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Running Head: 1
Identification of an Epiallele of RAV6 in rice 2
3
Corresponding Author: 4
Xianwei Song 5
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, 6
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 7
Beijing 100101, China. 8
Tel: 86-10-64869203 9
Fax: 86-10-6487342 10
Email: [email protected] 11
12
Research Area: 13
Genes, Development and Evolution (Epigenetics and Gene Regulation) 14
15 16
Plant Physiology Preview. Published on September 8, 2015, as DOI:10.1104/pp.15.00836
Copyright 2015 by the American Society of Plant Biologists
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Title 17
Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 18
19
Authors 20
Xiangqian Zhang1,#, Jing Sun2, # , Xiaofeng Cao2,3 and Xianwei Song2, * 21
#These authors contributed equally to this work. 22
* Corresponding author 23
24
1 Guangdong Engineering Research Center of Grassland Science, College of Forestry and 25
Landscape Architecture, South China Agricultural University, Guangzhou 510642, China. 26
2 State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute 27
of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 28
3 Collaborative Innovation Center of Genetics and Development, Shanghai 200433, China. 29
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One-sentence Summary 31
Epigenetic modification of rice RAV6, which encodes a B3 transcription factor, alters 32
rice leaf angle via modulation of brassinosteroid homeostasis. 33
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Footnotes 43
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Financial sources: 45
This work was supported by the National Natural Science Foundation of China (grant 46
nos. 31300987 to X. Song and 30900884 to X. Zhang); by Genetically Modified 47
Breeding Major Projects (grant no. 2014ZX08010-002 to X. Cao); by Natural Science 48
Foundation of Guangdong Province, China (grant no. 2014A030313457), the 49
postdoctoral fellowship to (J. Sun) and the State Key Laboratory of Plant Genomics. 50
51
Corresponding Author: 52
Xianwei Song 53
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, 54
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 55
Beijing 100101, China. 56
Tel: 86-10-64869203 57
Fax: 86-10-6487342 58
Email: [email protected] 59
60
Author contributions: 61
X.Z designed and performed experiments, J.S analysed data and modified the paper. 62
X.C designed experiments and modified the papers. X.S designed and performed 63
experiments and wrote the paper. 64
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ABSTRACT 71
Heritable epigenetic variants of genes, termed epialleles, can broaden genetic and phenotypic 72
diversity in eukaryotes. Epialleles may also provide a new source of beneficial traits for crop 73
breeding, but very few epialleles related to agricultural traits have been identified in crops. 74
Here, we identified Epi-rav6, a gain-of-function epiallele of rice RELATED TO ABI3 AND 75
VP1 6 (RAV6), which encodes a B3 DNA binding domain-containing protein. The Epi-rav6 76
plants show larger lamina inclination and smaller grain size; these agronomicially important 77
phenotypes are inherited in a semi-dominant manner. We did not find nucleotide sequence 78
variation of RAV6. Instead, we found hypomethylation in the promoter region of RAV6, which 79
caused ectopic expression of RAV6 in Epi-rav6 plants. Bisulfite analysis revealed that 80
cytosine methylation of 4 CG and 2 CNG loci within a continuous 96-bp region plays 81
essential roles in regulating RAV6 expression; this region contains a conserved MITE 82
transposon insertion in cultivated rice genomes. Overexpression of RAV6 in wild type 83
phenocopied the Epi-rav6 phenotype. The brassinosteroid (BR) receptor 84
BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BR biosynthetic genes EBISU DWARF 85
(D2), DWARF11 (D11), and BR-DEFICIENT DWARF1 (BRD1) were ectopically expressed in 86
Epi-rav6 plants. Also, treatment with a BR biosynthesis inhibitor restored the leaf angle 87
defects of Epi-rav6 plants. This indicates that RAV6 affects rice leaf angle by modulating BR 88
homeostasis and demonstrates an essential regulatory role of epigenetic modification on a key 89
gene controlling important agricultural traits. Thus, our work identifies a novel rice epiallele, 90
which may represent a common phenomenon in complex crop genomes. 91
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Keywords: rice, epiallele, DNA methylation, B3 DNA binding protein, brassinosteroid 93
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INTRODUCTION 101
Epigenetic gene variants (epialleles) carry heritable changes in gene expression that do not 102
result from alterations in the underlying DNA sequence (Kakutani, 2002). In eukaryotes, 103
cytosine DNA methylation, a conserved epigenetic mark, plays essential roles in the silencing 104
of transposable elements (TEs) and genes (Law and Jacobsen, 2010). In higher plants, the few 105
known epialleles involve alterations in DNA methylation, indicating that this epigenetic 106
marker makes a large contribution to epigenetic diversity. The Arabidopsis thaliana clark kent 107
(clk) epiallele, which has hypermethylated cytosines at the SUPERMAN locus, causes 108
increased numbers of stamens and carpels (Jacobsen and Meyerowitz, 1997). Also, DNA 109
hypomethylation at two direct repeats in the promoter region of FLOWERING 110
WAGENINGEN (FWA) (Soppe et al., 2000) causes the late-flowering phenotype in 111
Arabidopsis plants carrying the fwa epiallele. Plants carrying the natural epiallele 112
hypermethylated at Linaria cyc-like (Lcyc) show altered floral symmetry, from bilateral to 113
radial, in Linaria vulgaris (Cubas et al., 1999). In tomato, the Colorless non-ripening (Cnr) 114
phenotype results from hypermethylation at the promoter of SQUAMOSA promoter binding 115
protein-like (SBP-box) (Manning et al., 2006). In melon (Cucumis melo L.J), the transition 116
from male to female flowers results from DNA hypermethylation in the promoter of CmWIP1, 117
as mediated by a transposon insertion in gynoecious varieties (Martin et al., 2009). 118
Work in rice has found only two epialleles, Epi-d1 and Epi-df, both of which show a 119
dwarf phenotype (Miura et al., 2009; Zhang et al., 2012). Epi-d1 is a spontaneous epiallele 120
that shows a metastable dwarf phenotype caused by DNA hypermethylation in the promoter 121
