The protein modifications of O-GlcNAcylation and ... · 3 49. Introduction. 50. O-linked...

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Short title: Protein modifications in vernalization. 1

The protein modifications of O-GlcNAcylation and phosphorylation mediate 2

vernalization response for flowering in winter wheat 3

Shujuan Xua,c, Jun Xiaoa,d, Fang Yina,c, Xiaoyu Guoa,c, Lijing Xinga, Yunyuan Xua, Kang Chonga,b,c* 4

aKey Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 5

Beijing 100093, China. 6

bNational Center for Plant Gene Research, Beijing 100093, China. 7

cUniversity of Chinese Academy of Sciences, Beijing 100049, China. 8

dKey Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental 9

Biology, Chinese Academy of Sciences, Beijing 100101, China. 10

*To whom correspondence should be addressed. E-mail: [email protected]; Fax: +86-10-62836517 11

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Author contributions 13

SJ.X and K.C designed the project. SJ.X. and F. Y performed the experiments. J.X., YY. X., XY. G. 14

and LJ.X discussed the data analysis and polished language of the manuscript. SJ.X., J.X., and 15

K.C. analyzed the data. SJ.X., and K.C. wrote the article. 16

Funding 17

This work was supported by the National Key Research and Development Program of China 18

(2016YFD0101004) and the China Postdoctoral Science Foundation. 19

One-sentence summary: The dynamic modifications of O-GlcNAcylation and phosphorylation 20

on the key proteins mediate vernalization for winter wheat flowering. 21

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Plant Physiology Preview. Published on May 6, 2019, as DOI:10.1104/pp.19.00081

Copyright 2019 by the American Society of Plant Biologists

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Abstract 24

O-GlcNAcylation and phosphorylation are two post-translational modifications that 25

antagonistically regulate protein function. However, the regulation of and the crosstalk between 26

these two protein modifications are poorly understood in plants. Here we investigated the role of 27

O-GlcNAcylation during vernalization, a process whereby prolonged cold exposure promotes 28

flowering in winter wheat (Triticum aestivum), and analyzed the dynamic profile of 29

O-GlcNAcylated and phosphorylated proteins in response to vernalization. Altering O-GlcNAc 30

signaling by chemical inhibitors affected the vernalization response, modifying the expression of 31

VRN genes and subsequently affecting flowering transition. Over a vernalization time-course, 32

O-GlcNAcylated and phosphorylated peptides were enriched from winter wheat plumules by 33

Lectin weak affinity chromatography (LWAC) and iTRAQ-TiO2, respectively. Subsequent mass 34

spectrometry and GO term enrichment analysis identified 168 O-GlcNAcylated proteins that are 35

mainly involved in responses to abiotic stimulus and hormones, metabolic processing and gene 36

expression; and 124 differentially expressed phosphorylated proteins that participate in translation, 37

transcription and metabolic processing. Of note, 31 vernalization-associated proteins were 38

identified that carried both phosphorylation and O-GlcNAcylation modifications, of which the 39

majority (97%) exhibited the coexisting module and the remainder exhibited the potential 40

competitive module. Among these, TaGRP2 was decorated with dynamic O-GlcNAcylation (S87) 41

and phosphorylation (S152) modifications, and the mutation of S87 and S152 affected the binding 42

of TaGRP2 to the RIP3 motif of TaVRN1 in vitro. Our data suggest that a dynamic network of 43

O-GlcNAcylation and phosphorylation at key pathway nodes regulate the vernalization response 44

and mediate flowering in wheat. 45

Key Words: Vernalization, O-GlcNAcylation modification, phosphorylation modification, 46

proteomics, winter wheat 47

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Introduction 49

O-linked N-acetylglucosamine (O-GlcNAc) is a simple monosaccharide modification on the 50

side chain of serine and threonine that is involved in the regulation of multiple biological 51

processes. It cycles rapidly during cellular activity (Wells et al., 2001; Wang et al., 2008; Shimoji 52

et al., 2010; Zeidan and Hart, 2010; Hart et al., 2011). Uridine diphosphate-N-acetylglucosamine 53

(UDP-GlcNAc), generated from the hexosamine biosynthesis pathway (HBP) derived from 54

glucose catabolism, is the direct donor of O-GlcNAc (Hanover et al., 2010). Two conserved 55

enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) mediate the reversible addition 56

and removal of O-GlcNAc (Macauley and Vocadlo, 2010; Wang, 2013; Nagel and Ball, 2014). 57

Their activity can be inhibited specifically by chemical inhibitors such as alloxon and PUGNase, 58

respectively (Macauley and Vocadlo, 2010; Trapannone et al., 2016). Many proteins have been 59

identified that carry this modification, including transcription factors, cytoskeletal proteins, and 60

nuclear pore proteins. O-GlcNAc modification can affect protein phosphorylation status, stability, 61

localization and/or interaction with other partners (Wells and Hart, 2003; Slawson et al., 2006; 62

Zachara and Hart, 2006; Ozcan et al., 2010; Liu et al., 2015). O-GlcNAc signaling is implicated in 63

human diseases such as cancer, diabetes and neurodegeneration (Copeland et al., 2008; Singh et 64

al., 2015; Banerjee et al., 2016). In Arabidopsis thaliana, there are two putative OGTs, SECRET 65

AGENT (SEC) and SPINDLY (SPY). O-GlcNAc signaling is reported to function in response to 66

hormones (such as GA and CK), environmental signals, circadian rhythms and developmental 67

stage (Silverstone et al., 2007; Olszewski et al., 2010). A recent study suggested that SEC 68

catalyzes RGA O-GlcNAcylation, regulating its activity and impacting multiple signalling 69

pathways in Arabidopsis (Zentella et al., 2016). O-GlcNAcylation on histone methytransferase 70

ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) by SEC impacted transcription of 71

FLOWERING LOCUS C (FLC) involved in flowering (Xing et al., 2018). However, the big 72

challenge for studying O-GlcNAcylation signaling is the technical difficulty of monitoring its 73

dynamics due to its instability. Recently, some strategies have being developed, such as LWAC 74

(lectin weak affinity chromatography) and chemical derivatization approaches (Xu et al., 2012; 75

