running head: novel dna methylation profiles in …...92 c, t or a), which seems to be distinct from...

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Running head: Novel DNA methylation profiles in dicot seeds 1

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Corresponding author: 4

Aizhong Liu 5

Department of Economic Plants and Biotechnology, Yunnan Key 6

Laboratory for Wild Plant Resources, Kunming Institute of Botany, 7

Chinese Academy of Sciences, Kunming, 132 Lanhei Road, Kunming, 8

650204, China 9

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Plant Physiology Preview. Published on April 28, 2016, as DOI:10.1104/pp.16.00056

Copyright 2016 by the American Society of Plant Biologists

www.plant.org on April 30, 2016 - Published by www.plantphysiol.orgDownloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

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Genomic DNA methylation analyses reveal the distinct profiles in 23

castor bean seeds with persistent endosperms 24

Wei Xu1,2,3,†, Tianquan Yang,2,4,†, Xue Dong1, De-Zhu Li1, Aizhong Liu1,* 25

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1Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for 27

Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, 28

Kunming, 132 Lanhei Road, Kunming, 650204, China 29

2University of the Chinese Academy of Sciences, Beijing, 100049, China 30

3College of Life Sciences, Yunnan University, 650091, Kunming, China 31

4Key Laboratory of Tropical Plant Resource Science, Xishuangbanna Tropical 32

Botanical Garden, Chinese Academy of Sciences, 88 Xuefu Road, Kunming, 650223, 33

China 34

One-sentence summary: Dicotyledonous castor seeds with persistent 35

endosperms exhibit novel genomic DNA methylation profiles mediated by 24-nt 36

siRNAs. 37

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Authors' contributions: 39

AL and WX conceived and designed the experiments; WX and TY performed the 40

experiments; WX, TY, DL and XD analyzed the data; AL and WX wrote the paper. 41

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Founding information 43

This work was jointly supported by Chinese National Key Technology R & D 44

Program (2015BAD15B02), National Key Basic Research Program of China 45

(2014CB954100) and NSFC (31501034). 46

Corresponding author 47

E-mail: [email protected] 48 † These authors contributed equally to this work 49




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ABSTRACT 50

Investigations of genomic DNA methylation in seeds have been restricted to a few 51

model plants. The endosperm genomic DNA hypo-methylation has been identified in 52

angiosperm, but it is difficult to dissect the mechanism how this hypo-methylation is 53

established and maintained because endosperm is ephemeral and disappears with seed 54

development in most of dicots. Castor bean (Ricinus communis), unlike Arabidopsis, 55

its endosperm is persistent throughout seed development, providing an excellent 56

model to dissect the mechanism of endosperm genomic hypo-methylation in dicots. 57

We characterized the DNA methylation-related genes encoding DNA 58

methyltransferases and demethylases, and analyzed their expression profiles in 59

different tissues. We examined the genomic methylation including CG, CHG and 60

CHH contexts in endosperm and embryo tissues using bisulfite sequencing and 61

revealed that the CHH methylation extent in endosperm and embryo was, 62

unexpectedly, substantially higher than known plants, irrespective of the CHH 63

percentage in their genomes. In particular, we found that the endosperm exhibited a 64

global reduction in the CG and CHG methylation extents relative to the embryo, 65

markedly switching the global gene expression. However, the CHH methylation 66

occurring in endosperm did not exhibit a significant reduction. Combining with the 67

expression of 24-nt siRNAs mapped within TE regions and genes involved in the 68

RdDM pathway, we demonstrated that the 24-nt siRNAs played a critical role in 69

maintaining CHH methylation and repressing the activation of TEs in endosperm 70

persistent development. This study discovered a novel genomic DNA methylation 71

pattern and proposed the potential mechanism occurring in dicot seeds with persistent 72

endosperm. 73

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Keywords: DNA methylation, TEs, 24-nt siRNA, DMRs, endosperm, Ricinus 75

communis 76

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INTRODUCTION 79

Methylated cytosine residue, also called the fifth nucleotide, is one of the most well 80

studied epigenetic mark extensively found in eukaryotes (Feng et al., 2010; Zemach et 81

al., 2010a), in spite of the fact that the levels and patterns of cytosine DNA 82

methylation appear to exhibit drastic changes among different eukaryotic organisms 83

(Zhu, 2009; Zemach et al., 2010a). DNA methylation has shown broad-ranging 84

functions, including regulation of gene expression, involvement in chromatin 85

organization and protection of genome from invading and mobile DNA elements 86

(Heard and Disteche, 2006; Klose and Bird, 2006; Feinberg, 2007). Usually, DNA 87

methylation can be maintained through mitotic cell division, thus it is relatively stable 88

and heritable epigenetic marker (Henderson and Jacobsen, 2007). In plants, DNA 89

methylation generally refers to three different nucleotide sequence contexts: 90

symmetric CG and CHG methylation and asymmetric CHH methylation (where H = 91

C, T or A), which seems to be distinct from that in mammalians where they are mostly 92

limited to the CG context. 93

Studies have been made into elucidating the mechanisms that result in DNA 94

methylation levels and patterns at the genome level in plants. Generally, the CG 95

methylation is generated by the conserved DNA methyltransferase MET1 96

(METHYLTRANSFERASE 1) (Vongs et al., 1993; Genger et al., 1999; Kankel et al., 97

2003; Law and Jacobsen, 2010), and the CHG methylation is produced by the 98

plant-specific DNA methyltransferase CMT3 (CHROMOMETHYLASE 3) (Cao and 99

Jacobsen, 2002a; Law and Jacobsen, 2010; Zemach et al., 2010a), whereas CHH de 100

novo methylation is established by 24-nt siRNA-directed DNA methylation (RdDM) 101

pathway to guide the DNA methyltransferase DRM1/2 (DOMAINS REARRANGED 102

METHYLTRANSFERASE 1 and 2) in plants (Cao and Jacobsen, 2002b; Law and 103

Jacobsen, 2010; Mosher and Melnyk, 2010). Also, several studies have demonstrated 104

that de novo CHH methylation could be established by the plant-specific DNA 105

methyltransferase CMT2 in an independent RdDM pathway (Zemach et al., 2013; 106

Stroud et al., 2014). In addition, the DNA methyltransferase-like Dnmt2, firstly 107


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identified from bacteria, was also detected in plants though its function in regulating 108

DNA methylation remained largely unknown (Hermann et al., 2003; Mund et al., 109

2004; Ponger and Li, 2005). According to the genomic DNA methylation observation, 110

there is generally a substantial amount of CG methylation and a small amount of 111

non-CG methylation, though the genomic DNA methylation level broadly varies in 112

plants tested (Zemach et al., 2010a). Besides, it has been known that active DNA 113

demethylation mainly, in Arabidospsis, depends upon activity of the DNA glycosylase 114

DEMETER (DME) (Kinoshita et al., 2004; Gehring et al., 2006), which is able to 115

recognize and remove methylated cytosines by a base excision-repair pathway (Law 116

and Jacobsen, 2010). In particular, studies have revealed that DME is specifically 117

expressed in the central cell of the female gametophyte (Choi et al., 2002), leading to 118

a tissue-specific removal of DNA methylation marks on the maternal genome and the 119

establishment of genomic imprinting in Arabidospsis (Gehring et al., 2009). DNA 120

glycosylases such as REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 121

(DML2) and DEMETER-LIKE 3 (DML3) are also involved in the removal of 122

5-methylated cytosines in Arabidopsis (Gong et al., 2002), preventing from the 123

hypermethylation of specific genome regions (Penterman et al., 2007). 124

Loss-of-function mutants of ROS1, DML2 and DML3 would result in a significant 125

change of DNA methylation level in all sequence contexts, particularly CG 126

dinucleotide in Arabidopsis (Penterman et al., 2007). These studies clearly suggest 127

that the extents and patterns of genomic DNA methylation be maintained by the 128

interaction of DNA methyltransferases and demethylases. 129

Increasing evidences demonstrate that DNA methylation plays crucial roles in the 130

control of seed development and seed size (Xiao et al., 2006a,b; Hu et al., 2014; Xing 131

et al., 2015). In particular, DNA methylation was considered as a major regulator of 132

gene imprinting that mainly manifests itself in the endosperm of angiosperm plants, 133

closely associated with the endosperm and seed development, such as imprinted genes 134

MEA (Grossniklaus et al., 1998; Köhler et al., 2003), FWA (Kinoshita et al., 2004) 135

and FIS2 (Jullien et al., 2006). Investigations on genomic DNA methylation in the 136

seeds of Arabidopsis (Gehring et al., 2009; Hsieh et al., 2009), rice (Zemach et al., 137