region of DWARF 1 (Miura et al., 2009). Epi-df is a gain-of-function epiallele caused by 122
hypomethylation in the 5’ region of FERTILIZATION-INDEPENDENT ENDOSPERM1 123
(FIE1). The ectopic expression of FIE1 in Epi-df results in dwarf and various floral defects 124
that are inherited in a dominant manner (Zhang et al., 2012). 125
In Arabidopsis, DNA methylation occurs in three sequence contexts: CG, CHG, and 126
CHH (where H= A, C, or T) catalyzed by the de novo DNA methyltransferase DRM2 (Cao 127
and Jacobsen, 2002). In the symmetrical contexts, DNA methyltransferases 1 (MET1) and 128
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CHROMOMETHYLASE3 (CMT3) maintain methylation in the CG and CHG contexts, 129
respectively (Law and Jacobsen, 2010; Lindroth et al., 2001). Small interfering RNAs 130
(siRNAs) trigger de novo methylation in all sequence contexts and also trigger the 131
maintenance of CHH methylation, via RNA-directed DNA methylation predominately 132
mediated by DRM2 (Cao and Jacobsen, 2002; Law and Jacobsen, 2010). 133
DNA methylation may be more prevalent and important in rice than in Arabidopsis, 134
owing to the large numbers of TEs in the rice genome. In fact, Arabidopsis mutants of various 135
DNA methyltransferases or DDM1 show few or no developmental defects (Cao and Jacobsen, 136
2002; Lindroth et al., 2001; Saze et al., 2003; Vongs et al., 1993). By contrast, the rice met1a 137
null mutant shows either viviparous germination or early embryonic lethality (Hu et al., 2014; 138
Yamauchi et al., 2014). Moreover, impairment of the RNA-directed DNA methylation 139
pathway proteins DCL3a and DRM2 causes drastic and pleiotropic developmental 140
phenotypes (Moritoh et al., 2012; Wei et al., 2014). 141
Leaf angle is an important agronomic trait that directly affects crop architecture and 142
grain yields (Sinclair and Sheehy, 1999). Crops with erect leaves capture more light for 143
photosynthesis and are suitable for dense planting, all of which increase yields (Sakamoto et 144
al., 2006). In rice, the brassinosteroid (BR) phytohormones participate in the determination of 145
leaf angle (Tong and Chu, 2012; Zhang et al., 2014). BR-deficient or BR-insensitive mutants 146
display erect leaves, while overexpression of BR biosynthesis genes or signaling components 147
results in less-erect leaves with large leaf inclination (Bai et al., 2007; Hong et al., 2005; 148
Yamamuro et al., 2000). For example, loss-of-function mutants of rice OsBRI1 and EBISU 149
DWARF (D2), which encode the rice BR receptor kinase and a BR synthesis enzyme, 150
respectively, show erect leaves (Hong et al., 2003; Yamamuro et al., 2000). In rice, 151
RAV-LIKE 1 (RAVL1), a B3 DNA binding domain-containing protein, maintains BR 152
homeostasis via the coordinated activation of BRI1 and BR biosynthetic genes, D2, 153
DWARF11 (D11), and BR-DEFICIENT DWARF1 (BRD1) (Je et al., 2010). The ravl1 mutant 154
and RAVL1 overexpression lines showed erect leaves and large leaf angles, respectively (Je et 155
al., 2010). 156
Here, we identified a natural epiallele of rice RAV6 and found that plants carrying this 157
epiallele show increased leaf angle and small grains, as well as hypomethylation in the RAV6 158
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promoter region and ectopic expression of RAV6. Our work revealed that epigenetic 159
modification of RAV6 plays an essential role in the regulation of important agricultural traits 160
in rice and found that RAV6 acts via BR homeostasis. 161
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RESULTS 164
A Semi-dominant Mutant Shows Large Lamina Inclination and Small Seed Size 165
A spontaneously occurring rice mutant with large leaf angle and small seeds was isolated 166
from the japonica variety Zhonghua 11 and was named Epi-rav6 based on our subsequent 167
characterization (see below). Compared with the wild type (Zhonghua11), almost all leaf 168
angles throughout all developmental stages were larger in Epi-rav6 plants (Fig. 1A). The 169
mutant had almost the same amount of rachis as wild type but had obviously smaller grains 170
(Supplemental Fig. S1; Fig. 1B-D). Compared with the wild type, the Epi-rav6 seeds were 171
markedly smaller, by 12.8% in length (average 7.8 mm in wild type to 6.8 mm in Epi-rav6) 172
(Fig. 1C and 1D), 25% in width (average 3.6 mm in wild type to 2.7 mm in Epi-rav6) (Fig. 173
1B and 1D) and 20% in thickness (average 2.4 mm in wild type to 1.9 mm in Epi-rav6) (Fig. 174
1E). Thus, the 1,000-grain weight of Epi-rav6 seeds was only 57% of that of wild type (Fig. 175
1E). Together, these findings indicate that Epi-rav6 influences leaf angle and grain size. 176
To determine the inheritance of Epi-rav6, we examined the phenotypes of 260 progeny 177
of self-pollinated Epi-rav6 plants. Three phenotypes segregated in these progeny: 62 were 178
like wild type, 143 were like Epi-rav6, and 55 had severe defects, including fewer tillers, very 179
large leaf angles, and sterility (Fig. 1A). The ratio of wild-type to Epi-rav6-like to severe 180
phenotypes was 1:2.3:0.89 (χ2=2.98, P>0.05). In addition, 27 F1 plants derived from the cross 181
between Epi-rav6 and Zhonghua 11 showed nearly a 1:1 ratio of wild-type progeny and 182
Epi-rav6-like progeny (Supplemental Fig. S2). This genetic evidence demonstrated that the 183
mutant is controlled by a single, semi-dominant locus and the derived allele is heterozygous. 184
Thus, we regarded Epi-rav6-like progeny as heterozygotes and refer to them as Epi-rav6 (+/-); 185
we also regarded the progeny with severe defects as homozygotes and refer to them as 186
Epi-rav6 (-/-). 187
188
Cloning and Characterization of RAV6 189
To explore the molecular mechanism responsible for the Epi-rav6 phenotype, we used 190
map-based cloning to isolate the causal gene. Using an F2 population derived from a cross 191
between the Epi-rav6 mutant and Huajingxian74 (indica), we mapped Epi-rav6 to an 18.5-kb 192
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region on chromosome 2. Based on the MSU7.0 annotation (http://rice.plantbiology.msu.edu/), 193
this region contains only the promoter region of a putative gene, Os02g45850 (Fig. 2A). 194
However, we found no nucleotide sequence difference between the mutant and wild type in 195
this region. We next investigated the expression level of Os02g45850, the only annotated gene 196