Kim, 2015), which provide a possibility for exploring the global O-GlcNAcylation map, 76

particularly in plants. 77



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Protein phosphorylation is a major post-translational modification that regulates diverse 78

cellular processes and functions in various signaling transduction in cells. Protein kinases catalyze 79

the addition of a phosphate group to three amino acids: serine (Ser), threonine (Thr) and tyrosine 80

(Tyr), and the added phosphate group can be subsequently removed by protein phosphatases 81

(Olsen et al., 2006; Thingholm et al., 2009). The dynamic phosphorylation status of proteins plays 82

an important role in endogenous hormone perception and transduction, and environmental stress 83

sensing and response (Osakabe et al., 2013; Yu et al., 2014). There are hundreds of predicted 84

protein kinases and phosphatases in both plants or animals, suggesting complicated 85

phosphorylation networks in signaling transduction. By contrast, only one or two genes encode 86

O-GlcNAc transferases in cells (Singh et al., 2015). O-GlcNAcylation and O-phosphorylation can 87

both modify serine and threonine residues, leading to a “yin-yang” model with antagonistic effects 88

at the global proteome level and on specific amino acids of particular proteins (Butkinaree et al., 89

2010). However, so far, little is known about their collaborative function in cellular processes in 90

response to environmental cues, especially in plants. 91

Winter annual plants from temperate regions are sown in autumn, but flower in spring of the 92

next year only after experiencing prolonged exposure to low temperatures during the winter, a 93

process termed vernalization (Dennis et al., 1996; Wilson and Dean, 1996; Xu and Chong, 2018; 94

Gauley and Boden, 2019; Koppolu and Schnurbusch, 2019). Many genes involved in the 95

vernalization response have been identified in cereal crops, such as wheat (Triticum spp.) and 96

barley (Hordeum vulgare), as well as in Arabidopsis (Dubcovsky et al., 1998; Minorsky, 2002; 97

Shindo and Sasakuma, 2002; Henderson et al., 2003). In Arabidopsis, vernalization promotes 98

flowering through epigenetic silencing of FLC, a key flowering repressor. Long noncoding RNA 99

and PHD-PRC2 (Polycomb repressive complex) mediate the silencing of FLC through the 100

switching of chromatin states and accumulation of H3K27me3 at the nucleation region during 101

cold exposure (Bastow et al., 2004; Questa et al., 2016; Yuan et al., 2016; Zhou et al., 2018). 102

However, in temperate crops such as wheat, a central regulator is TaVRN1, which promotes 103

flowering and is activated during vernalization through a complex transcriptional regulation 104

network (Yan et al., 2003; Trevaskis et al., 2006; Distelfeld et al., 2009; Trevaskis, 2010; Kippes 105

et al., 2015). Several putative vernalization memory-related genes were identified at the 106



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transcriptional level through transcriptome analysis in Brachypodium (Huan et al., 2013). Our 107

previous studies have reported that diverse metabolic changes occur sequentially during the 108

different stages of vernalization, and sucrose addition at an early stage of cold exposure can 109

promote flowering, substituting the requirement, to some extent, of vernalization treatment (Zhao 110

et al., 1998; Yong et al., 1999). This may be linked to the accumulation of metabolic intermediates 111

from sucrose, such as UDP-GlcNAc (Hanover et al., 2010). 112

We previously cloned the vernalization-induced gene VER2, which encodes a Jacalin-like 113

lectin in winter wheat (Zhao et al., 1998; Yong et al., 1999; Xu et al., 2004). Knockdown of VER2 114

caused delayed flowering whereas its overexpression partly replaced the necessity of vernalization 115

for winter wheat to flower (Zhong et al., 1995; Chong et al., 1998; Yong et al., 2003). VER2 can 116

specifically bind to GlcNAc, and vernalization induces an increase in O-GlcNAcylated proteins at 117

the global level (Xing et al., 2009). VER2 interacts with a glycine-rich RNA-binding protein 118

TaGRP2, which directly binds to TaVRN1 pre-mRNA to repress its expression. During 119

vernalization, gradually increased O-GlcNAc modification was detected for TaGRP2, thus 120

allowing phosphorylated VER2 to recognize O-GlcNAcylated TaGRP2 and repress its 121

accumulation in the nucleus and attenuate its binding to TaVRN1, thereby releasing the repression 122

of TaVRN1, ultimately promoting flowering (Xiao et al., 2014). These results illustrate the 123

involvement of O-GlcNAc signaling in vernalization-promoted flowering in winter wheat. 124

In this study, we investigated dynamic O-GlcNAcylation during the process of vernalization 125

and further explored the importance of O-GlcNAcylation signaling in mediating the vernalization 126

response by using a chemical inhibitor that modifies the enzymatic activity of OGA. In addition, 127

we identified hundreds of proteins with dynamic O-GlcNAcylation or phosphorylation during 128

vernalization. These proteins are mainly involved in metabolic processing, cellular processing and 129

response to stimulus. We also identified 31 proteins with both O-GlcNAc and phosphorylation, of 130

which some may indeed play an important role in mediating the vernalization response. Our data 131

suggests that O-GlcNAcylation and phosphorylation protein modifications may act in the 132

vernalization response and regulate the transcriptional network of VRNs for flowering in wheat. 133

Results 134

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O-GlcNAc signaling accelerates vernalization-promoted flowering by regulating VRNs 136

To address the physiological function of O-GlcNAc signaling, PUGNAc 137

(O-(2-Acetamido-2-deoxy-D-glucopyranosy lidenamino) N-phenylcarbamate), the inhibitor of 138

O-GlcNAcase (OGA), was used to treat V0 (non-vernalization), V14 (vernalization for 14 days) 139

and V21 (vernalization for 21 days) winter wheat cultivar Jingdong1 (JD1). As expected, the 140

global O-GlcNAcylated proteins increased as detected by anti-O-GlcNAc antibody in the different 141

vernalized wheat following PUGNAc treatment (Fig. S1). Morphologically, the appearance of a 142

double ridge at the shoot apex is a clear marker for initiation of flowering (Fig.1A). 143

PUGNAc-treated winter wheat flowers earlier than control plants under V14 and V21 conditions, 144

but such effect was not observed in wheat without vernalization (V0) (Fig.1A, B, C and Fig. S2). 145

Winter wheat treated with PUGNAc under V14 flowers at a similar time as the control plant under 146