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2010b) and maize (Zhang et al., 2014; Lu et al., 2015) revealed extensive 138

hypo-methylation in the endosperm genome. However, the mechanism about how the 139

extent and pattern of DNA methylation is established and whether the extent and 140

pattern of DNA methylation is conserved in plant seeds are largely unclear. 141

Specifically, in most dicots including Arabidopsis their endosperms are ephemeral 142

and gradually consumed by the embryo tissue during seed development (Sreenivasulu 143

and Wobus, 2013). Due to this reason it appears difficult to dissect the potential 144

mechanism of genomic DNA methylation occurring in dicot’s seeds. In contrast to 145

Arabidopsis, castor bean (Ricinus communis L.), a member of Euphorbiaceae family, 146

has relatively large and persistent endosperm throughout seed development 147

(Greenwood and Bewley, 1982). It has been considered as a model plant for studies 148

on endosperm and seed development among dicots (Houston et al., 2009; Nogueira et 149

al., 2012). Also, castor bean is one of the most important non-edible oilseed crops, 150

and its seed oil stored in endosperm is broadly used in industry (Ogunniyi, 2006). 151

Importantly, the castor bean genome data have been completed (Chan et al., 2010), 152

which facilitate the genome-wide investigation of DNA methylation in castor bean 153

seed. In this study, we investigated the genomic DNA methylome occurring in 154

endosperm and embryo of castor bean by deep bisulfite sequencing, and revealed a 155

conserved and distinct profile. Specifically, the extensive hypo-methylation of CG and 156

CHG in the endosperm genome could significantly influence the expression of 157

flanking genes. However, the methylation of CHH did not exhibit a significant 158

reduction in endosperm relative to the embryo. With the integration of expressions of 159

the small RNAs (mediating DNA methylation) and DNA methylation-related genes, 160

we proposed a potential model to dissect that how the DNA methylation (particularly 161

for the rich CHH methylation identified) was established and maintained in embryo 162

and endosperm of castor bean. 163

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RESULTS 166

Identification and characterization of DNA methylation-related genes 167

Genomic DNA methylation may be maintained by the interaction of DNA 168

methylation and DNA demethylation processes in a given tissue. The main 169

components involved in DNA methylation and demethylation processes have been 170

well-studied in Arabidopsis. To dissect the mechanism of DNA methylation in castor 171

bean we globally inspected the methylation-related genes encoding DNA 172

methyltransferase and demethlylases at the genome scale. In total, we identified and 173

characterized the sequence structures of eight genes encoding DNA methyltransferase 174

and three genes encoding demethlylase (see Table 1). Similar to the amino acid 175

sequences in Arabidopsis, identified DNA methyltransferases (including MET1, DRM, 176

CMT, DNMT2) and demethylases (including DME, ROS1, DML2 and DML3) from 177

castor bean shared the conserved domains (see Supplemental Figs. S1-S5), as 178

previously reported in other plants (Finnegan and Dennis, 1993; Mok et al., 2010; 179

Qian et al., 2014). Based on the structural features of these genes, in particular, the 180

presence and distribution of domains and motifs (see Supplemental Fig. S6, A and B), 181

the DNA methyltransferase genes could be easily identified as RcMET1-1 and 182

RcMET1-2 in the MET group, RcCMT1 and RcCMT2 in the CMT group, RcDRM1, 183

RcDRM2 and RcDRM3 in the DRM group and RcDnmt2 in the Dnmt2 group 184

(Supplemental Fig. S6C). By analogy, three DNA demethylase genes were identified 185

as RcDME, RcROS1 and RcDML3, respectively (Supplemental Fig. S6D). 186

As shown in Fig. S6C, the phylogenetic analysis of DNA methyltransferase genes 187

generated four distinct clades corresponding to MET, CMT, DRM and Dnmt2 groups 188

with well-supported bootstrap values. It seemed that each group displayed a 189

monophyly with an evolutionary lineage from dicots to monocots in consistent with 190

previous studies (Zemach et al., 2010a; Jurkowski and Jeltsch, 2011). Within the 191

DNA demethylase genes, phylogenetic analysis generated six clades (I-VI). The DME 192

homologues were restricted to dicots and absent in monocots, confirming previous 193

observations (Zemach et al., 2010b; Zhang et al., 2011). The ROS1 genes were 194




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clustered into the clades II (nested by the homologues from dicots), III and IV (both 195

nested by the homologues from monocots). However, both clade I and clade II were 196

rooted by the monkey flower (Mimulus guttatus), a basal dicotyledonous plant 197

(Zemach et al., 2010b), suggesting that the DME probably be phylogenetically 198

monophyletic in dicots after gene duplication arising in the early angiosperm. In 199

addition, the DML3 genes were clustered into the clades V and VI nested by the 200

homologues from dicots and monocots, respectively. 201

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Expression profiles of DNA methylation-related genes 203

To understand how the DNA methylation is established and maintained, we assayed 204

the expression profiles of DNA methylation-related genes (including DNA 205

methyltransferase and demethylase genes) in different tissues and the developing 206

seeds at the early and late stages. As shown in Fig. 1, within the MET1 genes, 207

RcMET1-1 was highly expressed in leaf, stem apex, pollen and early embryo, but 208

lowly expressed in ovule and developing endosperms (including the early and late 209

stages); while RcMET1-2 was expressed only in developing embryo tissue 210

(particularly, at the late stage). Similarly, within the CMT genes, responsible for 211

mediating CHG methylation, RcCMT1 was highly expressed in leaf, stem apex, pollen 212

and early embryo, but lowly expressed in ovule and developing endosperms 213

(including the early and late stages); while the RcCMT2 was mainly expressed in the 214

developing embryo and early endosperm tissues. The substantial reduction of 215

transcript levels of both RcMET1 and RcCMT genes in castor bean endosperm 216

compared to embryo was consistent with the report in Arabidopsis (Jullien et al., 217

2012). Within the DRM genes, responsible for guiding CHH methylation, it seemed 218

that the RcDRM1 was specifically expressed in ovule similar to the DRM1 expression 219

in Arabidopsis (Jullien et al., 2012), while RcDRM2 and RcDRM3 were mainly 220

expressed in developing embryo and endosperm tissues that were distinctly different 221

from their expression in Arabidopsis. In Arabidopsis, DRM2 was strongly transcribed 222

in the embryo tissue but not detected in endosperm (Jullien et al., 2012). In addition, 223

the RcDnmt2 gene, in spite of its functional uncertainty, was broadly expressed in 224




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diverse tissues. 225

For the DNA demethylase genes, intriguingly, the RcDME gene was broadly 226

expressed in diverse tissues, particularly in both embryo and endosperm tissues. This 227




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observation is distinct from the expression patterns of the DME gene in Arabidopsis, 228

which was specifically expressed in the central cell of the female gametophyte or the 229

vegetative cell of the male gametophyte (Choi et al., 2002; Schoft et al., 2011). Both 230

RcROS1 and RcDML3, which may be involved in mediating DNA demethylation, 231

were extensively expressed in diverse tissues. Also, using our previous RNA-seq data 232

(Xu et al., 2014), we re-assayed the expression profiles of DNA methylation-related 233

genes in endosperm and embryo tissues and obtained similar results (see 234

Supplemental Fig. S7). In addition, we noted that the expression levels of most genes 235

were distinct between the early and late stages of endosperm and embryo 236

development, implying that genomic DNA methylation level within endosperm and 237

embryo might be dynamic with seed development. 238

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Genomic DNA methylation in castor bean seed 240

To obtain the genomic DNA methylation profiles at single nucleotide, we examined 241

the DNA methylation extent and pattern in endosperm and embryo tissues by high 242

throughput bisulfite-treated DNA sequencing techniques, which had been broadly 243

applied in genomic DNA methylation studies (Feng et al., 2010; Zemach et al., 2010a). 244

Our bisulfite sequencing yielded about 120 million paired-end reads and 88 % reads 245

were mapped into castor bean genome with an effective depth of 31× coverage 246

(Supplemental Table S1). Moreover, there were about 83% of cytosines covered by at 247

least two reads in the castor bean genome. As the sequencing depth reached 30×, the 248

number of detected cytosine reached saturation (Supplemental Fig. S8), meaning that 249

our sequencing data was sufficient for further analysis. 250

After sequencing data were mapped into castor bean genome, we identified, in total, 251

19,444,574 and 21,157,129 methylated cytosine residues from endosperm and embryo, 252

respectively. Global DNA methylation profiles exhibited that all three sequence 253

contexts CG, CHG and CHH were, as an example shown in Fig. 2, heavily methylated 254

in the TE-rich regions (Fig. 2A), whereas the methylation patterns of three sequence 255

contexts appeared to be quite different in the gene-rich regions (Fig. 2B). The density 256

peaks of CG methylation were mainly found in the gene body regions but the peaks of 257




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both CHG and CHH methylation were mainly found in the TE regions and sparely 258

distributed in the gene body regions. Clearly, these observations displayed that the 259

DNA methylation mainly occurred in the TE-rich regions consistent with previous 260