in the mapped region. Compared with its low expression level in wild-type leaves, 197
Os02g45850 showed much higher expression in Epi-rav6 (+/-) and Epi-rav6 (-/-) leaves (Fig. 198
2B and 2C). Moreover, the degree of up-regulation was much higher in homozygous than 199
heterozygous lines (Fig. 2B and 2C). 200
To further confirm that the ectopic expression of Os02g45850 is responsible for the 201
developmental defects in Epi-rav6, we over-expressed Os02g45850 in wild type under the 202
control of the maize (Zea mays) Ubiquitin promoter. All the positive transformants with high 203
expression of Os02g45850 displayed large leaf inclination and small grains (Fig. 3). Thus, we 204
concluded that the abnormal phenotype in this mutant results from high expression of 205
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Os02g45850. Os02g45850 encodes a B3 DNA binding domain-containing protein, which 206
belongs to the related to ABI3/VP1 (RAV) family and was named RAV6 (Romanel et al., 207
2009). Therefore, we named the allele Epi-rav6. 208
209
RAV6 Regulates Leaf Angle via Mediating Brassinosteroid Homeostasis 210
The plant-specific RAV family belongs to the B3 transcription factor superfamily and consists 211
of 12 members in rice, including RAV6 (Romanel et al., 2009). Phylogenetic analysis showed 212
that the RAV6 (encoded by Os02g45850) is more closely related to RAVL1 (encoded by 213
Os04g49230) than to other members of the RAV family (Fig. 4A). Comparison of deduced 214
amino acid sequences suggested that RAV6 exhibits a high degree of sequence identity with 215
RAVL1, especially in the B3 DNA binding domain (Fig. 4B). These observations indicate that 216
RAV6 and RAVL1 may share similar functions. RAVL1 maintains BR homeostasis via the 217
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coordinated activation of BR synthetic and receptor genes in rice (Je et al., 2010). In addition, 218
plants over-expressing RAVL1 showed increased lamina inclination, like the Epi-rav6 mutants 219
(Je et al., 2010), suggesting that RAV6 may also be involved in the BR pathway via activation 220
of BR biosynthetic and signaling genes. To confirm this, we measured expression of 221
representative genes encoding BR biosynthesis enzymes and the BR receptor, known RAVL1 222
targets, in Epi-rav6 mutants. As expected, expression of BR synthetic genes including D2, 223
D11, and BRD1, was dramatically up-regulated in the Epi-rav6 mutant (Fig. 5A). Similarly, 224
we also observed higher levels of induction for the BR receptor gene BRI1 in Epi-rav6 mutants. 225
These data suggest that RAV6 affects the expression of genes involved in BR signaling and 226
BR biosynthesis. 227
To further confirm that the large lamina inclination of rav6 mutant results from BR 228
overdose, we postulated that treatment with a BR inhibitor would restore, at least partially, the 229
mutant phenotype. As expected, in the presence of Propiconazole (Pcz), a BR biosynthesis 230
inhibitor (Hartwig et al., 2012), the lamina inclination of Epi-rav6 mutant was restored (Fig. 231
5B). Taken together, these results suggest that RAV6 is involved in BR-mediated 232
developmental processes. 233
234
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Hypomethylation of the Promoter Region of RAV6 in Epi-rav6 plants 235
We found no nucleotide sequence difference between the wild type and Epi-rav6, but we did 236
observe alteration of RAV6 expression levels in the Epi-rav6 plants, suggesting that the 237
mutation may result from an epigenetic modification. We therefore investigated the DNA 238
methylation status of the RAV6 locus. The promoter region of RAV6 contains many TEs 239
including an adh-5-like MITE, a SINE, two unclassified retrotransposons, and a Snabo-like 240
MITE (Fig. 6A; Supplemental Fig. S3A). TEs, especially those proximal to genes, can act as 241
epigenetic mediators to influence nearby gene expression (Hollister and Gaut, 2009; Hollister 242
et al., 2011; Wei et al., 2014). So we performed bisulfite sequencing to analyze methylation in 243
a 2,358-bp genomic region consisting of 573-bp of gene body, 1,321-bp of upstream region 244
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and 464-bp of 5’ distal retrotransposon sequence (Supplemental Fig. S3A). In wild type and 245
homozygous Epi- rav6 mutants, we found no DNA methylation in all three sequence contexts 246
in the 573-bp gene body of RAV6. However, we observed higher CG and CHG but not CHH 247
DNA methylation in proximal and distal upstream regions of RAV6 in wild type compared 248
with the homozygous Epi- rav6 mutant. All the changed methylation sites occurred in a 249
contiguous 96-bp region at -600-bp to -504-bp relative to the transcriptional start site 250
including the closest MITE (adh5-like MITE). This region is hypermethylated in the wild type 251
but hypomethylated in Epi- rav6, containing four CG sites and two CHG sites (Fig. 6B; 252
Supplemental Fig. S3B). To further confirm that the ectopic expression of RAV6 in Epi- rav6 253
results in hypomethylation of the promoter region, we treated the wild-type seeds with 5-aza-2’ 254
deoxycytidine (5-aza-dC), an inhibitor of DNA methylation (Chang and Pikaard, 2005). The 255
expression levels of RAV6 were measured in 7-day-old seedlings with or without 5-aza-dC 256
treatment. Treatment with 5-aza-dC up-regulated RAV6 expression to varying degrees in three 257
cultivated rice strains, including two japonica (Zhonghua11 and Nipponbare) and one indica 258
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(Kasalath) accessions (Figure 6C). These three cultivated accessions also contain the MITE 259
(see below); therefore, these results indicate that the DNA methylation mediated by the TE in 260
the 5’ upstream sequence of RAV6 plays an essential role in the regulation of RAV6 261
expression. 262
263
The effect of MITE-mediated DNA methylation on RAV6 expression is conserved in 264
cultivated rice. 265
MITEs are short (less than 600-bp), nonautonomous DNA transposons. As the TEs with the 266
highest copy numbers in the rice genome, MITEs mainly occur in the chromosomal arms, 267
especially in the vicinity of genes (Jiang et al., 2004). According to the Plant Repeat Database, 268
the O. sativa L. spp. japonica cv. Nipponbare genome contains 2984 copies of the adh5-like 269
MITE (Ouyang and Buell, 2004). The adh5-like MITE in the RAV6 promoter region is 100-bp 270