V21 (Fig. 1C), with an even higher heading rate (Fig. 1B), suggesting that PUGNAc treatment 147

could partly substitute cold exposure. In addition, the transcription level of the key flowering 148

genes TaVRN1, TaVRN2 and TaFT1 were monitored at different cold exposure durations with or 149

without PUGNAc treatment. The expression of two flowering promoting genes TaVRN1 and TaFT 150

was increased when treated with PUGNAc at V7, V14 and V21 as compared to that in non-treated 151

wheat, but no difference was seen at V0 (Fig. 1D). The expression of TaVRN2, a repressor of 152

flowering, was decreased in wheat treated with PUGNAc at V7, V14 and V21 but no change was 153

observed at V0 (Fig. 1D). Therefore, O-GlcNAc signaling possibly modulates wheat vernalization 154

to impact flowering through regulating vernalization response genes such as TaVRN1, TaVRN2 155

and TaFT. Of note, the effects of O-GlcNAc signaling only apply at specific periods of the 156

vernalization process, which fits the previous report that glucose addition accelerates flowering 157

but only at certain time windows during vernalization (Yong et al., 2003). 158

A global map of proteins with O-GlcNAcylation and phosphorylation during vernalization 159

Our previous study shows that the global levels of O-GlcNAcylated proteins are significantly 160

different prior to and after vernalization treatment (Xing et al., 2009). Here, phosphorylated 161

proteins at different stages of the vernalization process were monitored by immunoblotting using 162

an antibody recognizing Phos-tag-Biotin. As expected, a dynamic phosphoprotein pattern was 163

detected during vernalization (Fig.S3A). In order to understand the global profile of 164

O-GlcNAcylated proteins and phosphorylated proteins participating in vernalization, a proteomic 165



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approach was used to enrich and identify such proteins. Wheat plumules at non-vernalized (V0), 166

vernalized for 2 days (V2), vernalized for 21 days (V21), and de-vernalized (V21+5, abbreviated 167

as V21*), of which the seedlings vernalized for 21 days were subsequently exposed at 35℃ for 5 168

days leading to elimination of vernalization, were used for comparison (Fig. 2A). An approach 169

with LWAC (Lectin Weak Affinity Chromatography) and LC-MS/MS was used to enrich and 170

identify O-GlcNAcylated peptides (Fig. 2B). As a result, a total of 201 O-GlcNAcylated peptides 171

representing 111 O-GlcNAcylated proteins were identified in the non-vernalized wheat sample, 172

and 143 O-GlcNAcylated peptides from 91 proteins were found in vernalized samples (Table S1 173

and S2). Among these O-GlcNAcylated proteins, only 34 proteins were present in both 174

non-vernalized and vernalized wheat plumules (Fig. 2D). As for phosphorylated proteins, after 175

protein extraction and digestion with Trypsin, iTRAQ labeling was used for quantification of the 176

different samples, and then TiO2 column was used to enrich phosphorylated peptides which was 177

followed by LC-MS/MS analysis (Fig. 2C). 332 proteins that were significantly changed in 178

phosphorylation level (SCPL) between V21 and V0 were identified. There were still 263 SCPL 179

proteins after deducting the SCPL proteins that arose in response to cold stress (V2/V0). A total of 180

205 unique phosphopeptides, representing 124 SCPL proteins (44 up-regulated proteins and 80 181

down-regulated proteins respectively), were found to arise in response to vernalization after 182

subtracting that of V2/V0 (SCPL in response to cold stress) and V21+5/V0 (SCPL in response to 183

devernalization) (Fig. 2E, Fig. S3B and table S3). This suggests that O-GlcNAcylation and 184

phosphorylation dynamically modify numerous proteins during the process of vernalization. 185

186

Functional categorization of O-GlcNAcylated proteins and SCPL proteins 187

In order to explore the functions of the identified O-GlcNAcylated and SCPL proteins, gene 188

ontology (GO) analysis was performed using agriGO (http://bioinfo.cau.edu.cn/agriGO/). The 189

identified O-GlcNAcylated proteins in either non-vernalized or vernalized wheat plumules are 190

involved in a series of biological processes, such as response to abiotic stimulus, response to 191

hormone, gene expression, nucleoside metabolic process, and developmental process (Fig. 3A). 192

The O-GlcNAcylated proteins identified in non-vernalized (V0) wheat were enriched in GO terms 193

such as shoot system development, plant organ development and response to abscisic acid (Fig. 194

3B), whereas the vernalization-specific (V21) O-GlcNAcylated proteins were enriched in response 195


http://bioinfo.cau.edu.cn/agriGO/


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to cytokinin, signal transduction, reproductive process (Fig. 3C). Of note, the enrichment of GO 196

terms is different in V0- and V21-specific O-GlcNAcylated proteins. This suggests that 197

O-GlcNAcylated proteins may mediate vernalization through integrative processes such as 198

hormone signaling, shoot and reproductive development and gene expression. 199

Further analysis showed that these O-GlcNAcylated or phosphorylated proteins in response 200

to vernalization belong to four clusters as follow (Fig. 3D): The first cluster is hormone response, 201

including several hormones response factors such as ARF3 (AUXIN RESPONSE 202

TRANSCRIPTION FACTOR 3), ARR10 (ARABIDOPSIS RESPONSE REGULATOR 10), PIN5 203

(PIN-FORMED 5), ETR2 (ETHYLENE RESPONSE 2) and ABCG37/40 (ATP-BINDING 204

CASSETTE G 37/40), which are O-GlcNAcylated after vernalization. The second cluster is stress 205

response, comprising PTR3 (PEPTIDE TRANSPORTER 3), RD22 (RESPONSIVE TO 206

DESICCATION 22), UPL3 (UBIQUITIN-PROTEIN LIGASE 3), PER64 (PEROXIDASE 64), 207

APG9 (AUTOPHAGY 9) and RZFP (RING/FYVE/PHD zinc finger superfamily protein) with 208

changeable O-GlcNAcylation or phosphorylation status during vernalization. The third cluster is 209

involved in energy and carbohydrate metabolism such as FBA5/6 (FRUCTOSE-BISPHOSPHATE 210

ALDOLASE 5/6), GAPCP2 (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE OF 211