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reports from other plants (Gehring et al., 2009; Zemach et al., 2010b), and the global 261

methylation patterns between CG and non-CG contexts were differentiated in castor 262

bean. 263

While investigating the genomic methylation percentage of CG, CHG and CHH in 264

embryo and endosperm tissues, we found that the methylation of CG, CHG and CHH 265

were respectively 30.3%, 18.3%, and 11.2% within the endosperm, and 40.7%, 24.0%, 266

and 12.7% within the embryo tissues. Obviously, the endosperm exhibited a 267

hypo-methylation with a reduction of 10.4% CG methylation and 5.7% CHG 268

methylation, and a nearly equal CHH methylation, comparing to the embryo (as 269

shown in Fig. 3A). Compared with the genomic DNA methylation levels in 270

Arabidopsis (Hsieh et al., 2009), rice (Zemach et al., 2010b) and maize (Zhang et al., 271

2014; Lu et al., 2015), we found that the genomic DNA hypo-methylation of 272

endosperm tissues relative to embryo tissues appeared to be extensive in angiosperm, 273

particularly for dicotyledons (Fig. 3A), though the biological roles of endosperm 274

hypo-methylation remain unknown. Interestingly, the global CHH methylation of 275

endosperm was not substantially reduced compared to the embryo in castor bean, 276

distinct from that in Arabidopsis (Hsieh et al., 2009) and rice (Zemach et al., 2010b). 277

In contrast to other plants tested, in which CG methylation is the most abundant, 278

castor bean showed rich CHH methylation (68%) in both endosperm and embryo (see 279

Fig. 3B). When inspecting the proportions of CG, CHG and CHH in genomes of 280

Arabidopsis, rice, maize and castor bean, we found that they did not display a 281

significant difference (Supplemental Table S2). This meant that the higher proportion 282

of CHH methylation occurring in castor bean endosperm and embryo was not resulted 283

from a higher CHH content in its genome comparing to Arabidopsis, rice and maize. 284

In addition, it is a remarkable fact that the methylated level of CHH context 285

apparently exhibited a more uniform distribution between 30-100% compared with 286

CG and CHG contexts, though the CHH methylation is predominant in number (see 287

Fig. 3C). While inspecting the methylated ratios of the CG and CHG sites, we found 288

that the majority of CG sites (>80%) were highly methylated in both embryo and 289

endosperm. The methylated ratios of the major CHG sites displayed a similar pattern 290




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to the CG sites (see Fig. 3C), consistent with the observation from cassava leaves 291

(Wang et al., 2015). This observation seems to be distinct from the previous report in 292

Arabidopsis (Greaves et al., 2012), where major CHG sites are not highly methylated. 293




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Besides, we noted that the methylated ratios of the major CG and CHG sites were 294

slightly reduced in endosperm compared to the embryo. Further, we examined the 295

average methylation levels of CG, CHG and CHH in TEs and found that about 6,228 296

and 9,941 TEs contained CG and CHG methylation, respectively, whereas about 297

47,943 TEs harbored CHH methylation. In particular, more than 80% of TEs with 298

very short sequence in length (< 1,000 bp) were heavily methylated (about 80%) in 299

CG and CHG contexts (see Fig. 3D). Most of TEs with CHH methylation (about 300

51.9%) were methylated in the medium range. Moreover, a large number of TEs were 301

more densely methylated in CHH than CG and CHG. The CHH context in long TEs (> 302

1,000 bp) was more heavily methylated (see Fig. 3D). These observations show that 303

CG and CHG methylation are more effectively maintained in short TEs than CHH 304

methylation during the DNA replication, while CHH methylation is more randomly 305

distributed across short and long TEs with a relatively low level of methylation 306

compared with CG and CHG in both embryo and endosperm. Compared to known 307

plants such as Arabidopsis (Hsieh et al., 2009), rice (Zemach et al., 2010b), maize 308

(Zhang et al., 2014; Lu et al., 2015) and soybean (Song et al., 2013), the genomic 309

methylation profiles in castor bean seems to be distinct. 310

Taken together, although there are general features in the methylation patterns 311

among different plant species, castor bean exhibits some specific patterns such as a 312

very high proportion of CHH methylation, more robust maintenance of CHG 313

methylation at a high level in TE regions and a very similar level of CHH methylation 314

arising between endosperm and embryo. 315

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DNA methylation profiles in gene and TE regions 317

While inspecting the distribution of CG, CHG and CHH methylations in gentic and 318

TE regions, we observed that the CG methylation occurred preferentially in the gene 319

body regions relative to the flanking regions, similar to previous reports in other 320

plants (Hsieh et al., 2009; Zemach et al., 2010b; Song et al., 2013; Lu et al., 2015; 321

Wang et al., 2015). Whereas the extents of CHG and CHH methylation were low in 322

gene body regions and relatively higher in flanking regions (as shown in Fig. 4A) in 323




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consistent with their global DNA methylation patterns (Fig. 2). The DNA methylation 324

extents of CG, CHG and CHH contexts were very low near transcriptional start sites 325

and transcriptional end sites but gradually increased when departing from these sites 326




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(Fig. 4A). In contrast to gene body regions, the TEs were highly methylated in all CG, 327

CHG and CHH sequence contexts (Fig. 4B) similar to previous studies in Arabidopsis 328

(Hsieh et al., 2009), rice (Zemach et al., 2010b) and soybean (Song et al., 2013), 329

suggesting that DNA methylation in transposon silencing might be conserved in 330

plants. Further, we noted that most of the methylated TEs belonged to the Class I 331

(retrotransposons), especially for LTR/Gypsy and LTR/Copia, in consistent with their 332

abundances in castor bean genome. Among the Class II (DNA transposons), the TE 333

type DNA/MuDR was more frequently methylated than others (Supplemental Fig. 334

S9A). 335

Unsurprisingly, the endosperm clearly exhibited the CG, CHG and CHH 336

hypo-methylation in both gene body and flanking regions compared to the embryo. 337

Moreover, the endosperm hypo-methylation of CG and CHG was shown in TE 338

regions. The endosperm exhibited a higher CHH methylation level in TE regions than 339

the embryo (Fig. 4B). Consistent with the reduced methylation extent of TEs in 340

endosperm, the average methylation extents of both Class I and II TEs were slightly 341

lower in CG and CHG contexts relative to the embryo. The TEs within Class I 342

exhibited a higher methylation extent than that within Class II. The CHH methylation 343

extent of the Class I TEs in endosperm, however, appeared to be slightly higher 344

compared to the embryo, and the CHH methylation extents of the Class II TEs 345

appeared to equal in endosperm and embryo (see Supplemental Fig. S9B). 346

347

Characterization of endosperm DMRs 348

To further characterize the genomic hypo-methylation regions in endosperm, we 349

analyzed the differential methylation regions (DMRs) within a sliding window by 350

subtracting the endosperm methylation loci from embryo methylation loci. Initially, 351

we identified 64,733 CG, 70,074 CHG and 3,298 CHH discreet DMRs between 352

endosperm and embryo, respectively. To sort out the substantial and significant 353

difference of DNA methylation level at DMRs, we applied a stringent standard, i.e., at 354

least a 40% difference (Fisher's exact test P < 0.05) with at least either methylation 355

level more than 60% for the CG and CHG contexts, and at least a 20% difference 356




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(Fisher's exact test P < 0.05) with at least either methylation level more than 40% for 357

the CHH context between endosperm and embryo. In total, 20,464 CG and 25,940 358

CHG DMRs were identified, of which 99.9% of loci were significantly 359

hypo-methylated in endosperm (Table 2 and Supplemental Table S3). However, only 360

2,727 CHH DMRs were identified, of which 55.9% loci exhibited a hypo-methylation 361

in endosperm, and 44.1% loci exhibited a hypo-methylation in embryo (Table 2 and 362

Supplemental Table S3). 363

Among these CG DMRs, there were approximately 30.8% DMRs located in the 364

gene body, 27.9% and 25.6% DMRs in 2 kb upstream and downstream regions of 365

gene respectively, while only 15.7% DMRs were located in TE regions. In the 366

contrary, most of CHG DMRs (about 42.0%) were found in TE regions, while just 367

10.1% DMRs were found in the gene body regions and 27.4% and 20.6% were found 368

in upstream and downstream regions respectively. For the CHH DMRs, most of them 369

were located in the upstream and downstream regions of gene (47.3% and 30.1%, 370

respectively), while only 6.9% DMRs were located in gene body and 15.7 % in TE 371

regions (Supplemental Fig. S10). These analyses provided a clear landscape of 372

genomic methylation differentiation between embryo and endosperm (see Fig. 4), i.e., 373

almost all of DMRs were linked to the genomic hypo-methylation of endosperm and 374

mainly resulted from the reduction of CG and CHG methylation. The CG DMRs were 375

mainly located in the gene body regions while most CHG DMRs were found in the 376

TEs regions. 377

Effects of DNA methylation on gene expression 378

To determine whether genomic DNA methylation level might affect global gene 379

expression in the castor bean seeds, we initially investigated the methylation changes 380

of CG, CHG and CHH contexts for all transcripts obtained from previous studies (Xu 381

et al., 2014) in embryo tissues. Based on their expression levels genes were divided 382

into four classes, i.e., no expression (RPKM ≤ 1), low (1 < RPKM ≤ 10), middle 383