long and located 198-bp upstream of the RAV6 gene body. 271
To further explore the evolutionary significance of the epigenetic regulation of RAV6, we 272
investigated the natural variation of the MITE in the promoter region of the RAV6 locus in 40 273
accessions of cultivated rice, which represent all of the major groups of Asian cultivated rice 274
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(Xu et al., 2012). The results showed that the TE insertion in the promoter region of RAV6 is 275
conserved in all cultivated rice accessions (data not shown). To further confirm this, we 276
aligned the sequences of the MITEs, as well as the 5’ region of RAV6, in four known 277
assembled rice genomes including three cultivated rice strains, Nipponbare, 93-11, Kasalath, 278
and wild rice Oryza brachyantha (Chen et al., 2013; Rice, 2005; Sakai et al., 2014; Yu et al., 279
2002). The MITE insertion was conserved in the cultivated rice genomes but was absent in 280
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Oryza brachyantha (Figure 7A). Consistent with this, the sequence of the 5’ promoter region 281
of RAV6 is also more conserved in cultivated rice, especially the 6 cytosine methylation sites, 282
which are essential for regulation of RAV6 expression (Figure 7A). 283
To further determine the conserved effect of the MITE-mediated DNA methylation on 284
RAV6 expression in cultivated rice accessions, we measured RAV6 transcript levels and DNA 285
methylation patterns in four cultivated rice strains, including three japonica (Nipponbare, 286
Kongyu131, and Koshihikari) and one indica (Kasalath) accessions. Compared with the high 287
expression of RAV6 observed in Epi- rav6, we observed little or no RAV6 expression in leaves 288
at the tillering stage in all cultivated rice strains (Figure 7B). Like the pattern of wild-type 289
Zhonghua11, the other four cultivated rice accessions also showed higher DNA methylation 290
in the contiguous 96-bp region of RAV6 promoter, in contrast with very low DNA 291
methylation in Epi- rav6 (Figure 7C; Supplemental Fig. S4). Thus, it is reasonable to predict 292
that the TE insertion occurred prior to divergence of indica and japonica rice and that the 293
TE-mediated epigenetic regulation of RAV6 expression is evolutionarily conserved in 294
cultivated rice. 295
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DISCUSSION 298
In this work, we identified an epiallele of rice RAV6; this epiallele shows hypomethylation of 299
the RAV6 promoter region, causing ectopic RAV6 expression, larger lamina inclination, and 300
smaller grains. We found that RAV6 expression is associated with extensive methylation of a 301
MITE along with the nearby sequence in the 5’ region of this gene. RAV6 encodes a B3 DNA 302
binding domain-containing protein that has the most sequence similarity to RAVL1, which 303
can maintain BR homeostasis to control rice development (Je et al., 2010). Functional 304
analysis revealed that RAV6 coordinately activates the BR receptor BRI1 and BR 305
biosynthetic genes to control rice leaf angle. This suggests that RAV6 performs a similar 306
function to its homolog, RAVL1. 307
308
The Rice Mutant Epi-rav6 Shows Hypomethylation of DNA at RAV6 309
Ectopic expression of RAV6 in Epi-rav6 mutants indicates that it is a gain-of-function allele 310
of RAV6. Although we found no DNA sequence changes in RAV6, we did find that Epi-rav6 311
shows a loss of cytosine DNA methylation in a MITE along with the nearby sequence located 312
in its 5’ promoter region (Fig. 6A; Supplemental Fig. S3A). Thus, Epi-rav6 has similar 313
features to the Arabidopsis fwa mutant, the first DNA hypomethylated mutant identified in 314
plants, with hypomethylation at two direct repeats in the 5’ region of FWA and ectopic 315
expression of FWA (Soppe et al., 2000). In rice, only two epialleles, Epi-d1 and Epi-df, were 316
identified previously, with hyper- and hypomethylation at the causal genes, respectively 317
(Miura et al., 2009; Zhang et al., 2012). Although both Epi-df and Epi-rav6 are 318
hypomethylated, the methylation sites of the two epi-alleles differ. Epi-df shows 319
hypomethylation mainly in the coding region and not associated with repeated sequences 320
(Zhang et al., 2012). However, the gain of methylation at the D1 locus in Epi-d1 is associated 321
with the upstream repeat sequences (Miura et al., 2009), similar to fwa and Epi-rav6. 322
Therefore, these repeat elements around the causal genes in Epi-d1, fwa, and Epi-rav6 might 323
function as epigenetic mediators to influence nearby gene expression, further confirming 324
previous findings (Hollister et al., 2011; Wei et al., 2014). 325
The phenotypes of epi-alleles generally show metastable inheritance with a low 326
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percentage of revertants, such as in Epi-d1 and Epi-df. Epi-rav6 is a semi-dominant mutant 327
and the homozygous lines are sterile. Therefore, it is impossible to identify authentic 328
revertants between generations (Fig. 1). 329
330
RAV6 Controls Rice Lamina Inclination via Mediating BR Homeostasis 331
The B3 DNA binding domain-containing proteins are plant-specific transcription factors 332
that play important roles in growth, development, flowering time, seed development, and seed 333
maturation (Romanel et al., 2009; Swaminathan et al., 2008). The B3 domain can bind DNA 334
in a sequence-specific or non-specific manner and activate or repress the transcription of 335
specific target genes (Je et al., 2010). Both RAV6 and RAVL1 belong to the RAV (Related to 336
ABI3/VP1) family and have higher sequence similarity to each other than to other members 337
of the RAV family (Figure 4). RAVL1 functions as a transcription factor, directly binding BR 338
biosynthetic and receptor genes and thus maintaining BR homeostasis (Je et al., 2010). In 339
Epi-rav6, the direct targets of RAVL1, including BRI1, D2, D11, and BRD1, showed 340
increased expression (Fig. 5A), indicating that RAV6 might also target these genes. The lines 341
overexpressing RAVL1 showed larger lamina inclination and hypersensitivity to BR (Je et al., 342
2010). Consistent with this, increased leaf angles were observed in Epi-rav6 mutants and 343
RAV6 overexpression lines (Fig. 3A). Moreover, treatment with BR inhibitor fully rescued the 344