PLASTID 2), BGLU13 (BETA GLUCOSIDASE 13), UXS (UDP-XYLOSE SYNTHASE) and 212

AAC2 (ADP/ATP CARRIER 2). This may explain how prolonged low temperature changes the 213

cellular nutrient status by shaping the metabolic patterns of energy and carbohydrate metabolites 214

through dynamic Yin-Yang modification of the related enzymes. The fourth cluster is enriched in 215

genetic information processing factors, such as proteins related to RNA splicing, epigenetic 216

modification, translation and transcription, with O-GlcNAcylation and/or phosphorylation 217

modification during vernalization. Such proteins are GRP2 (GLYCINE-RICH RNA-BINDING 218

PROTEIN 2), PABP8 (POLY(A) BINDING PROTEIN 8), GBF4 (G-BOX BINDING FACTOR 219

4), CPN60B (CHAPERONIN 60 BETA) and H1.2 (HISTONE 1.). Most of the dynamic 220

phosphoproteins identified during different vernalization treatment were involved in several 221

processes such as protein folding, nucleosome and chromatin assembly, translation, protein 222

metabolic process, regulation of RNA biosynthetic process, transcription and primary metabolic 223

process (Fig. S4). In the modification profiling, there were 31 proteins carrying both 224



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O-GlcNAcylation and phosphorylation modifications (Table 1). Most of these 31 proteins are 225

involved in metabolic processing, and some are involved in response to stress (Table 1). These 226

proteins may play an important role in mediating flowering during vernalization through multiple 227

processes. 228

The possible correlation between phosphorylation and O-GlcNAcylation modification 229

Considering the crosstalk modification module proposed in animal studies (Gupta and Brunak, 230

2002; Leney et al., 2017), we further explored the possible correlation between the modified sites 231

and the computationally defined structures of the 31 proteins with both O-GlcNAcylation and 232

phosphorylation. The results suggested that there were two major patterns between the 233

O-GlcNAcylation and phosphorylation modification: coexisting or competitive (Fig. 4A). For 234

example, the phosphorylation modified site (S205) and the O-GlcNAc modified site (T213) of 235

glyceraldehydes-3-phosphate dehydrogenase (GAPD) were coexisting in non-vernalization 236

samples and erased (O-GlcNAcylation on T213) or reduced (Phosphorylation on S205) during 237

vernalization. This suggests that both modifications are coexisting and coordinately regulated. 238

Although the linear distance between S205 and T213 is very close, they are spatially far away 239

from each other (located at the two terminals of β-sheet), representing 39% of the identified 240

proteins with O-GlcNAcylation and phosphorylation (Fig. 4A). Meanwhile, the coexisting relation 241

was also found between the long linear distant sites on Enolase protein, which are physically close 242

to each other in the three-dimensional structure. This represents more than 58% of such bivalent 243

modified proteins (Fig. 4A). Interestingly, we found that the fructose-bisphosphatealdolase (FBA) 244

with the O-GlcNAcylation and phosphorylation at the same site S350, which existed in only 3% of 245

the proteins with both modifications identified here (Fig. 4A). The level of phosphorylation at 246

S350 was decreased during vernalization, whereas the O-GlcNAcylation at S350 was present after 247

vernalization. This illustrates the potential competitive relation (Yin-Yang model) of 248

O-GlcNAcylation and phosphorylation during vernalization. These predicted results will help 249

guide further studies in the crosstalk of O-GlcNAcylation and phosphorylation modifications on 250

key proteins that regulate vernalization response in the future. 251

To validate the identified O-GlcNAcylation on these proteins by MS, an in vitro 252

O-GlcNAcylation assay was used. His-tagged GAPD, Enolase, FBA and FBA-m (mutation of the 253



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three identified O-GlcNAcylated sites T35, T320 and S350) were expressed in Escherichia coli 254

and affinity purified, as well as the truncated version of SEC△N with proofed OGT activity in 255

vitro (Xing et al., 2018). Incubation with SEC△N GAPD, Enolase and FBA could be recognized 256

by the O-GlcNAcylation-specific antibody CTD110.6, whereas FBA-m was not recognized (Fig. 257

4B and 4C). This further verifies our observation by MS. 258

Mutations of the O-GlcNAcylation and phosphorylation modified sites on TaGRP2 may 259

impact its binding to TaVRN1-RIP3. 260

Among the 31 proteins with both O-GlcNAcylation and phosphorylation, TaGRP2, a RNA 261

binding protein, was chosen for further study to test that the identified proteins with 262

post-translational modifications may play vital roles in vernalization (Table 1). From the MS data, 263

Ser87 of TaGRP2 was identified as an O-GlcNAcylation site whereas Ser152 was a 264

phosphorylation site (Fig. 5A-C). The linear distance between Ser87 and Ser152 was far, but they 265

are close to each other in the higher structure (Fig. 5A, D, E). This suggests the possibility of 266

crosstalk between O-GlcNAcylation and phosphorylation. Sequence alignment showed that the 267

S87 of TaGRP2 is relatively conserved, and is either serine (50%) or glycine (50%) in different 268

species (Figure S5 and S6). However, the residues corresponding to S152 of TaGRP2 are variable 269

(e.g., S, G, R and I) in different species (Figure S5). This pattern indicates that the conserved S87 270

might be important in regulating TaGRP2 function. To test the effect of O-GlcNAcylated and 271

phosphorylated sites on the function of TaGRP2, RNA-electrophoretic mobility shift assay 272

(RNA-EMSA) was used to analyze the binding of TaGRP2 and TaGRP2 mutants to the 273

TaVRN1-RIP3. TaVRN1-RIP3 was the target binding-motif of TaGRP2 (Xiao et al., 2014). 274

GST-tagged TaGRP2, GRP2-T17m (T17 of TaGRP2 is detected to be modified by 275

O-GlcNAcylation in the previous study (Xiao et al., 2014)), TaGRP2-S87m, TaGRP2-S152m, 276

TaGRP2-SS2m (mutation of the identified O-GlcNAcylated sites S87 and the phosphorylated site 277

S152), TaGRP2-TSS3m (mutation of the two identified O-GlcNAcylated sites T17 and S87, the 278

identified phosphorylated site S152) were expressed in Escherichia coli and affinity purified. The 279