(10 < RPKM ≤ 100) and high expression level (RPKM > 100) according to Lu et 384

al.’s criteria (2015). As shown in Supplemental Fig. S11, it seemed that the CG 385




18

methylation levels occurring in the up-stream and down-stream regions of genes were 386

negatively correlated with their gene expressions, but in the gene body regions the 387

moderately expressed genes showed the highest CG methylation level, though it 388

might be difficult to predict that the interaction between the methylation extents 389

(occurring in the up-stream, down-stream, or gene body regions) and gene expression 390

levels. However, it appeared to be clear that the non-expressed (or rarely expressed) 391

genes exhibited the highest CHG and CHH methylation levels in the up-stream, 392

down-stream and gene body regions, though the changes of methylation levels were 393

slight across the different expression levels. These observations implied that DNA 394

methylation, in a way, repressed gene expression in castor bean embryo tissues. 395

To characterize the effect of hypo-methylation regions in endosperm on gene 396

expression, we collected the genes with substantially reduced DNA methylation 397

within 2 kb of either the 5' or 3' end and examined their expression between 398

endosperm and embryo based on our previous mRNA-seq data from embryo and 399

endosperm (Xu et al., 2014). Results showed that genes with reduced CG and CHG 400

methylation in the upstream of the start of transcription were, slightly but not 401

significantly, upregulated their expression in the endosperm relative to embryo, 402

whereas genes with hypo-methylation occurring in the 3' end did not alter their 403

expression level (Fig. 5, A and B). In particular, expression levels of genes exhibiting 404

reduced CHH methylation in the 5' and 3' ends were not affected (Fig. 5C). However, 405

we investigated the transcriptome of endosperm and embryo and identified subsets of 406

the genes that were preferentially expressed in endosperm with at least fourfold higher 407

than embryo (Supplemental Table S4). By calculating the average methylation levels 408

in the gene body and the 5' and 3' end, we found that these genes exhibiting 409

endosperm preferential expression showed significantly low levels of CG and CHG 410

methylation (Fisher's exact test, P < 0.05), but not in CHH loci of endosperm 411

compared with embryo. Clearly, it was observed that these genes, which were 412

preferentially up-expressed in endosperm relative to embryo, exhibited a reduced CG 413

and CHG methylation in endosperm throughout the 5', gene body, 3' (Fig. 5, D and E), 414

and a tiny CHH methylation reduction around these genes as well, if any (Fig. 5F). 415


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Indeed, several studies also showed DNA hypo-methylation could alter gene 416

preferential expression in endosperm (Hsieh et al., 2009; Zemach et al., 2010). As 417

illustrated in Fig. 6, endosperm hypo-methylation in some specific loci markedly 418




20

promoted the gene expression, whereas embryo hypermethylation near the promoter 419

regions repressed the gene transcription. For example, the gene 28629.m000565 420

encoding a beta-fructofuranosidase involved in the carbohydrate metabolic processes 421




21

was strongly expressed in endosperm. The gene 30093.m000370, homologous to the 422

AtYUC10 (AT1G48910) involved in regulating seed development in Arabidopsis 423

(Cheng et al., 2007), was strongly expressed in endosperm. The two genes exhibited 424

substantially lower DNA methylation extents and higher expression levels in 425

endosperm than that in embryo. Further, the DNA methylation extents and gene 426

expression levels of the two genes were experimentally validated by using the 427

combination method of bisulfite treatment and cloning sequencing (see Fig. 6, B and 428

C). In addition, gene ontology (GO) analysis showed significant evidence for 429

functional enrichment of genes affected by endosperm hypo-methylation DMRs 430

(Supplemental Fig. S12). The GO terms including binding, catalytic, biological 431

regulation, cellular and metabolic processes were substantially enriched (χ2 test, P < 432

0.05). These evidences strongly imply that the endosperm hypo-methylation are likely 433

involved in the regulation of specific endospermogenesis and storage materials 434

biosynthesis in endosperm, though its potential mechanism is unclear yet. In short, 435

our data indicated that the CG and CHG hypo-methylation might be the potential 436

mechanism for altering gene preferentially expressed in castor bean endosperm, 437

particularly when the CG and CHG hypo-methylations occur near or within a gene. 438

439

The association analyses of small RNAs expression and DNA methylation 440

Increasing evidences have indicated that small RNAs can direct de novo DNA 441

methylation at their target loci through RdDM (Law and Jacobsen, 2010; Mosher and 442

Melnyk, 2010). Here, we investigated the small RNA expression profiles in 443

endosperm and embryo tissues by high-throughput deep sequencing. A total number 444

of 10,323,788 and 12,382,489 clean reads were obtained from embryo and endosperm 445

libraries, respectively. After filtration of raw data (see Materials and methods), we 446

found that the 24-nt class was the most abundant group of small RNAs in castor bean 447

seeds based on their distribution in length (Fig. 7A), consistent with our previous 448

study in castor bean (Xu et al., 2013). 449

Considering that the function of different small RNAs in mediating DNA 450

methylation was unclear in castor bean seeds, the base composition and 451




22

strand-specific DNA methylation state of bisulfite converted genomic DNA sequences 452

were analyzed within the regions homologous to all 21–24 nt unique smRNAs 453

sequenced from castor bean seeds. Theoretically, the cytosine distribution pattern of 454




23

small RNA sequence could influence the methylated cytosine distribution of the DNA 455

strand identical to the sRNA sequence (termed the sense strand, mC), and guanine in 456

sRNAs could influence the methylated cytosine distribution of the DNA strand 457

complementary to the small RNA sequence (termed the antisense strand, mC*). We 458

investigated all uniquely mapping sRNA loci and counted the number of methylated 459

cytosines on either DNA strand of the nuclear genome. As a result, we found that the 460

changes of methylated cytosines on the sense and antisense strand were strongly 461

associated with the altered cytosine and guanine residue in 24-nt siRNAs (Fig. 7B), 462

whereas this tendency seem to be not obvious in mapping regions of 21–23 nt sRNAs 463

(Supplemental Fig. S13, A-C), suggesting DNA methylation might be closely linked 464

to the expression of 24-nt siRNAs in castor bean seed. Therefore, we focused on the 465

24-nt siRNAs mapping regions for subsequent analyses. 466

We next sought to investigate the average methylation levels between 24-siRNA 467

mapping regions and no mapping regions at each methylation sequence contexts. 468

Interestingly, the methylation levels of both CHG and CHH contexts were 469

significantly higher in siRNA covered regions than the regions without siRNA 470

mapping (Fisher's exact test, P < 0.05), whereas the CG methylation level of siRNA 471

regions did not resulted in a substantial increase compared to the regions without 472

siRNA (see Fig. 7C). Potentially, the increase of CHH methylation level in 24-siRNA 473

mapped regions might be caused by the activation of RdDM pathway. To test this, we 474

identified five genes, including RcPoI IV (30190.m011113), RcPoI V 475

(30131.m007197), RcRDR2 (30147.m013981), RcDCL3 (29739.m003642) and 476

RcAGO4 (29684.m000322), involved in the RdDM pathway from castor bean based 477

on genome data, and examined their expression levels in different tissues including 478

leaf, embryo and endosperm. Compared to leaf tissue, these five genes were 479

up-regulated in endosperm and embryo tissues. Further comparing their expression 480

levels between endosperm and embryo, we found that these five genes were markedly 481

down-regulated in endosperm relative to embryo (see Supplemental Fig. S13D). 482

These evidences suggest that genes involved in the RdDM pathway be participated in 483

altering non-CG methylation in castor bean endosperm and embryo. 484




24

In addition, we found that the 24-nt siRNA abundance of the gene body was 485

smaller than the upstream and downstream flanking sequence regions. More siRNAs 486

located in the upstream rather than in the downstream sequence were identified (Fig. 487

8A). In particular, the abundance of siRNAs in the flanking regions in endosperm was 488

substantially lower than that in embryo, consistent with the methylation pattern of 489

non-CG contexts in the gene region (Fig. 4). Although the distribution pattern of 490

siRNAs in the TE and its flanking regions was similar to that in gene regions, the 491

abundance of siRNAs in TE regions was significantly higher in number than gene 492

regions, consistent with the denser and heavier methylation level occurring in TE 493

regions (Fig. 8B). Interestingly, a large number of TEs with siRNA coverage (85.2%) 494

were methylated at the moderate level in CHH context, whereas only 7.8% and 15.0% 495

of TEs were methylated in CG and CHG contexts, respectively (Fig. 8C). Also, we 496

found the significant evidence of the enrichment of these TEs around the genes within 497

the 4 kb regions (Fisher's exact test, P < 0.01). In particular, we found that there were 498

more 24-siRNAs enriched in CHH hyper-methylated regions but fewer siRNAs in the 499