larger lamina inclination in Epi-rav6 (Fig. 5B). All these results indicated that both RAV6 and 345
RAVL1 control leaf angles via maintaining BR homeostasis. 346
Besides leaf angle, BRs also influence rice grain size (Zhang et al., 2014). Generally, 347
BR-deficient and BR-insensitive mutants, such as d2, d11, and brd1, showed small grains, 348
while overexpression lines such as Increase Leaf Inclination 1 (OsILI1) and 349
BRI1-SUPPRESSOR 1 (OsBU1) showed large lamina joints and increased grain size (Hong et 350
al., 2002; Hong et al., 2003; Tanabe et al., 2005; Tanaka et al., 2009). The Epi-rav6 plants 351
showed activation of BR signaling and large leaf angles, but small grains, which differs from 352
typical mutants affecting BR. We proposed that decreased grain size in Epi-rav6 may result 353
from an integrated effect of changes in expression of multiple target genes. Since RAV6 354
encodes a B3 DNA binding domain-containing protein, lots of target genes involved multiple 355
developmental processes might be influenced by the gain-of-function change of RAV6 in 356
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Epi-rav6. This also can be confirmed as the Epi-rav6 homozygotes showed pleiotropic 357
developmental defects (Fig. 1A). 358
359
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MATERIALS AND METHODS 360
Plant Materials and Growth Conditions 361
A spontaneously occurring rice mutant Epi-rav6 was isolated from a Zhonghua 11 (Oryza 362
sativa L. ssp. japonica) population in Guangzhou, China in 2006. Transgenic plants 363
overexpressing RAV6 were produced by introducing the respective genes driven by the maize 364
(Zea mays) Ubiquitin promoter into wild-type plants. Briefly, the 1,239-bp coding sequence 365
of RAV6 was amplified with the primers OsRAV-1BXF and OsRAV-1239RNS 366
(Supplemental Table S1) and the PCR product was inserted into the BamHI/SpeI sites of 367
pCUbi1390, resulting in a plant expression vector driven by the Ubiquitin promoter, which 368
was then transformed into rice. Plants were grown in the field under natural conditions. 369
370
Map-Based Cloning 371
For map-based cloning of OsRAV6, 725 recessive individual plants showing normal leaf 372
inclinations were selected from an F2 population derived from a cross between the Epi-rav6 373
mutant and indica var Huajingxian74. Simple sequence repeat (SSR) markers and 374
insertion/deletion (InDel) markers on chromosome 2 were used for fine mapping. The RAV6 375
gene was selected from an approximately 18-kb region as the candidate gene. To find out the 376
mutation site, we amplified the corresponding fragments from the Epi-rav6 mutant and 377
wild-type plants, respectively. Primers used in the map-based cloning are listed in 378
Supplemental Table S1. 379
380
RT-PCR and Real-Time PCR 381
Real-time PCR analysis was performed using the CFX96 Real-Time PCR System (Bio-Rad) 382
and SYBR Green I (S-7567; Invitrogen). PCR was performed using hot-start Taq DNA 383
polymerase (DR007B; Takara Bio Inc., http://www.takara-bio.com/). For each sample, 384
quantifications were made in triplicate. Melting curves were read at the end of each 385
amplification by steps of 0.3°C from 65°C to 95°C to ensure that the quantifications were 386
derived from real PCR products, and not primer dimers. The primers used for Fig. 5A are 387
listed in Supplemental Table S1. 388
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389
Bisulfite Sequencing 390
Genomic DNAs were isolated from 3-week-old rice seedlings or the leaf at tillering stage. 391
Bisulfite treatment of genomic DNA was conducted using the Methylation-Gold kit according 392
to the manufacturer’s instructions (ZYMO Research Corporation). Treated DNAs were then 393
used for PCR amplification using hot-start Taq DNA polymerase (DR007B; TaKaRa). PCR 394
conditions were as follows: 95°C, 5 min; 35 cycles of (95°C, 30 s; 50°C, 30 s; 72°C, 40 s); 72°C, 395
10 min. The PCR products were cloned into the pEASY-T1 cloning vector (TransGen Biotech 396
Co., Ltd.), and individual clones were sequenced. The analysis of sequencing data was 397
performed using KISMETH (Gruntman et al., 2008). The primers used for bisulfite sequencing 398
are listed in Supplemental Table S1. 399
400
5-Aza-2’Deoxycytidine Treatment 401
Rice seeds were soaked in water at 37°C for 16 h. The seeds were then immersed in 20 mM 402
Tris–HCl, pH 7.5, with or without 0.3 mM 5-aza-2’deoxycytidine (A3656, Sigma) at room 403
temperature for 48 hours in the dark. After washing, seeds were planted in soil. 7-day-old 404
seedlings were sampled for further analysis. 405
406
Phylogenetic Analysis and Alignment 407
Full-length protein sequences were used for phylogenetic analysis. The sequence of OsRAV6 408
was downloaded from TIGR, release 7 (http://rice.plantbiology.msu.edu/) and used to search 409
the proteome database of rice available at TIGR using BLAST. Alignments of protein 410
sequences were performed using MUSCLE (Edgar, 2004) with default parameters (gap 411
opening, -2.9; gap extension, 0; hydrophobicity multiplier, 1.2; max iteration, 8). The 412
maximum likelihood (ML) tree was constructed using MEGA6 (Tamura et al., 2013). 413
Forty-eight different amino acid substitution models were tested and the JTT+G+F model 414
(Jones et al., 1992) was considered as the best model with the lowest BIC (Bayesian 415
Information Criterion) scores. All sites of the alignment were used for model testing. The –Ln 416
likelihood was 10996.08. Nonparametric bootstrap analyses (Sanderson and Wojciechowski, 417
2000) consisted of 1000 replicates. All 4 known genome sequences of Oryza were collected, 418
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22
including Oryza. sativa ssp. japonica cv. Nipponbare (Rice, 2005), O. sativa ssp. indica cv. 419
93-11 (Yu et al., 2002), O. sativa ssp. aus cv. Kasalath (Sakai et al., 2014), and O. 420
brachyantha (wild rice) (Chen et al., 2013). The genomic DNA sequence of the OsRAV6 gene 421
body and 230 bp upstream region was used as a query sequence, and homologous regions 422
were searched the in other 3 genomes of Oryza using FASTA (v36.3.5d) (Pearson and 423
Lipman, 1988). Multiple sequence alignment was performed using ClustalW (v2.1) (Larkin et 424
al., 2007; Thompson et al., 1994). 425
426
Propiconazole Treatment 427
In this study, propiconazole (Pcz), a brassinosteroid biosynthesis inhibitor (Hartwig et al., 2012) 428