RNA-EMSA results showed that mutation of the O-GlcNAcylated sites (T17 and S87) or the 280

phosphorylated site (S152) of TaGRP2 changed the signal density of the TaGRP2-TaVRN1-RIP3 281

complex bands (Fig. 6 and Fig. S7). Changing S87 to A87 enhanced the signal, whereas mutant 282



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S152 weakened it. Mutating both S87 and S152 reduced the signal slightly, but mutating the three 283

sites T17, S87 and S152 clearly abated the signal. These results suggest that the three modified 284

sites may be important for the function of TaGRP2 to bind RNA. The TaGRP2-OE transgenic 285

wheat lines and wild type were vernalized for 0, 21 and 28 days to test the function of TaGRP2 286

during vernalization. The results showed that the shift from the single ridge to double ridge stage 287

of apex development in TaGRP2-OE transgenic wheat lines was slower than that in wild type in 288

either V21 or V28 treatment (Fig. S8), which is consistent with the previous report that TaGRP2 289

represses flowering tranistion (Xiao et al., 2014). The O-GlcNAcylated and phosphorylated sites 290

of TaGRP2 may be involved in vernalization regulation in wheat. 291

Discussion 292

O-GlcNAc is a protein modification that regulates vernalization 293

294

As sessile organisms, plants are constantly exposed to various environmental stresses (Qi et 295

al., 2018; Wang et al., 2018). Low temperature constitutes a key factor influencing plant growth, 296

development, crop productivity and geographic distribution (Guo et al., 2018; Liu et al., 2018). In 297

responding to cold, plants could rapidly change the metabolism in existing tissue, and metabolome 298

analyses revealed that the levels of mono-saccharides such as glucose and fructose from the starch 299

degradation and sucrose metabolism were significantly higher in cold-treated plants (Maruyama et 300

al., 2014; Zhang et al., 2016), and the glucose addition can reduce a requirement of winter wheat 301

for vernalization (Yong et al., 2003). However, little is known about which metabolite of glucose 302

participates in the transduction of signaling during vernalization. 303

O-GlcNAcylation is an abundant nutrient-driven modification linked to cellular signaling and 304

regulation of gene expression (Zachara and Hart, 2004; Butkinaree et al., 2010; Lewis and 305

Hanover, 2014). In Drosophila, O-GlcNAc signaling is extremely important in developmental 306

regulation, stem cell maintenance, circadian regulation and responses to ambient temperature 307

(Gambetta et al., 2009; Sinclair et al., 2009; Kaasik et al., 2013; Radermacher et al., 2014). Our 308

results showed that the addition of an inhibitor of OGA can reduce the requirement of 309

vernalization in winter wheat, which suggests that elevation of O-GlcNAcylation level can partly 310

mimic vernalization treatment to promote wheat flowering and regulate the expression of VRNs 311



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(Fig.1 and Fig. S2), thus indicating that O-GlcNAc signaling plays an important role in regulating 312

vernalization response. 313

There were 168 O-GlcNAc modified proteins identified in our data (Fig. 2 and Table S1 and 314

S2). Many of them shared the expression patterns observed in animal cells, such as H4, H2B, PFK 315

and HSP70 (Guinez et al., 2006; Singh et al., 2015). Histones are subject to post-translational 316

modification, and these modifications are important parts of regulatory circuits that control 317

chromatin dynamics and the activities of DNA. Numerous reports have shown that histones 318

possess lots of post-translational modifications such as methylation, phosphorylation, 319

ubiquitination and acetylation (Yun et al., 2011). Recent research also reported that histone H3 320

lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) at VRN3 321

regulated the epigenetic memory of vernalization in Brachypodium distachyon (Huan et al., 2018). 322

However, there is a poor understanding of the O-GlcNAcylation on histones that are involved in 323

regulating vernalization; the O-GlcNAcylation modification of histones will be an attractive 324

research direction in the future. About 15% of the identified O-GlcNAcylated proteins in wheat 325

plumules have close homologues as SEC interactors in Arabidopsis (Table S5). Although SEC is 326

highly conserved between monocotyledons and dicotyledons, the O-GlcNAcylated target proteins 327

were very diverse. Recently, a report has showed the profile of the O-GlcNAcylated proteins in 328

Arabidopsis (Xu et al., 2017); however, only a few proteins were identified as O-GlcNAcylated 329

proteins in our data, such as TCP, ARF, TIC, 60S and PAB8. A possible explanation for this is that 330

the samples and treatments were very different between the Arabidopsis inflorescence tissues used 331

in the previous study and the wheat plumules used here. However, the cellular processes related to 332

O-GlcNAc-modified proteins were similar among wheat, Arabidopsis and animals, and mainly 333

involved in signal transduction, translation, transcription, and metabolic process (Liu et al., 2015; 334

Xu et al., 2017). In our study, the O-GlcNAc-modified proteins from vernalized samples were 335

associated with amino acid and nucleotide metabolism and translation (Fig. S9). O-GlcNAc may 336

be a protein modification that regulates vernalization in winter wheat. 337

The possible correlation between O-GlcNAcylation and phosphorylation 338

modifications during vernalization 339



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340

O-GlcNAcylation and O-phosphorylation both modify serine and threonine residues, which 341

leads to the “yin-yang” regulatory theory (Hu et al., 2010). Based on our data, 31 proteins were 342

detected to have both O-GlcNAc modification and phosphorylation modification at the same time 343

(Table 1), but co-occurrence of the two modifications in the same peptide was rare. The FBA was 344

identified to have O-GlcNAcylation and phosphorylation modification at the same site S350. In 345

addition, the two modifications existed competitively during vernalization (Fig. 4A). The 346

Yin-Yang relationship may regulate flowering through mediating vernalization response. TaGRP2 347

was gradually O-GlcNAcylated during vernalization (Xiao et al., 2014). Meanwhile, TaGRP2 can 348

be phosphorylated before vernalization, and the spatial distance of the two modifications on 349

TaGRP2 protein was very close (Fig. 5). The O-GlcNAc and phosphorylation modification of 350

TaGRP2 may antagonistically mediate the function of TaGRP2 to bind RNA (such as 351