CHH hypo-methylated regions in endosperm and embryo tissues (Wilcoxon rank-sum 500

test, P < 0.01) (Fig. 8D). These results clearly indicated that 24-siRNAs could 501

significantly induce the increase of DNA methylation level in non-CG sequence 502

contexts, special for CHH methylation which might be guided by canonical RdDM 503

pathway. Also, we noted that the number of CHH methylation guided by the RdDM 504

pathway just made up about 27% of total CHH methylation loci in castor bean 505

endosperm. 506

507

508




25

DISCUSSION 509

In current study, we globally identified the DNA methylation-related genes, 510

comprehensively investigated DNA methylation levels and patterns in castor bean 511




26

endosperm and embryo integrated with the expression profiles of small RNA and 512

mRNA, and found a new landscape of genomic DNA methylation from a 513

dicotyledonous seeds with persistent endosperm throughout seed development. As 514

reported above, our observations showed that key methyltransferases and 515

demethylases were independently evolved in monocotyledons and dicotyledons, 516

though these methylation-related genes were conserved among species. However, our 517

current study still leaves an open question of whether global methylation or 518

demethylation has a common evolutionary origin among angiosperms, though the 519

overall process of extensive DNA methylation or demethylation is likely conserved 520

between monocots and dicots. 521

Genome-wide bisulfite sequencing reveals that rich CHH methylation occurred in 522

castor bean endosperm and embryo tissues, which appears to be distinct from 523

previous reports from other plants. The higher proportion of CHH methylation arose 524

in both embryo and endosperm of castor bean, in a way, consistent with the high 525

proportion of CHH methylation identified from the leaves of cassava (about 58%) 526

(Wang et al., 2015). In spite of the global CG and CHG DNA hypo-methylation arisen 527

in endosperm relative to embryo, the CHH methylation did not exhibit a significant 528

reduction in endosperm. Comparing to the genomic DNA methylation in seeds of 529

angiosperm such as Arabidopsis (Hsieh et al., 2009), rice (Zemach et al., 2010b), 530

maize (Lu et al., 2015) and soybean (Song et al., 2013), our observation on genomic 531

DNA methylation of castor bean endosperm and embryo exhibits a novel pattern, 532

implying a distinct mechanism of maintaining DNA methylation occurs in dicot castor 533

bean seeds with persistent endosperm. We do not rule out, however, whether the 534

non-reduction of CHH methylation arisen in endosperm relative to embryo in castor 535

bean was resulted from the different stages of seed development in this study, 536

compared to Arabidopsis (in which its endosperm exists only at the early stage of seed 537

development). 538

Generally, the low methylation level in the upstream of genes could promote the 539

gene expression and the presence of DNA methylation inhibits transcriptional 540

initiation. In present study, we noted that relationship between DNA methylation and 541




27

global gene expression is not strong in castor bean seeds tissues consistent with 542

previous reports in Arabidopsis (Hsieh et al., 2009) and rice (He et al., 2010). Our 543

investigations appeared to demonstrate that DNA methylation, in a way, mainly 544

repressed gene expression in castor bean seeds, though the associations were variable, 545

depending on the methylated regions (such as up-stream, down-stream and gene body) 546

and different methylation sequence contexts. For the genes with preferential 547

expression in endosperm, however, there is a significant decrease of CG and CHG 548

methylation level in the promoter region of genes relative to in embryo tissues. These 549

genes affected by hypo-methylation in endosperm involved into some specific 550

development processes. 551

It is well-known that the final levels and patterns of DNA methylation were 552

determined by the activity of both DNA methyltransferase and demethlylases. While 553

inspecting the expression profiles of these DNA methylation-related genes, we found 554

a distinct expression pattern of RcDME and RcDRM genes in castor bean seeds. In 555

Arabidopsis, the DME gene was specifically expressed in central cell of the female 556

gametophyte (Choi et al., 2002), which probably may play a critical role in 557

maintaining the genomic DNA methylation extent and resulting in the global genomic 558

DNA hypo-methylation in the developing endosperm (Hsieh et al., 2009). Current 559

results demonstrated that, as shown in Fig. 1, the RcDME were broadly expressed in 560

various tissues of castor bean, strongly suggesting that the mechanism of maintaining 561

genomic DNA methylation might be different in castor bean from that of Arabidopsis. 562

Besides, the expressions of RcMET1 and RcCMT, encoding CG and CHG 563

methyltransferase, were substantially lower in the ovule or the developing endosperm 564

relative to embryo, implying that activities of CG and CHG methyltransferase might 565

be insufficient in endosperm which might result in a CG and CHG hypo-methylation 566

in some regions. As shown in Supplemental Fig. 7, the expression level of RcDRM3, 567

however, was higher than RcMET1 and RcCMT genes in endosperm and was not 568

repressed (unlike in Arabidopsis, this gene were repressed in endosperm, Jullien et al., 569

2012) suggesting the potential roles of RcDRM3 in castor bean seed development with 570

persistent endosperm. Indeed, there are evidences that loss-of-function of DRM could 571




28

result in abnormal reproductive growth and seed morphology (Garcia-Aguilar et al., 572

2010; Moritoh et al., 2012). 573

In the RdDM pathway, the 24-nt siRNAs can direct DRM2 to methylate CHH 574

sequence context (Law and Jacobsen, 2010; Mosher and Melnyk, 2010). Our current 575

study revealed that a large number of 24-nt siRNAs were transcribed in endosperm 576

and embryo, and were mapped with TEs regions where CHH methylation arisen more 577

densely and heavily. In addition, the expression levels of genes (RcPoI IV, RcPoI V, 578

RcRDR2, RcDCL3, RcAGO4 and RcDRM3), involved in activating the RdDM 579

pathway, were significantly up-regulated in endosperm and embryo tissues than that 580

in leaf tissue, clearly suggesting the RdDM pathway might be activated and play a 581

critical role in directing CHH methylation in endosperm and embryo. Although the 582

biological interests of the rich CHH methylation in castor bean endosperm remain 583

unclear, it is possible that when the CG and CHG demethylation occurs extensively in 584

endosperm genome, the maintenance of CHH methylation would help to repress the 585

movement of many TEs, safeguarding global gene expressions and endosperm 586

persistent development. However, it should be noted that the CHH methylation 587

directed by the RdDM pathway just made up about 27% of total CHH methylation 588

loci in castor bean endosperm. This implies that the rich CHH methylation in 589

endosperm might be, not only, established and maintained by the RdDM pathway, and 590

other uncertain factors or pathways are, probably, involved in mediating the CHH 591

methylation in castor bean seeds. 592

Taken together, as shown in Fig. 9, we propose a potential scenario to explain how 593

the DNA methylation was maintained in castor bean seed. The repression of RcMET1 594

and RcCMT expression and activity of RcDME in endosperm result in genomic 595

hypo-methylation of CG and CHG at some discrete loci, which may be a conserved 596

mechanism for the establishment of endosperm hypo-methylation among flowing 597

plants, at least in Arabidopsis, rice and castor bean. In contrast with RcMET1 and 598

RcCMT, the expressions of RcDRM3 and the abundant 24-nt siRNAs in endosperm 599

might result in an effective maintenance of CHH methylation within siRNAs mapping 600

regions during the endosperm development in castor bean. Similarly, the relatively 601




29

higher expressions of RcMET1, RcCMT and RcDRM genes maintain the genomic CG, 602

CHG and CHH methylation extents in embryo development. Although this scenario 603

just provides a preliminary hypothesis in explaining the potential mechanism of 604




30

genomic DNA methylation in castor bean seed, the current study adds new insights 605

into understanding the molecular mechanism of genomic DNA methylation in embryo 606

and endosperm of angiosperms. 607

608

CONCLUSION 609

We globally characterized the DNA methylation-related genes encoding DNA 610

methyltransferase and demethylases and comprehensively dissected the associations 611

of genomic DNA methylation and global gene expression in castor bean seeds. Our 612

finding revealed that the CHH methylation extent in endosperm and embryo was, 613

unexpectedly, substantially higher than known plants, irrespective of the CHH 614

percentage in their genomes. In particular, we found that the global hypo-methylation 615

of CG and CHG in endosperm markedly switched the global gene expression and was 616

closely associated with the endosperm development. Curiously enough, within the 617

siRNAs coverage regions, the CHH methylation occurring in endosperm tightly 618

associated with the expression of 24-nt siRNAs. The rich CHH methylation occurring 619

in castor bean seeds and the non-reduction of CHH methylation in endosperm relative 620

to embryo result from, at least partially, the RdDM pathway, where the 24-nt siRNAs 621

play a critical role in repressing the activation of TEs around the genes by guiding 622

CHH methylation in TE regions. These results reveal a novel genomic DNA 623

methylation pattern in dicot seeds with persistent endosperms. 624

625

MATERIALS AND METHODS 626

Identification of DNA methylation-related genes 627

An extensive search was carried out to detect all DNA methylation-related genes 628

encoding DNA methyltransferases and demethylases based on castor bean genome 629

database (downloaded from the website http://castorbean.jcvi.org/index.php). The 630

sequences of Arabidopsis DNA methylation-related genes were obtained from the 631

website (https://www.arabidopsis.org). The amino acid sequences obtained from 632