was used to measure the mutants’ growth and morphological responses to BR by observing 429
inclination of lamina joints after exposure to Pcz. For Pcz treatment, the plants were grown in 430
the fields after incubating seeds for 3 days at 30°C to ensure synchronized germination. The 431
30-d-old seedlings were sprayed with 10 mM Pcz (Banner Maxx, Syngenta) every three days. 432
After 15 days, plants were transplanted into pots and photographed. Control plants were grown 433
and not treated with Pcz. 434
435
Supplemental Data 436
Supplemental Figure S1. The panicle and seeds of WT and Epi-rav6 (+/-) plants. 437
Supplemental Figure S2. The WT, Epi-rav6 (+/-), and hybrid F1 plants at the heading stage. 438
Supplemental Figure S3. Analyses of DNA methylation levels at RAV6 in WT and mutants 439
by bisulfite sequencing. 440
Supplemental Figure S4. Analyses of DNA methylation levels at RAV6 in WT, Epi-rav6 (-/-) 441
and four cultivated rice strains by bisulfite sequencing. 442
Supplemental Table S1. List of primers used in this paper. 443
444
ACKNOWLEDGEMENTS 445
This work was supported by the National Natural Science Foundation of China (grant nos. 446
31300987 to X. Song and 30900884 to X. Zhang); by Genetically Modified Breeding Major 447
Projects (grant no. 2014ZX08010-002 to X. Cao), the postdoctoral fellowship to (J. Sun) and the 448
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23
State Key Laboratory of Plant Genomics (2014B0227-01;2015B0129-01). 449
450
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Figure legends 451
Figure 1. Characterization of a semi-dominant rice mutant with larger leaf angle and smaller 452
seeds. A, The wild-type (WT), heterozygous Epi-rav6 (+/-) and homozygous Epi-rav6 (-/-) 453
plants at tilling stage. Bar = 20cm. B and C, Comparison of grain and seed width (B) and 454
length (C) between WT and Epi-rav6 (+/-). Bar = 1 cm. D, Characterization of seed weight, 455
seed length, seed width, and seed thickness in WT and the Epi-rav6 mutant. Values are means 456
± SD. In each graph, statistically significant differences are indicated by double asterisks 457
(**P<0.01). 458
459
Figure 2. Molecular cloning and expression analysis of RAV6. A, Map-based cloning of 460
RAV6. The RAV6 locus was mapped to the long arm of rice chromosome 2 between markers 461
RM3512 and RM318. The gene was further delimited to an 18-kb genomic region between the 462
markers ID8-26 and ID5-3 within the BAC clones AP004178 and AP004255. The number of 463
recombinants is marked corresponding to the molecular markers. This 18-kb interval contains 464
the candidate gene Os02g45850. Grey and black boxes indicate the untranslated regions and 465
the coding sequence, respectively. B and C, Expression analysis of RAV6 by RT-PCR (B) and 466
real-time RT-PCR (C) in WT, Epi-rav6 (+/-) and Epi-rav6 (-/-). OsEF1a was used as a control. 467
Values are means ±SD of three biological replicates. 468
469
Figure 3. Over-expression of RAV6 phenocopies the Epi-rav6 mutant phenotype. A, The 470
morphologies of WT, Epi-rav6 (+/-), and transgenic plants overexpressing (OV) RAV6 at 471
seedling stages. Bar = 1 cm. B, Relative expression analysis of RAV6 in WT, Epi-rav6 (+/-), 472
and the RAV6-overexpressing (OV) plants at seedling stage. C and D, The grain morphologies 473
of the transformed WT plants. OV-1, 2: transgenic plants overexpressing RAV6. Bars = 2 cm. 474
475
Figure 4. Evolutionary relationship between RAV6 and RAVL1 in rice. A, Phylogenetic 476
relationship of the RAV subfamily of B3 DNA binding domain-containing proteins in rice. 477
The bar indicates substitutions per site. B, Amino acid sequence alignment of rice RAV6 and 478
RAVL1. Identical and similar amino acids are boxed in black and grey, respectively. The 479
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25
signature B3 binding domain is underlined. 480
481
Figure 5. RAV6 regulates leaf angle by controlling genes affecting BR homeostasis. A, 482
Real-time RT-PCR analyses of BRD1, D2, D11, and BRI1 in WT, Epi-rav6 (+/-), and 483
Epi-rav6 (-/-) plants. 25S rRNA was used as a control. Values are means ±SD of three 484
biological replicates. B, Lamina inclination of 45-day-old seedlings in WT, Epi-rav6 (+/-), 485
and Epi-rav6 (-/-) plants subjected to propiconazole (Pcz) treatment for 15 days. Bar = 20 cm. 486
487
Figure 6. DNA methylation analysis of the RAV6 locus. A, Schematic representation of RAV6 488
with putative MITE, MITE-adh-5-like DNA transposon. Boxes indicate exon (black), UTR 489
regions (white) and MITE (green). The red line shows the region used for analysis of DNA 490
methylation by bisulfite sequencing (B). B, DNA methylation status of bisulfite-sequenced 491
region (as indicated in A) in WT and Epi-rav6 (-/-) plants. Histograms represent the 492
percentage of CG (red), CNG (blue), and CHH (green). C, Real-time RT-PCR analyses of 493
RAV6 expression in 7-day-old seedlings treated with (+) or without (-) 5-aza-2’ deoxycytidine, 494
an inhibitor of DNA methylation. OsEF1a was used as a control. Values are means ±SD of 495
three biological replicates. 496
497
Figure 7. Genome sequence-, gene expression- and DNA methylation analysis of the RAV6 498
locus in different rice accessions. A, Comparative sequence analysis of the RAV6 locus in 4 499
assembled rice genomes. The synteny of the RAV6 locus in Nipponbare, 93-11, Kasalath, and 500
O. brachyantha is shown in the top panel. The bottom panel shows an alignment of the DNA 501
sequence 258-bp upstream and the 5’ gene body of RAV6 in four assembled rice genomes. 502
The MITE region and 5’ gene body are underlined by green and black lines, respectively. The 503
“*” indicates the cytosine region, whose degree of methylation was reduced in Epi-rav6 (-/-), 504
as shown in Figure 6B. B, Expression analysis of RAV6 by RT-PCR (top) and real-time 505
RT-PCR (bottom) in cultivated rice strains and Epi-rav6 (-/-). Zhonghua11, Nipponbare, 506
Kongyu131 and Koshihikari are japonica cultivars; Kasalath is an indica cultivar. OsEF1a 507
was used as a control. Values are means ± SD of three biological replicates. C, DNA 508
methylation status of the bisulfite-sequenced region (as indicated in Figure 6A) in WT 509
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26
(Zhonghua11), Epi-rav6 (-/-) and four cultivated rice strains. Histograms represent the 510
percentage of methylation in the CG (red), CNG (blue), and CHH (green) contexts. 511
