled rice genomes, including three cultivated rice strains, cv Nipponbare, 93-11, and Kasalath, and the wild rice Oryza brachyantha (Yu et al., 2002; International Rice Genome Sequencing Project, 2005; Chen et al., 2013; Sakai et al., 2014). The MITE insertion was conserved in the cultivated rice genomes but was absent in O. brachyantha (Fig. 7A). Consistent with this, the sequence of the 5′ promoter region of RAV6 is also more conserved in cultivated rice, especially the six cytosine methylation sites, which are essential for regulation of RAV6 expression (Fig. 7A).

Figure 7.

Figure 7.

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Genome sequence, gene expression, and DNA methylation analysis of the RAV6 locus in different rice accessions. A, Comparative sequence analysis of the RAV6 locus in four assembled rice genomes. The synteny of the RAV6 locus in cv Nipponbare, 93-11, and Kasalath and O. brachyantha is shown in the top section. The bottom section shows an alignment of the DNA sequence 258 bp upstream and the 5′ gene body of RAV6 in four assembled rice genomes. The MITE region and 5′ gene body are underlined by green and black lines, respectively. The asterisk indicates the cytosine region, whose degree of methylation was reduced in Epi-rav6 (–/–), as shown in Figure 6B. B, Expression analysis of RAV6 by reverse transcription-PCR (top) and real-time reverse transcription-PCR (bottom) in cultivated rice strains and Epi-rav6 (–/–). Zhonghua11, Nipponbare, Kongyu131, and Koshihikari are japonica cultivars, and Kasalath is an indica cultivar. Rice elongation factor 1a (OsEF1a) was used as a control. Values are means ± sd of three biological replicates. C, DNA methylation status of the bisulfite-sequenced region (as indicated in Fig. 6A) in the wild type (WT; cv Zhonghua11), Epi-rav6 (–/–), and four cultivated rice strains. Histograms represent the percentage of methylation in the CG (red), CNG (blue), and CHH (green) contexts.

To further determine the conserved effect of the MITE-mediated DNA methylation on RAV6 expression in cultivated rice accessions, we measured RAV6 transcript levels and DNA methylation patterns in four cultivated rice strains, including three japonica (cv Nipponbare, Kongyu131, and Koshihikari) and one indica (cv Kasalath) accessions. Compared with the high expression of RAV6 observed in Epi-rav6, we observed little or no RAV6 expression in leaves at the tillering stage in all cultivated rice strains (Fig. 7B). Like the pattern of wild-type cv Zhonghua11, the other four cultivated rice accessions also showed higher DNA methylation in the contiguous 96-bp region of RAV6 promoter, in contrast with very low DNA methylation in Epi-rav6 (Fig. 7C; Supplemental Fig. S4). Thus, it is reasonable to predict that the TE insertion occurred prior to divergence of indica and japonica rice and that the TE-mediated epigenetic regulation of RAV6 expression is evolutionarily conserved in cultivated rice.

DISCUSSION

In this work, we identified an epiallele of rice RAV6; this epiallele shows hypomethylation of the RAV6 promoter region, causing ectopic RAV6 expression, larger lamina inclination, and smaller grains. We found that RAV6 expression is associated with extensive methylation of a MITE along with the nearby sequence in the 5′ region of this gene. RAV6 encodes a B3 DNA-binding domain-containing protein that has the most sequence similarity to RAVL1, which can maintain BR homeostasis to control rice development (Je et al., 2010). Functional analysis revealed that RAV6 coordinately activates the BR receptor BRI1 and BR biosynthetic genes to control rice leaf angle. This suggests that RAV6 performs a similar function to its homolog, RAVL1.

The Rice Mutant Epi-rav6 Shows Hypomethylation of DNA at RAV6

Ectopic expression of RAV6 in Epi-rav6 mutants indicates that it is a gain-of-function allele of RAV6. Although we found no DNA sequence changes in RAV6, we did find that Epi-rav6 shows a loss of cytosine DNA methylation in a MITE along with the nearby sequence located in its 5′ promoter region (Fig. 6A; Supplemental Fig. S3A). Thus, Epi-rav6 has similar features to the Arabidopsis fwa mutant, the first DNA-hypomethylated mutant identified in plants, with hypomethylation at two direct repeats in the 5′ region of FWA and ectopic expression of FWA (Soppe et al., 2000). In rice, only two epialleles, Epi-d1 and Epi-df, were identified previously, with hyper- and hypomethylation at the causal genes, respectively (Miura et al., 2009; Zhang et al., 2012). Although both Epi-df and Epi-rav6 are hypomethylated, the methylation sites of the two epialleles differ. Epi-df shows hypomethylation mainly in the coding region and not associated with repeated sequences (Zhang et al., 2012). However, the gain of methylation at the D1 locus in Epi-d1 is associated with the upstream repeat sequences (Miura et al., 2009), similar to fwa and Epi-rav6. Therefore, these repeat elements around the causal genes in Epi-d1, fwa, and Epi-rav6 might function as epigenetic mediators to influence nearby gene expression, further confirming previous findings (Hollister et al., 2011; Wei et al., 2014).