Arabidopsis were designed as queries, respectively, against the castor bean protein 633




31

database by running the NCBI-blast-2.2.24+ software. Subsequently, all hit amino 634

acid sequences were extracted using a custom Perl script based on their ID number, 635

and then these sequences were confirmed by presenting the characterized domain in 636

the candidate sequences by SMART tools (http://smart.embl-heidelberg.de/) and 637

InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/). Multiple alignments of amino 638

acid sequences were carried out, respectively, using Clustal W (Larkin et al., 2007) 639

and conserved motifs and doamin of each group were identified. Besides, we 640

constructed an un-rooted phylogenetic tree using neighbor-joining criteria in MEGA 641

5.0 (Tamura et al., 2011) with 10,000 bootstrap replicates. 642

643

Expression profiles analysis of DNA methylation-related genes by qPCR 644

In order to investigate the expression profiles of DNA methylation-related genes, we 645

collected the diverse tissues including tender leaf, stem apex, pollen, ovule, early 646

embryo and endosperm (25 DAP, days after pollination), late embryo and endosperm 647

(40 DAP) from the inbred ZB107 (kindly provided by Zibo Academy of Agricultural 648

Sciences, Shandong, China). Total mRNA was isolated from the various tissues using 649

RNAprep Pure Plant Kit (Tiangen, Beijing, China). The mRNA with DNA enzyme 650

digestion was used for first-strand cDNA synthesis using PrimeScript™ RT reagent 651

Kit (TaKaRa, Dalian, China), and then these cDNAs were subjected to real-time 652

quantitative PCR (qPCR) using SYBR Green Master Mix (TaKaRa, Dalian, China). 653

The qPCR for each tissue was performed with three independent biological 654

replications in the Bio-Rad CFX Manager system, as follows: precycling steps of 95℃ 655

for 2 min and then followed by 40 cycles of 95℃ for 15 sec, 58℃ for 20 sec and 72℃ 656

for 30 sec. The RcACTIN2 gene was used as internal reference to normalize the 657

relative amount of mRNAs for all samples. Also, the expressions of genes involved 658

into the RdDM pathway were examined by qPCR, as described above. All primers 659

used in this study are listed in the Supplemental Table S5. 660

661

Bisulfite sequencing 662

Based on previous morphological descriptions (Greenwood and Bewley, 1982), 663


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developing seeds at the early stage of cotyledon formation (35 DAP, inbred ZB107) 664

were collected and dissected. Bisulfite sequencing (bis-seq) from five replicates of 665

endosperm and embryo tissues was performed as described in our previous study (Xu 666

et al., 2014). Firstly, high-quality genomic DNA (about 5 μg) from ten replicates of 667

35-DAF endosperm and embryo was isolated using the Plant genomic DNA kit 668

(Tiangen, Beijing, China). Secondly, genomic DNA was fragmented between 300–669

500bp and end repaired. Thirdly, custom Illumina adapters were ligated following the 670

manufacturer’s protocols, and then fragments with adapters were bisulfite converted 671

using EpiTech Bisulfite Kits (Qiagen, Valencia, CA, USA). Finally, the converted 672

DNA fragments were PCR amplified with Pfu DNA polymerase (TaKaRa, Dalian, 673

China) and sequenced at BGI (Shenzhen, China), generating paired-end 90 bp reads 674

from each library. 675

676

Small RNA library construction and sequencing data 677

Small RNA sequencing with three replicates was performed as described in our 678

previous study (Xu et al., 2013) using 35-DAP endosperm and embryo tissues. Briefly, 679

after total RNA isolation and polyacrylamide gel electrophoresis, the small RNAs 680

(16–30 nt) were isolated and purified by sRNA gel extraction Kit (TaKaRa, Dalian, 681

China). Then, adapters were respectively ligated to the 5' and 3' termini of small 682

RNAs and reserve transcription was performed followed by low cycle PCR. Finally, 683

small RNA libraries were constructed and sequenced using an Illumina Genome 684

Analyzer. 685

After sequencing, we clipped the adapter sequences and filtered out the low quality 686

tags. The reads with identical sequence including rRNA, tRNA, snRNA and snoRNA 687

were removed from the raw data. The clean small RNA sequences that ranged from 16 688

to 30 nt were collected and mapped to castor bean genome using SOAP 2.0 program 689

(Li et al., 2009). The unique sequences were subjected to subsequent analysis. 690

691

Analysis of differentially methylated regions 692

The clean data we obtained were mapped to the castor bean Hale reference genome 693


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using the BSMAP software (Xi and Li, 2009) allowing up to two mismatches. Then 694

we used a custom Perl scripts to call methylated cytosine and calculate its methylation 695

level based on cytosine percentage in a given position. The methylation profile at 696

flanking 2 kb regions and gene body (or transposable elements) is plotted based on the 697

average methylation levels for each 100-bp interval. Additionally, we identified 698

differentially methylated regions (DMRs) for each sequence context (CG, CHG and 699

CHH) between endosperm and embryo tissue by using the tDMR package (Rakyan et 700

al., 2008) with the following stringent criteria: a) at least five methylated cytosine 701

sites; b) more than average ten reads coverage; c) the distance between adjacent 702

methylated sites < 200 bp; d) more than 20% methylation level at least one sample; 703

and e) fisher's exact test P < 0.05 and false discovery rate FDR < 0.05. The putative 704

DMRs overlapping at adjacent 2 kb (upstream or downstream) or body regions of 705

genes or transposable elements (TEs) were sorted out for further study. 706

707

Effect of DNA methylation on gene expression 708

We used publically available global gene expression data (downloaded from our 709

previous study under accession number SRX485027) for endosperm and embryo at 710

the same stage. The gene's expression levels were normalized and differentially 711

expressed genes were also identified between endosperm and embryo using the 712

TopHat software package (Trapnell et al., 2012). Expression scores for genes with 713

DMRs in the endosperm within 2 kb of either the 5' or 3' end were used in the next 714

analysis. 715

716

Validation of the DMRs and DMR-related gene expression 717

To experimental confirm whether the differential DNA methylation alters the gene 718

expression, we picked up two hypo-methylated DMRs identified in endosperm and 719

tested the expressions of adjacent genes in endosperm and embryo tissues. About 500 720

ng genomic DNA were extracted from 35-DAP endosperm and embryo and were 721

subjected to bisulfite treatment using the EZ DNA Methylation-Lightning Kit (ZYMO 722

Research) according to the manufacturer's instructions. The bisulfite converted DNA 723


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was PCR amplified with AceTaq DNA Polymerase (Vazyme, Nanjing, China). For the 724

DMR identified at the upstream region of gene 28629.m000565, primers 725

(5'-TGGTTGGTTTTGGTGYAATTAAATAYTGGTGG-3' and 726

5'-TTACATTATTTRACATATRTCAAACTTTCATC-3' were used; for the DMR 727

identified within the first exon of gene 30093.m000370, primers 728

5'-TAGGGTTTATTTTGAAAAATGAAATGTAGT-3' and 729

5'-AAATACACACACACTCTCTCTCTCATA-3' were used in this study designed by 730

Kismeth software (http://katahdin.mssm.edu/kismeth) (Gruntman et al., 2008). The 731

purified PCR products were cloned into the pGEM-T vector and 15 clones were 732

randomly selected for sequencing. The final sequencing results were summarized and 733

visualized with MATLAB software. 734

In addition, relative expression levels of these two genes tested in endosperm and 735

embryo tissue were carried out by qPCR as described above. For the genes 736

28629.m000565 and 30093.m000370, primer pairs 737

5'-AGCCCAAAGACAATCCGGTA-3' and 5'- ATACCAGTTTCCGCTTTCGC-3', 738

and 5'-ATCGTCACAGCAATGGATCG-3' and 739

5'-CCAGAGCCAACAACCAAGAC-3' were used for qPCR amplification in this 740

study, respectively. 741

742

Accession numbers 743

The raw data used for this study have been submitted to the NCBI Sequence Read 744