512
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Parsed CitationsBai, M.Y., Zhang, L.Y., Gampala, S.S., Zhu, S.W., Song, W.Y., Chong, K., and Wang, Z.Y. (2007). Functions of OsBZR1 and 14-3-3proteins in brassinosteroid signaling in rice. Proc Natl Acad Sci U S A 104, 13839-13844.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cao, X., and Jacobsen, S.E. (2002). Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and genesilencing. Curr Biol 12, 1138-1144.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chang, S., and Pikaard, C.S. (2005). Transcript profiling in Arabidopsis reveals complex responses to global inhibition of DNAmethylation and histone deacetylation. J Biol Chem 280, 796-804.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen, J., Huang, Q., Gao, D., Wang, J., Lang, Y., Liu, T., Li, B., Bai, Z., Luis Goicoechea, J., Liang, C., et al. (2013). Whole-genomesequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome evolution. Nat Commun 4, 1595.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cubas, P., Vincent, C., and Coen, E. (1999). An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401,157-161.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Edgar, R.C. (2004). MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics5, 113.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Gruntman, E., Qi, Y., Slotkin, R.K., Roeder, T., Martienssen, R.A., and Sachidanandam, R. (2008). Kismeth: analyzer of plantmethylation states through bisulfite sequencing. BMC Bioinformatics 9, 371.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hartwig, T., Corvalan, C., Best, N.B., Budka, J.S., Zhu, J.-Y., Choe, S., and Schulz, B. (2012). Propiconazole Is a Specific andAccessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize. PLoS ONE 7, e36625.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hollister, J.D., and Gaut, B.S. (2009). Epigenetic silencing of transposable elements: a trade-off between reduced transpositionand deleterious effects on neighboring gene expression. Genome Res 19, 1419-1428.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hollister, J.D., Smith, L.M., Guo, Y.L., Ott, F., Weigel, D., and Gaut, B.S. (2011). Transposable elements and small RNAs contributeto gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata. Proc Natl Acad Sci U S A 108, 2322-2327.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hong, Z., Ueguchi-Tanaka, M., Fujioka, S., Takatsuto, S., Yoshida, S., Hasegawa, Y., Ashikari, M., Kitano, H., and Matsuoka, M.(2005). The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, isrescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone. Plant Cell 17, 2243-2254.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hong, Z., Ueguchi-Tanaka, M., Shimizu-Sato, S., Inukai, Y., Fujioka, S., Shimada, Y., Takatsuto, S., Agetsuma, M., Yoshida, S.,Watanabe, Y., et al. (2002). Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organizedarrangement and polar elongation of cells in the leaves and stem. Plant J 32, 495-508.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hong, Z., Ueguchi-Tanaka, M., Umemura, K., Uozu, S., Fujioka, S., Takatsuto, S., Yoshida, S., Ashikari, M., Kitano, H., and Matsuoka,M. (2003). A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochromeP450. Plant Cell 15, 2900-2910.
www.plantphysiol.orgon May 31, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hu, L., Li, N., Xu, C., Zhong, S., Lin, X., Yang, J., Zhou, T., Yuliang, A., Wu, Y., Chen, Y.R., et al. (2014). Mutation of a major CGmethylase in rice causes genome-wide hypomethylation, dysregulated genome expression, and seedling lethality. Proc Natl AcadSci U S A 111, 10642-10647.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jacobsen, S.E., and Meyerowitz, E.M. (1997). Hypermethylated SUPERMAN epigenetic alleles in arabidopsis. Science 277, 1100-1103.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Je, B.I., Piao, H.L., Park, S.J., Park, S.H., Kim, C.M., Xuan, Y.H., Huang, J., Do Choi, Y., An, G., Wong, H.L., et al. (2010). RAV-Like1maintains brassinosteroid homeostasis via the coordinated activation of BRI1 and biosynthetic genes in rice. Plant Cell 22, 1777-1791.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jiang, N., Feschotte, C., Zhang, X., and Wessler, S.R. (2004). Using rice to understand the origin and amplification of miniatureinverted repeat transposable elements (MITEs). Curr Opin Plant Biol 7, 115-119.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Johannes, F., Porcher, E., Teixeira, F.K., Saliba-Colombani, V., Simon, M., Agier, N., Bulski, A., Albuisson, J., Heredia, F., Audigier,P., et al. (2009). Assessing the impact of transgenerational epigenetic variation on complex traits. PLoS Genet 5, e1000530.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jones, D.T., Taylor, W.R., and Thornton, J.M. (1992). The rapid generation of mutation data matrices from protein sequences.Comput Appl Biosci 8, 275-282.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kakutani, T. (2002). Epi-alleles in plants: inheritance of epigenetic information over generations. Plant Cell Physiol 43, 1106-1111.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez,R., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining and modifying DNA methylation patterns in plants and animals. NatRev Genet 11, 204-220.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lindroth, A.M., Cao, X., Jackson, J.P., Zilberman, D., McCallum, C.M., Henikoff, S., and Jacobsen, S.E. (2001). Requirement ofCHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292, 2077-2080.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Manning, K., Tor, M., Poole, M., Hong, Y., Thompson, A.J., King, G.J., Giovannoni, J.J., and Seymour, G.B. (2006). A naturallyoccurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38, 948-952.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Martin, A., Troadec, C., Boualem, A., Rajab, M., Fernandez, R., Morin, H., Pitrat, M., Dogimont, C., and Bendahmane, A. (2009). Atransposon-induced epigenetic change leads to sex determination in melon. Nature 461, 1135-1138.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Miura, K., Agetsuma, M., Kitano, H., Yoshimura, A., Matsuoka, M., Jacobsen, S.E., and Ashikari, M. (2009). A metastable DWARF1epigenetic mutant affecting plant stature in rice. Proc Natl Acad Sci U S A 106, 11218-11223.