TaVRN1-RIP3) through changing GRP2’s structure (Fig. 6). According to a previous study (Leney 352

et al., 2017; van der Laarse et al., 2018), the correlation of the two modifications also existed 353

between the function of phosphorylation and O-GlcNAcylation on different proteins during 354

vernalization. Vernalization increases the O-GlcNAc modification of TaGRP2 (a repressor in 355

vernalization) in the nucleus and the phosphorylation of VER2 (an activator in vernalization) in 356

cytoplasm, which antagonistically regulated the expression of TaVRN1 to mediate flowering in 357

winter wheat (Xiao et al., 2014). The study of vernalization has mainly been focused on the 358

regulation and function of VRNs so far. But it is unclear how wheat transduces the vernalization 359

signaling, which is of vital importance for vernalization. Our data here suggest that the O-GlcNAc 360

signaling play a role in transducing vernalization signal and the possible correlation between 361

O-GlcNAcylation and phosphorylation modifications may participate in regulation of wheat 362

flowering through affecting vernalization, which will attract us to continue the follow-up research 363

in future. In summary, the approaches of LWAC and iTRAQ-TiO2 were used to detect and 364

identify a series of O-GlcNAcylated and alternatively changed phosphorylated proteins in 365

different vernalized wheat. Functional analysis showed that the O-GlcNAcylated proteins 366

identified in vernalized wheat mediated the vernalization response through the four main 367

processes, such as hormone response (such as ARF3, ARR10, PIN5, and ETR2), response to stress 368



14

(such as PTR3, RD22, UPL3, and PER64), energy and carbohydrate metabolism (such as AAC2, 369

FBA5, UXS, and BGLU13) and genetic information processing (such as GRP2, PABP8, GBF4, 370

and CPN60B), and some of the O-GlcNAcylated proteins also were modified by phosphorylation, 371

which indicated that the crosstalk of the two modifications may involve in vernalization regulation 372

(Fig. 3D). The results of OGA inhibitor treatment showed that O-GlcNAc signaling during 373

vernalization accelerated flowering transition in winter wheat. O-GlcNAc as an abundant 374

nutrient-driven modification may measure the time horizon to initiate vernalization in winter 375

wheat. Taken together, O-GlcNAcylation and phosphorylation modification may act as signals to 376

mediate vernalization response and regulate the network of VRNs for flowering in wheat. 377

Methods and materials 378

Plant materials and growth conditions 379

JD1 and JH9 were Chinese winter wheat (Triticum aestivum) cultivars. TaGRP2 380

overexpression (TaGRP2-OE) and RNA interference (TaGRP2- RNAi) transgenic wheat were 381

generated in JH9 accession by microprojectile bombardment-mediated transformation. Seeds of 382

winter wheat (JD1, JH9 and TaGRP2 transgenic lines) were surface sterilized in 2% v/v NaClO 383

for 20 min, then rinsed overnight with flowing water. After that, the seeds were germinated on 384

moist filter paper under gradient time (14, 21 and 28 days, as V14, V21, V28) of 4℃ treatment in 385

the dark (V), or grown at 25℃ for 3 days (V0), and 20 μM PUGNAc (the inhibitor of OGA) was 386

used to treat JD1 during the vernalization. Then, transferred to soil and grown in greenhouse 387

(20–22℃, 16 h light/8 h dark) for 70 days. Finally, we used a dissecting mirror to dissect the 388

wheat to observe the flowering phenotype. 389

The methods of inhibitor PUGNAc of OGA treated Plant materials 390

The seeds were germinated on moist filter paper under gradient time 14 and 21 days (as V14, 391

V21) of 4℃ treatment in the dark, or grown at 25℃ for 3 days as non-vernalization (V0). Then, 392

transferred to soil and grown in greenhouse (20–22℃, 16 h light/8 h dark) for 70 days. Then the 393

dissecting mirror was used to dissect the wheat to observe the phenotype of apex development, 14 394

to 16 seedlings of each treatment were dissected, the showed one was the representative image in 395

each treatment 396



15

Protein extracted, trypsin digestion and labeling the peptides with the iTRAQ reagents 397

Total proteins from the wheat plumules (V0, V2, V21 and V21+5) were extracted in 398

homogenization buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA, 10% v/v glycerol, 399

0.2% v/v Triton X-100, 1 mM PMSF, Protease inhibitor cocktail, Phosphatase Inhibitor Cocktail). 400

The mixture was thoroughly vortexed for 1 min and centrifugated at 16,000 g and 4°C for 30 min. 401

The supernatant was pipetted into fresh 10-mL tubes and three fold volumes of cold TCA-acetone 402

were added, -20℃ to precipitate 2 h. And then centrifugated at 16,000 g and 4°C for 30 min, the 403

supernatant was carefully discarded and the precipitated proteins were washed twice with cold 404

acetone. Finally, the precipitated proteins were dissolved in lysis buffer (8 M urea，30 mM 405

HEPES，2 mM Na3VO4, 2 mM NaF and 2 mM ß- sodium glycerophosphate), then centrifugated 406

at 16,000 g and 4°C for 30 min, the supernatant was pipetted into fresh 1.5-mL tubes, then 407

quantified of protein by Bradford method, bovine serumalbumin (BSA) (1 mg/mL) as the standard. 408

200 µg of protein from each sample were digested by 6.6 µg trypsin (m/m 1:30), incubated at 409

37℃, 16 h. Allow each vial of iTRAQ reagent required to reach room temperature, and spin to 410

bring the solution to the bottom of the tube. Add 70 µL of ethanol to each room-temperature 411

iTRAQ reagent vial. Vortex each vial to mix, then spin. Transfer the contents of one iTRAQ 412

reagent vial to one sample tube. iTRAQ reagent 113 vial to the sample V0-1 protein digest tube, 413

114 to V0-2, 115 toV2-1, 116 to V2-2, 117 to V21-1, 118 to V21-2, 119 to V21+5-1 and 121 to 414

V21+5-2 (followed the protocol of Applied Biosystems iTRAQ Reagents). 415

Phosphopeptide enrichment using TiO2 microcolumns and identification using Q-Exactive 416

The peptides labeling with the iTRAQ reagents were merged used 1 ml loading buffer (60% 417

v/v Acetonitrile (ACN), 2% v/v trifluoroacetic acid (TFA) pH 2.0), saturated with glutamic acid, 418

and then incubated with 3.2 mg TiO2 beads (GL Sciences, Tokyo, Japan) which were incubated in 419