Archive under accession number SRX1267331. 745

746

Table and Figure Legends 747

Table 1. Identification of genes encoding DNA methyltransferases and 748

demethylases in castor bean. 749

Gene loci Location CDS length (bp) Protein length (aa) Homologs Annotation

Cytosine-5 DNA methyltransferases

29983.m003308 29983:1075551-1081789 4,629 1,542 AT5G49160 MET1-1

29609.m000606 29609:246194-255135 4,755 1,584 AT5G49160 MET1-2

28582.m000332 28582:146814-156888 2,538 845 AT1G80740 CMT1




35

29827.m002677 29827:858418-869538 2,205 734 AT4G19020 CMT2

29848.m004665 29848:1192243-1195163 1,215 404 AT5G25480 Dnmt2

29631.m001043 29631:330007-332680 1,440 479 AT5G14620 DRM1

29889.m003366 29889:694336-699871 2,037 678 AT3G17310 DRM2

29917.m001982 29917:300487-305138 2,061 686 AT5G15380 DRM3

DNA Demethylases

29428.m000327 29428:105887-115737 5,621 1,876 AT5G04560 DME

29092.m000452 29092:87494-96521 4,905 1,634 AT2G36490 ROS1

29991.m000647 29991:222724-231238 5,139 1,712 AT4G34060 DML3

750

Table 2. Differentially methylated regions (DMRs) between embryo and 751

endosperm in castor bean. 752

DNA context Total number of

DMRs Hyper-methylation

in embryo Hyper-methylation in

endosperm CG 20,464 20,443 (99.9%) 21 (0.01%)

CHG 25,940 25,903 (99.9%) 37 (0.01%) CHH 2,727 1,526 (55.9%) 1,201 (44.1%)

753

Figure 1. The expression profiles of DNA methyltransferase and demethylase 754

genes in different tissues of castor bean. Castor bean ACTIN2 gene was used as the 755

internal reference for qRT-PCR analysis. The expression levels of each gene in 756

different tissues were normalized to the leaf. The error bars indicate the standard error 757

with three biological replicates. 758

759

Figure 2. The DNA methylation profiles in castor bean seeds. A, Global 760

distribution of three sequences methylation in Scaffold 29852 (TE-rich Scaffold). B, 761

Global distribution of three sequences methylation in Scaffold 29848 (gene-rich 762

Scaffold). Red and blue track represent the cytosine methylation in embryo (Em) and 763

endosperm (En), respectively. Genes and TEs oriented 5' to 3' and 3' to 5' are shown 764

above and below the line respectively. 765

766

Figure 3. DNA methylayion levels and distributions in castor bean seed. A, 767

Genomic methylation levels of CG, CHG and CHH in endosperm (En) and embryo 768

(Em) among different species. B, Relative proportion of CG, CHG and CHH 769




36

methylation contexts in endosperm and embryo among different species. C, 770

Histogram of methylation frequencies on the y axis for different levels (%) of CG, 771

CHG and CHH methylation (x axis) in endosperm and embryo tissues. D, Points 772

scatter of the correlation between TE length and average methylation level in CG, 773

CHG and CHH contexts. 774

775

Figure 4. Genomic DNA methylation profiles in endosperm (blue line) and 776

embryo (red line) tissues. A, DNA methylation patterns across genes. B, DNA 777

methylation patterns across TEs. Castor bean annotated genes and TEs were aligned at 778

the 5' end (Left) or the 3' end (Right). The average methylation levels for each 100-bp 779

interval are plotted. The dashed line represents the point of alignment. 780

781

Figure 5. Effect of hypo-DMR regions in endosperm on gene expression. A-C, 782

Box plots indicate the differences of expression level between endosperm and embryo 783

for genes with either 5' hypo-methylation or 3' hypo-methylation in endosperm, 784

comparing to the average of all genes. D-F, The average methylation levels for 785

endosperm-preferred genes with fourfold expression level relative to embryo are 786

plotted for each 100-bp interval. 787

788

789

Figure 6. Effect of two hypo-methylation regions in endosperm on gene 790

expression. A, DNA methylation level of all three sequence contexts in endosperm 791

and embryo. The grey box indicates the region that was experimentally confirmed . B, 792

Validation of DNA methylation level by bisulfite PCR sequencing. C, Relative 793

expression level of two genes (28629.m000565 and 30093.m000370) overlapping the 794

hypo-methylation regions by qPCR analysis. 795

796

Figure 7. The length distribution of small RNAs and relationship between 24-nt 797

siRNA and DNA methylation. A, The length size distribution of small RNAs (from 798

18-26 nt) in castor bean endosperm and embryo. B, Nucleotide frequency and 799




37

distribution of 24-nt siRNAs mapping region and within 10-nt flanking regions. mC 800

represents the methylcytosine on the sense strand; mC* represents the methylcytosine 801

on the antisense strand. C, Comparison of DNA methylation level between 24-siRNA 802

uniquely mapping region and without region in each sequence contexts. 803

804

Figure 8. The abundance of 24-siRNAs in castor bean embryo and endosperm 805

tissues. A, The number distribution of siRNAs in gene body from 5' end (Left) to the 806

3' end (Right) and flanking 2kb region. B, The number distribution of siRNAs in TE 807

and flanking 2kb region. C, Methylation level of all three sequence contexts in the 808

TEs within siRNAs coverage. D, Abundance of 24-siRNA located to the CHH 809

hyper-methylated and hypo-methylated regions. 810

811

Figure 9. A proposed model for explaining the DNA methylation pattern in castor 812

bean endosperm and embryo. RcDME or RcROS1 removes the methylated cytosine 813

at genome scale (blue lollipops for CG, green lollipops for CHG and red lollipops for 814

CHH methylation respectively), and demthylation leads to the generation of 24-nt 815

siRNAs that could guide de novo CHH DNA methylation by RcDRM2 in the 816

endosperm and embryo; the insufficient expressions of RcMET1 and RcCMT in the 817

endosperm tissue, which maintain the CG and CHG methylation levels (denoted by 818

looping arrow) result in the reduced CG and CHG methylation (denoted by 819

short-handled lollipops), compared with embryo (denoted by long-handled lollipops). 820

821

Supplemental Data 822

Supplemental Figure S1 Comparison of amino acid sequences of DNA 823

methyltransferases MET1 between Arabidopsis thaliana and castor bean. The blue 824

boxes underlines represent the conserved domain regions and motifs, respectively. 825

826


methyltransferases DRM between Arabidopsis thaliana and castor bean. The blue 828




38


830


methyltransferases CMT between Arabidopsis thaliana and castor bean. The blue 832


834


methyltransferases DNMT2 between Arabidopsis thaliana and castor bean. The blue 836

underlines represent the conserved motifs.between Arabidopsis thaliana and castor 837

bean. 838

839

Supplemental Figure S5 Comparison of amino acid sequences of the domains of 840

DNA demethylases between Arabidopsis thaliana and castor bean. The blue boxes 841

represent the conserved domain regions. 842

843

Supplemental Figure S6 Schematic structures of DNA methyltransferases and 844

demethylases (A and B) and phylogenetic analysis (C and D). Monocot and dicot 845

proteins are colored red and blue respectively. Aly, A. lyrata; Ath, A. thaliana; Bdi, 846

Brachpodium distachyon; Mes, Manihot esculenta; Mgu, Mimulus guttatus (monkey 847

flower); Osa, Oryza sativa (rice); Ptr, Populus trichocarpa (poplar); Rco, Ricinus 848

communis (castor bean); Sbi, Sorghum bicolor; Zma, Zea mays (maize). 849

850

Supplemental Figure S7 Heatmaps representing the expression profiles of 851

methyltransferase and demethylase genes in RNA-seq data in castor bean endosperm 852

and embryo tissues. 853

854

Supplemental Figure S8 Sequencing depth and saturation analysis of the embryo and 855

endosperm tissues in castor bean. The y axis representing the cytosine coverage 856

percentage as the sequencing depth (x axis). 857

858




39

Supplemental Figure S9 DNA methylation and TEs. A, Percentage of different TE 859

types with DNA methylation in castor bean endosperm and embryo. B, Methylation 860

levels in class I and class II TEs in all sequence contexts. 861

862

Supplemental Figure S10 The percentage of DMRs distribution in TE, gene and 2kb 863

flanking gene regions. 864

865

Supplemental Figure S11 Effect of DNA methylation on global gene expression in 866

castor bean embryo tissues. 867

868

Supplemental Figure S12 GO enrichment analysis of genes affected by endosperm 869

DMRs. * indicates significant enrichment of GO terms. 870

871

Supplemental Figure S13 Relationship between small RNAs and methylated 872

cytosines, and genes expression involved in RdDM pathway. A-C, Nucleotide 873

frequency and distribution of 21-23 nt small RNAs mapping region. mC represents 874

the methylcytosine on the sense strand; mC* represents the methylcytosine on the 875

antisense strand. D, Expression levels of genes participated in the RdDM pathway 876

were tested by qPCR. the dash line represents the expression level of leaf. 877

878

Supplemental Table S1 Information on bisulfite sequencing data. 879

880

Supplemental Table S2 The percentages of CG, CHG and CHH in genomes of 881

Arabidopsis (At), rice (Os), maize (Zm) and castor bean (Rc). 882

883

Supplemental Table S3 Substantial differentially methylated regions in CG, CHG 884

and CHH sequence contexts between embryo and endosperm. 885

886

Supplemental Table S4 Endosperm-preferred genes and the differentially methylated 887

regions in each sequence context. 888




40

889

Supplemental Table S5 Primers used in this study for gene expression profiles based 890

on qPCR. 891

892

Acknowledgements 893

This study was facilitated by the Germplasm Bank of Wild Species at the Kunming 894

Institute of Botany. We are grateful to the Guangzhou Gene denovo Biotechnology 895

Co., Ltd for assisting in bisulfite sequencing analysis. 896

897

Competing interests 898

The authors declare that they have no competing interests. 899

900

901




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http://scholar.google.com/scholar?as_q=BSMAP: whole genome bisulfite sequence MAPping program&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xi&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xiao W, Custard KD, Brown RC, Lemmon BE, Harada JJ, Goldberg RB, Fischer RL (2006b) DNA methylation is critical forArabidopsis embryogenesis and seed viability. Plant Cell 18: 805-814.