www.plantphysiol.orgon May 31, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Moritoh, S., Eun, C.H., Ono, A., Asao, H., Okano, Y., Yamaguchi, K., Shimatani, Z., Koizumi, A., and Terada, R. (2012). Targeteddisruption of an orthologue of DOMAINS REARRANGED METHYLASE 2, OsDRM2, impairs the growth of rice plants by abnormalDNA methylation. Plant J 71, 85-98.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ouyang, S., and Buell, C.R. (2004). The TIGR Plant Repeat Databases: a collective resource for the identification of repetitivesequences in plants. Nucleic Acids Research 32, D360-D363.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pearson, W.R., and Lipman, D.J. (1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444-2448.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rice, S.P.I. (2005). The map-based sequence of the rice genome. Nature 436, 793-800.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Romanel, E.A., Schrago, C.G., Counago, R.M., Russo, C.A., and Alves-Ferreira, M. (2009). Evolution of the B3 DNA bindingsuperfamily: new insights into REM family gene diversification. PLoS One 4, e5791.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sakai, H., Kanamori, H., Arai-Kichise, Y., Shibata-Hatta, M., Ebana, K., Oono, Y., Kurita, K., Fujisawa, H., Katagiri, S., Mukai, Y., et al.(2014). Construction of pseudomolecule sequences of the aus rice cultivar Kasalath for comparative genomics of Asian cultivatedrice. DNA Res 21, 397-405.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sakamoto, T., Morinaka, Y., Ohnishi, T., Sunohara, H., Fujioka, S., Ueguchi-Tanaka, M., Mizutani, M., Sakata, K., Takatsuto, S.,Yoshida, S., et al. (2006). Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice.Nat Biotechnol 24, 105-109.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sanderson, M.J., and Wojciechowski, M.F. (2000). Improved bootstrap confidence limits in large-scale phylogenies, with anexample from Neo-Astragalus (Leguminosae). Syst Biol 49, 671-685.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Saze, H., Mittelsten Scheid, O., and Paszkowski, J. (2003). Maintenance of CpG methylation is essential for epigenetic inheritanceduring plant gametogenesis. Nat Genet 34, 65-69.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sinclair, T.R., and Sheehy, J.E. (1999). Erect leaves and photosynthesis in rice. Science 283.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Song, X., Li, P., Zhai, J., Zhou, M., Ma, L., Liu, B., Jeong, D.H., Nakano, M., Cao, S., Liu, C., et al. (2012). Roles of DCL4 and DCL3b inrice phased small RNA biogenesis. Plant J 69, 462-474.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Soppe, W.J., Jacobsen, S.E., Alonso-Blanco, C., Jackson, J.P., Kakutani, T., Koornneef, M., and Peeters, A.J. (2000). The lateflowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol Cell 6, 791-802.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Swaminathan, K., Peterson, K., and Jack, T. (2008). The plant B3 superfamily. Trends in Plant Science 13, 647-655.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title www.plantphysiol.orgon May 31, 2018 - Published by Downloaded from
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version6.0. Mol Biol Evol 30, 2725-2729.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tanabe, S., Ashikari, M., Fujioka, S., Takatsuto, S., Yoshida, S., Yano, M., Yoshimura, A., Kitano, H., Matsuoka, M., Fujisawa, Y., et al.(2005). A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant,dwarf11, with reduced seed length. Plant Cell 17, 776-790.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tanaka, A., Nakagawa, H., Tomita, C., Shimatani, Z., Ohtake, M., Nomura, T., Jiang, C.J., Dubouzet, J.G., Kikuchi, S., Sekimoto, H., etal. (2009). BRASSINOSTEROID UPREGULATED1, encoding a helix-loop-helix protein, is a novel gene involved in brassinosteroidsignaling and controls bending of the lamina joint in rice. Plant Physiol 151, 669-680.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tong, H., and Chu, C. (2012). Brassinosteroid signaling and application in rice. J Genet Genomics 39, 3-9.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vongs, A., Kakutani, T., Martienssen, R.A., and Richards, E.J. (1993). Arabidopsis thaliana DNA methylation mutants. Science 260,1926-1928.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wei, L., Gu, L., Song, X., Cui, X., Lu, Z., Zhou, M., Wang, L., Hu, F., Zhai, J., Meyers, B.C., et al. (2014). Dicer-like 3 producestransposable element-associated 24-nt siRNAs that control agricultural traits in rice. Proc Natl Acad Sci U S A 111, 3877-3882.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, X., Liu, X., Ge, S., Jensen, J.D., Hu, F., Li, X., Dong, Y., Gutenkunst, R.N., Fang, L., Huang, L., et al. (2012). Resequencing 50accessions of cultivated and wild rice yields markers for identifying agronomically important genes. Nat Biotechnol 30, 105-111.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamamuro, C., Ihara, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto, S., Ashikari, M., Kitano, H., and Matsuoka, M. (2000). Loss offunction of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell12, 1591-1606.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamauchi, T., Johzuka-Hisatomi, Y., Terada, R., Nakamura, I., and Iida, S. (2014). The MET1b gene encoding a maintenance DNAmethyltransferase is indispensable for normal development in rice. Plant Mol Biol 85, 219-232.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yu, J., Hu, S., Wang, J., Wong, G.K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X., et al. (2002). A draft sequence of the ricegenome (Oryza sativa L. ssp. indica). Science 296, 79-92.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang, C., Bai, M.Y., and Chong, K. (2014). Brassinosteroid-mediated regulation of agronomic traits in rice. Plant Cell Rep 33, 683-696.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang, L., Cheng, Z., Qin, R., Qiu, Y., Wang, J.L., Cui, X., Gu, L., Zhang, X., Guo, X., Wang, D., et al. (2012). Identification andcharacterization of an epi-allele of FIE1 reveals a regulatory linkage between two epigenetic marks in rice. Plant Cell 24, 4407-4421.
Pubmed: Author and TitleCrossRef: Author and Title
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