The phenotypes of epialleles generally show metastable inheritance with a low percentage of revertants, such as in Epi-d1 and Epi-df. Epi-rav6 is a semidominant mutant, and the homozygous lines are sterile. Therefore, it is impossible to identify authentic revertants between generations (Fig. 1).

RAV6 Controls Rice Lamina Inclination via Mediating BR Homeostasis

The B3 DNA-binding domain-containing proteins are plant-specific transcription factors that play important roles in growth, development, flowering time, seed development, and seed maturation (Swaminathan et al., 2008; Romanel et al., 2009). The B3 domain can bind DNA in a sequence-specific or nonspecific manner and activate or repress the transcription of specific target genes (Je et al., 2010). Both RAV6 and RAVL1 belong to the RAV family and have higher sequence similarity to each other than to other members of the RAV family (Fig. 4). RAVL1 functions as a transcription factor, directly binding BR biosynthetic and receptor genes and thus maintaining BR homeostasis (Je et al., 2010). In Epi-rav6, the direct targets of RAVL1, including BRI1, D2, D11, and BRD1, showed increased expression (Fig. 5A), indicating that RAV6 might also target these genes. The lines overexpressing RAVL1 showed larger lamina inclination and hypersensitivity to BR (Je et al., 2010). Consistent with this, increased leaf angles were observed in Epi-rav6 mutants and RAV6 overexpression lines (Fig. 3A). Moreover, treatment with BR inhibitor fully rescued the larger lamina inclination in Epi-rav6 (Fig. 5B). All these results indicated that both RAV6 and RAVL1 control leaf angles via maintaining BR homeostasis.

In addition to leaf angle, BRs also influence rice grain size (Zhang et al., 2014). Generally, BR-deficient and BR-insensitive mutants, such as d2, d11, and brd1, showed small grains, while overexpression lines such as Increase Leaf Inclination1 and BRI1-SUPPRESSOR1 showed large lamina joints and increased grain size (Hong et al., 2002, 2003; Tanabe et al., 2005; Tanaka et al., 2009). The Epi-rav6 plants showed activation of BR signaling and large leaf angles, but small grains, which differs from typical mutants affecting BR. We proposed that decreased grain size in Epi-rav6 may result from an integrated effect of changes in expression of multiple target genes. Because RAV6 encodes a B3 DNA-binding domain-containing protein, lots of target genes involved in multiple developmental processes might be influenced by the gain-of-function change of RAV6 in Epi-rav6. This also can be confirmed, as the Epi-rav6 homozygotes showed pleiotropic developmental defects (Fig. 1A).

MATERIALS AND METHODS

Plant Materials and Growth Conditions

A spontaneously occurring rice (Oryza sativa) mutant Epi-rav6 was isolated from a japonica rice ‘Zhonghua11’ population in Guangzhou, China, in 2006. Transgenic plants overexpressing RAV6 were produced by introducing the respective genes driven by the maize (Zea mays) Ubiquitin promoter into wild-type plants. Briefly, the 1,239-bp coding sequence of RAV6 was amplified with the primers OsRAV-1BXF and OsRAV-1239RNS (Supplemental Table S1), and the PCR product was inserted into the BamHI/SpeI sites of pCUbi1390, resulting in a plant expression vector driven by the Ubiquitin promoter, which was then transformed into rice. Plants were grown in the field under natural conditions.

Map-Based Cloning

For map-based cloning of OsRAV6, 725 recessive individual plants showing normal leaf inclinations were selected from an F2 population derived from a cross between the Epi-rav6 mutant and indica var Huajingxian74. Simple sequence repeat markers and insertion/deletion markers on chromosome 2 were used for fine mapping. The RAV6 gene was selected from an approximately 18-kb region as the candidate gene. To find out the mutation site, we amplified the corresponding fragments from the Epi-rav6 mutant and wild-type plants, respectively. Primers used in the map-based cloning are listed in Supplemental Table S1.

Reverse Transcription-PCR and Real-Time PCR

Real-time PCR analysis was performed using the CFX96 Real-Time PCR System (Bio-Rad) and SYBR Green I (S-7567; Invitrogen). PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio). For each sample, quantifications were made in triplicate. Melting curves were read at the end of each amplification by steps of 0.3°C from 65°C to 95°C to ensure that the quantifications were derived from real PCR products and not primer dimers. The primers used for Figure 5A are listed in Supplemental Table S1.