500 μL loading buffer containing 60% v/v ACN, 2% v/v TFA pH 2.0, saturated with glutamic acid, 420

30 min at room temperature. After washing twice with 500 μL wash buffer I (60% v/v ACN, 0.5% 421

v/v TFA, pH 2.5) and 500 μL wash buffer II (60% v/v ACN, 0.1% v/v TFA, pH 3), the 422

phosphopeptides were eluted twice with 500 μL elution buffer I (50% v/v CAN, 300 mM NH4OH, 423

pH11) and 500 μL elution buffer II (50% v/v ACN, 500 mM NH4OH, pH11). The eluates were 424

dried and reconstituted in 30 μL 50% v/v TEAB for MS analysis. The enriched phosphopeptides 425



16

were identified using Q-Exactive, separated on a C18 chromatographic column (5 μm I.D., 100 426

mm length). Pump flow was split to obtain a flow rate of 1 mL/min for sample loading and 400 427

nL/min for the MS analysis. The mobile phases consisted of 0.1% v/v FA (Formic acid) (A), and 428

0.1% v/v FA and 80% v/v ACN (B). A five-step linear gradient of 3% to 30% B in 70 min, 30% to 429

80% B in 8 min, 80% B in 7 min, 80% to 5% B for 3 min, and 5% B for 7 min was employed. The 430

spray voltage was set to 1.8 kV, and the temperature of the heated capillary was set to 320°C. For 431

data acquisition, each MS scan was acquired at a resolution of 17,500, with the lock mass option 432

being enabled, and was followed by data-dependent top 10 MS/MS scans using higher energy 433

collisional dissociation (HCD). The threshold for precursor ion selection was 500, and the mass 434

window for precursor ion selection was set to 350–2000 Da. The raw files were processed using 435

Mascot (version 2.4.1), and were then searched against the uniprot_triticeae database. The fixed 436

modification is carbamidomethyl (C), and the variable modifications are oxidation (M), Gln 437

(N-termQ), phospho (ST), phospho (Y), iTRAQ8plex (K), iTRAQ 8 plex(Y) and iTRAQ 8 plex 438

(N-term). One missing cleavage point was allowed. Proteome Discoverer 1.3 (Thermo) was used 439

to extract the peak intensity within 15 ppm of each expected iTRAQ reporter ion from each 440

fragmentation spectrum. Only spectra in which all the expected iTRAQ reporter ions were 441

detected were used for quantification. The phosphopeptide ratios were normalized by dividing the 442

average value of all peptides identified. The false discovery rate (FDR) was set to < 1.0% for the 443

identification of both peptides and proteins and with PhosphoRS probability ≥0.75. Significant 444

changes in a phosphopeptide’s abundance were inferred where its abundance ratio was >1.2 or 445

<0.83, and p value < 0.05 which was derived from the Student’s t-test. The mass spectrometry 446

proteomics data obtained in this study have been deposited to the ProteomeXchange Consortium 447

(http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset 448

identifier PXD008298. 449

O-GlcNAcylated peptides enrichment and identification 450

Total proteins from the wheat plumules (V0 and V21) were extracted with NitroExtraTM 451

(Cat. PEX-001-250ML, N-Cell Technology) added 20 μM the inhibitor PUGNAc of OGA. The 452

mixture was thoroughly vortexed for 1 min and centrifugated at 16,000 g and 4°C for 1 h. After 453

centrifugation, protein was precipitated with 1:3 (sample to acetone) cold acetone at -20℃ 454



17

overnight. Precipitated proteins were washed twice with cold acetone and finally re-suspend in 8M 455

urea after protein precipitate has been air dried, and quantified of protein by Bradford method, 456

bovine serumalbumin (BSA) (1 mg/mL) as the standard. Appropriate amount of trypsin is then 457

added to the sample in an enzyme-to-substrate ratio of 1:30, and incubated at 37℃, 16 h. Digested 458

proteins were desalted by C18 column and dried in spin vacuum. N-glycopepetides were 459

de-glycosylated with 20 μM PNGase F (P7367-50UN, Sigma) and 10 μM PNGase A 460

(G0535-.005UN, Sigma) in 50 mM ammonium bicarbonate (pH 8.0) and 50 mM citrate-phosphate 461

buffer (pH 5.0) respectively for 24 h. The GlcNAcylated peptides were enriched from the sample 462

with a Wheat Germ Agglutinin Column. Enriched glycopeptides were dried in spin vacuum. Each 463

dried peptide sample is dissolved in 25 μL of 0.1% v/v FA. The sample was analyzed by 464

nanoLC-MS/MS using an UltiMate 3000 RSLCnano System (Thermo Scientific/ Dionex) coupled 465

to LTQ Velos Dual-Pressure Ion Trap (Thermo Scientific, Bremen, Germany). After sample was 466

loaded onto a reversed-phase 25 cm C18 PicoFrit column (New Objective, Woburn, MA) a linear 467

gradient of acetonitrile (3–36% v/v) in 0.1% v/v formic acid was used. The elution duration was 468

120 min at a flow rate of 0.3 μL/min. Eluted peptides from the PicoFrit column were ionized and 469

sprayed into the mass spectrometer, using a Nanospray Flex Ion Source ES071 (Thermo) under 470

the following settings: spray voltage, 1.6 kV, Capillary temperature 250℃. The LTQ instrument 471

was operated in the data dependent mode to automatically switch between full scan MS and 472

MS/MS acquisition. The 12 most intense multiply charged ions (z ≥2) were sequentially isolated 473

and fragmented with collisional induced dissociation (CID. The presence of HexNAc oxonium 474

ions (m/z 203, 101.5 and 67.67) will trigger the acquisition of an ETD fragmentation spectrum 475

(MS3) of the precursor ions. Raw data files were converted to MGF (Mascot Generic Format). 476