Xing MQ, Zhang YJ, Zhou SR, Hu WY, Wu XT, Ye YJ, Wu XX, Xiao YP, Li X, Xue HW (2015) Global Analysis Reveals the CrucialRoles of DNA Methylation during Rice Seed Development. Plant Physiol 168: 1417-1432.


Xu W, Cui Q, Li F, Liu A (2013) Transcriptome-wide identification and characterization of microRNAs from castor bean Ricinuscommunis L. PLoS One 8: e69995.


Xu W, Dai M, Li F, Liu A (2014) Genomic imprinting, methylation and parent-of-origin effects in reciprocal hybrid endosperm ofcastor bean. Nucleic Acids Res 42: 6987-6998.


Zemach A, Kim MY, Hsieh PH, Coleman-Derr D, Eshed-Williams L, Thao K, Harmer SL, Zilberman D (2013) The ArabidopsisNucleosome Remodeler DDM1 Allows DNA Methyltransferases to Access H1-Containing Heterochromatin. Cell 153: 193-205.


Zemach A, Kim MY, Silva P, Rodrigues JA, Dotson B, Brooks MD, Zilberman D (2010b) Local DNA hypomethylation activates genesin rice endosperm. Proc Natl Acad Sci U S A 107: 18729-18734.


Zemach A, McDaniel IE, Silva P, Zilberman D (2010a) Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science14: 916-919.


Zhang M, Xie S, Dong X, Zhao X, Zeng B, Chen J, Li H, Yang W, Zhao H, Wang G, et al (2014) Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize. Genome Res 224: 167-176.


Zhang M, Zhao H, Xie S, Chen J, Xu Y, Wang K, Zhao H, Guan H, Hu X, Jiao Y, et al (2011) Extensive, clustered parental imprintingof protein-coding and noncoding RNAs in developing maize endosperm. Proc Natl Acad Sci U S A 108: 20042-20047.


Zhu JK (2009) Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet 43: 143-166.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title


http://search.crossref.org/?page=1&rows=2&q=Xiao W, Brown RC, Lemmon BE, Harada JJ, Goldberg RB, Fischer RL (2006a) Regulation of Seed Size by Hypo-methylation of Maternal and Paternal Genomes. Plant Physiol 142: 1160-1168.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xiao&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of Seed Size by Hypo-methylation of Maternal and Paternal Genomes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of Seed Size by Hypo-methylation of Maternal and Paternal Genomes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xiao&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 18%5BVolume%5D AND 805%5BPagination%5D AND Xiao W%2C Custard KD%2C Brown RC%2C Lemmon BE%2C Harada JJ%2C Goldberg RB%2C Fischer RL%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Xiao W, Custard KD, Brown RC, Lemmon BE, Harada JJ, Goldberg RB, Fischer RL (2006b) DNA methylation is critical for Arabidopsis embryogenesis and seed viability. Plant Cell 18: 805-814.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xiao&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=DNA methylation is critical for Arabidopsis embryogenesis and seed viability&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=DNA methylation is critical for Arabidopsis embryogenesis and seed viability&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xiao&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Physiol%5BTitle%5D%29 AND 168%5BVolume%5D AND 1417%5BPagination%5D AND Xing MQ%2C Zhang YJ%2C Zhou SR%2C Hu WY%2C Wu XT%2C Ye YJ%2C Wu XX%2C Xiao YP%2C Li X%2C Xue HW%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Xing MQ, Zhang YJ, Zhou SR, Hu WY, Wu XT, Ye YJ, Wu XX, Xiao YP, Li X, Xue HW (2015) Global Analysis Reveals the Crucial Roles of DNA Methylation during Rice Seed Development. Plant Physiol 168: 1417-1432.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xing&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Global Analysis Reveals the Crucial Roles of DNA Methylation during Rice Seed Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Global Analysis Reveals the Crucial Roles of DNA Methylation during Rice Seed Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xing&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28PLoS One%5BTitle%5D%29 AND 8%5BVolume%5D AND 69995%5BPagination%5D AND Xu W%2C Cui Q%2C Li F%2C Liu A%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Xu W, Cui Q, Li F, Liu A (2013) Transcriptome-wide identification and characterization of microRNAs from castor bean Ricinus communis L. PLoS One 8: e69995.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Transcriptome-wide identification and characterization of microRNAs from castor bean Ricinus communis L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Transcriptome-wide identification and characterization of microRNAs from castor bean Ricinus communis L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Nucleic Acids Res%5BTitle%5D%29 AND 42%5BVolume%5D AND 6987%5BPagination%5D AND Xu W%2C Dai M%2C Li F%2C Liu A%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Xu W, Dai M, Li F, Liu A (2014) Genomic imprinting, methylation and parent-of-origin effects in reciprocal hybrid endosperm of castor bean. Nucleic Acids Res 42: 6987-6998.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Genomic imprinting, methylation and parent-of-origin effects in reciprocal hybrid endosperm of castor bean&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Genomic imprinting, methylation and parent-of-origin effects in reciprocal hybrid endosperm of castor bean&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Cell%5BTitle%5D%29 AND 153%5BVolume%5D AND 193%5BPagination%5D AND Zemach A%2C Kim MY%2C Hsieh PH%2C Coleman%2DDerr D%2C Eshed%2DWilliams L%2C Thao K%2C Harmer SL%2C Zilberman D%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zemach A, Kim MY, Hsieh PH, Coleman-Derr D, Eshed-Williams L, Thao K, Harmer SL, Zilberman D (2013) The Arabidopsis Nucleosome Remodeler DDM1 Allows DNA Methyltransferases to Access H1-Containing Heterochromatin. Cell 153: 193-205.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zemach&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The Arabidopsis Nucleosome Remodeler DDM1 Allows DNA Methyltransferases to Access H1-Containing Heterochromatin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The Arabidopsis Nucleosome Remodeler DDM1 Allows DNA Methyltransferases to Access H1-Containing Heterochromatin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zemach&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci U S A%5BTitle%5D%29 AND 107%5BVolume%5D AND 18729%5BPagination%5D AND Zemach A%2C Kim MY%2C Silva P%2C Rodrigues JA%2C Dotson B%2C Brooks MD%2C Zilberman D%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zemach A, Kim MY, Silva P, Rodrigues JA, Dotson B, Brooks MD, Zilberman D (2010b) Local DNA hypomethylation activates genes in rice endosperm. Proc Natl Acad Sci U S A 107: 18729-18734.


http://scholar.google.com/scholar?as_q=Local DNA hypomethylation activates genes in rice endosperm&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Local DNA hypomethylation activates genes in rice endosperm&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zemach&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Science%5BTitle%5D%29 AND 14%5BVolume%5D AND 916%5BPagination%5D AND Zemach A%2C McDaniel IE%2C Silva P%2C Zilberman D%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zemach A, McDaniel IE, Silva P, Zilberman D (2010a) Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 14: 916-919.


http://scholar.google.com/scholar?as_q=Genome-wide evolutionary analysis of eukaryotic DNA methylation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Genome-wide evolutionary analysis of eukaryotic DNA methylation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zemach&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Genome Res%5BTitle%5D%29 AND 224%5BVolume%5D AND 167%5BPagination%5D AND Zhang M%2C Xie S%2C Dong X%2C Zhao X%2C Zeng B%2C Chen J%2C Li H%2C Yang W%2C Zhao H%2C Wang G%2C et al%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci U S A%5BTitle%5D%29 AND 108%5BVolume%5D AND 20042%5BPagination%5D AND Zhang M%2C Zhao H%2C Xie S%2C Chen J%2C Xu Y%2C Wang K%2C Zhao H%2C Guan H%2C Hu X%2C Jiao Y%2C et al%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Extensive, clustered parental imprinting of protein-coding and noncoding RNAs in developing maize endosperm&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Extensive, clustered parental imprinting of protein-coding and noncoding RNAs in developing maize endosperm&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Annu Rev Genet%5BTitle%5D%29 AND 43%5BVolume%5D AND 143%5BPagination%5D AND Zhu JK%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhu JK (2009) Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet 43: 143-166.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Active DNA demethylation mediated by DNA glycosylases&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Active DNA demethylation mediated by DNA glycosylases&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1



running head: novel dna methylation profiles in …...92 c, t or a), which seems to be distinct from...

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