Bisulfite Sequencing

Genomic DNAs were isolated from 3-week-old rice seedlings or the leaf at tillering stage. Bisulfite treatment of genomic DNA was conducted using the Methylation Gold kit according to the manufacturer’s instructions (ZYMO Research Corporation). Treated DNAs were then used for PCR amplification using Hot-Start Taq DNA polymerase (DR007B; TaKaRa). PCR conditions were as follows: 95°C for 5 min; 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 40 s; and 72°C for 10 min. The PCR products were cloned into the pEASY-T1 cloning vector (TransGen Biotech), and individual clones were sequenced. The analysis of sequencing data was performed using KISMETH (Gruntman et al., 2008). The primers used for bisulfite sequencing are listed in Supplemental Table S1.

5-aza-dC Treatment

Rice seeds were soaked in water at 37°C for 16 h. The seeds were then immersed in 20 mm Tris-HCl, pH 7.5, with or without 0.3 mm 5-aza-dC (A3656; Sigma) at room temperature for 48 h in the dark. After washing, seeds were planted in soil. Seven-day-old seedlings were sampled for further analysis.

Phylogenetic Analysis and Alignment

Full-length protein sequences were used for phylogenetic analysis. The sequence of OsRAV6 was downloaded from TIGR (release 7; http://rice.plantbiology.msu.edu/) and used to search the proteome database of rice available at TIGR using BLAST. Alignments of protein sequences were performed using MUSCLE (Edgar, 2004) with default parameters (gap opening, –2.9; gap extension, 0; hydrophobicity multiplier, 1.2; and max iteration, 8). The maximum likelihood tree was constructed using MEGA6 (Tamura et al., 2013). Forty-eight different amino acid substitution models were tested, and the JTT+G+F model (Jones et al., 1992) was considered the best model with the lowest Bayesian Information Criterion scores. All sites of the alignment were used for model testing. The –ln likelihood was 10,996.08. Nonparametric bootstrap analyses (Sanderson and Wojciechowski, 2000) consisted of 1,000 replicates. All four known genome sequences of Oryza spp. were collected, including japonica rice ‘Nipponbare’ (International Rice Genome Sequencing Project, 2005), indica rice ‘93-11’ (Yu et al., 2002), aus rice ‘Kasalath’ (Sakai et al., 2014), and the wild rice Oryza brachyantha (Chen et al., 2013). The genomic DNA sequence of the OsRAV6 gene body and 230-bp upstream region was used as a query sequence, and homologous regions were searched in the other three genomes of Oryza spp. using FASTA (version 36.3.5d; Pearson and Lipman, 1988). Multiple sequence alignment was performed using ClustalW (version 2.1; Thompson et al., 1994; Larkin et al., 2007).

Pcz Treatment

In this study, Pcz, a BR biosynthesis inhibitor (Hartwig et al., 2012), was used to measure the mutants’ growth and morphological responses to BR by observing inclination of lamina joints after exposure to Pcz. For Pcz treatment, the plants were grown in the fields after incubating seeds for 3 d at 30°C to ensure synchronized germination. The 30-d-old seedlings were sprayed with 10 mm Pcz (Banner Maxx; Syngenta) every 3 d. After 15 d, plants were transplanted into pots and photographed. Control plants were grown and not treated with Pcz.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_169_3_2118__index.html (1.5KB, html)

Glossary

BR

brassinosteroid

TE

transposable element

Pcz

propiconazole

5-aza-dC

5-aza-2′ deoxycytidine

Footnotes

1

This work was supported by the National Natural Science Foundation of China (grant nos. 31300987 to X.S. and 30900884 to X.Z.), the Genetically Modified Breeding Major Projects (grant no. 2014ZX08010–002 to X.C.), the Natural Science Foundation of Guangdong Province, China (grant no. 2014A030313457), a postdoctoral fellowship (to J.S.), and the State Key Laboratory of Plant Genomics.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_169_3_2118__index.html (1.5KB, html)
supp_pp.15.00836_PP2015-00836R1_Supplemental_Materialltbl1.pdf (66.3KB, pdf)
supp_pp.15.00836_PP2015-00836R1_Supplemental_Material_fig1.pdf (5.5MB, pdf)
supp_pp.15.00836_PP2015-00836R1_Supplemental_Material_fig2.pdf (2.1MB, pdf)
supp_pp.15.00836_PP2015-00836R1_Supplemental_Material_fig3.pdf (1.3MB, pdf)
supp_pp.15.00836_PP2015-00836R1_Supplemental_Material_fig4.pdf (1.2MB, pdf)

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