The MGF files were searched against the UniProt, NCBI and common MS contaminant database 477

using Mascot Software (version 2.4.1). The tolerance for MS1 and MS2 error is 1 Da and 0.5 Da 478

respectively. Caramidomethylation (+57 Da) was added as fixed modification while Oxidation (M) 479

and O-GlcNAc (S/T) were added as variable modification. A maximum of 2 trypsin miss 480

cleavages was allowed. And the mass spectrometry proteomics data obtained in this study have 481

been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) 482

via the PRIDE partner repository with the dataset identifier PXD008285. 483



18

Determination of UDP-GlcNAc by UHPLC-MS 484

Seedlings (500 mg) of winter wheat (JD1) vernalized for 0, 3, 7, 10, 14, 21, 28 and 35 days 485

respectively were frozen in liquid nitrogen and stored at -70°C. Frozen samples were ground into 486

power, and the resultant powder was transferred to 0.5 ml of 75% v/v ice-cold ethanol. The extract 487

was vortexed and centrifuged at 16,000 × g for 10 min at 4°C to remove large chunks of debris. 488

The supernatant collected was filtered through a 0.44 μm filter (Millipore). Extracts (20 μl 489

injections) were separated using an Agilent 1290 Infinity UHPLC system consisting of a binary 490

pump, an autosampler, a thermostatted column compartment. The chromatography was performed 491

using an X BridgeTM HILIC column from Agilent Technologies (2.1 × 150 mm, 5 μm). The 492

mobile phases consisted of (A) H2O and (B) acetonitrile. The UHPLC eluting conditions were 493

optimized as follows: 95% B (0–5 min), 75% B (5–10 min), 55% B (10–15 min) and 95% B 494

(15–20 min). The flow rate was 0.4 mL/min. The column was maintained at 30°C. The 495

UDP-GlcNAc standard (Sigma) was used to determine UDP-GlcNAc concentration and 496

composition in seedlings extracts. Mass spectrometry was performed using an Agilent 6540 497

Q-TOF equipped with electrospray ionization (Rotini et al.) source operating in negative ion mode. 498

The nebulization gas was set to 35 psi. The drying gas was set to 10 L/min at temperature of 499

350°C; the sheath gas was set to 11 L/min at temperature of 350°C. The capillary voltage was set 500

to 3500 V. The Q-TOF acquisition rate was set to 0.5 s. 501

Western blot analysis 502

Total proteins were extracted from wheat plumules in homogenization buffer (20 mM 503

Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA, 10% v/v glycerol, 0.2% v/v Triton X-100, 1 mM 504

PMSF, Protease inhibitor cocktail) and quantified by Bradford assay, then separated by denaturing 505

polyacrylamide electrophoresis on 4–12% SDS-PAGE gels and electro-blotted onto PVDF 506

membranes. Phosphorylated proteins were detected using antibody Phos-tag-Biotin (BTL-111S1, 507

wako) in Tris-buffered saline with Tween 20 buffer with 5% v/v BSA at 1/1,000 dilution, and 508

O-GlcNAcylated proteins were detected using antibody CTD110.6 (9875S, CST) in Tris-buffered 509

saline with Tween 20 buffer with 5% v/v BSA at 1/2,000 dilution. Stabilized streptandin-HRP 510

Conjugate (Thermo Scientific) and HRP-Goat anti Mouse IgM (Proteintech) were used for 511

secondary detection at 1/10,000 and 1/1,000 dilution respectively and Supersignal West Dura 512



19

Substrate was used for signal detection. 513

Total RNA extraction and RT-qPCR 514

Total RNA of V0, V7, V14, V21 and V28 wheat plumules was extracted using a TRIzol RNA 515

extraction kit according to the user manual (Invitrogen). Total RNA was treated with DNase I 516

(Fermentas) and then 2 μg RNA was used to synthesize cDNA using AMV Reverse Transcriptase 517

(Promega). cDNA was diluted 30 fold to be used as template for RT-qPCR analysis. RT-qPCR 518

analyses were performed on an Mx3000P (Stratagene) Real-Time PCR System using the SYBR 519

Green Master Mix (TOYOBO) according to the manufacturer’s instructions. The expression levels 520

of the samples were normalized to that of Actin. The gene-specific primers used for RT-qPCR are 521

described in Supplemental Table S6. 522

O-GlcNAcylation assay in vitro 523

The O-GlcNAcylation assay in vitro with some modification was used as previously described 524

(Xing et al., 2018). 2 μg of recombinant expressed His-SECDN was incubated with 8 μg 525

His-GAPD, 20 μg His-Enolase, 5 μg His-FBA and 5 μg His-FBA-m, respectively and 50 μM 526

UDP-N-acetylglucosamine in 50 μL of reaction system for 1 h at 37°C. The reaction buffer 527

contained 12.5 mM MgCl2, 50 mM Tris–HCl, pH 7.5, and 1 mM DTT, pH 7.5. After reaction, the 528

mix were denatured at 95°C for 15 min in 5× loading buffer (100 mM Tris–HCl, pH 6.8, 4% w/v 529

SDS, 20% v/v glycerol, 200 mM DTT, and 0.2% w/v bromophenol blue) and electrophoresed by 530

SDS–PAGE. The antibody CTD110.6 specific to O-GlcNAc sites was used to detect O-GlcNAc 531

modification of proteins in immunoblot analysis. 532

RNA-EMSA 533

Biotin-tagged RNA probe was synthesized by Invitrogen company. RNA-EMSA was performed 534

according to the kit instructions (Pierce). Purified RNase-free GST-TaGRP2 and GST-TaGRP2m 535

were used. The probe sequence is listed in Supplementary Table 6. 536

Statistical analyses 537

Statistical differences were assessed by one-way ANOVA. Different letters in graphs indicate the 538

significant treatment difference at p-value <0.05. 539

Accession numbers 540



20

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession 541

numbers Enolase (AGH20062.1), FBA (EMS58841.1), GAPD (EMS68847.1), TaGRP2 542

(BAF30986.1). 543

Supplemental Data 544

Supplemental Figure S1. Detection of global O-GlcNAcylated proteins in different 545

vernalized-winter-wheat with or without PUGNAc treatment by immunoprecipitation, Actin serves as 546

loading control 547

Supplemental Figure S2. Alteration of O-GlcNAc signaling affects vernalization accelerated 548

flowering transition in winter wheat JD1, bar=0.5 mm. 549

Supplemental Figure S3. Dynamic phosphorylated proteins during vernalization 550

Supplemental Figure S4. Biological function enrichment of alternative phosphorylated proteins 551

during vernailization. 552

Supplemental Figure S5. Sequence alignment of GRP2 protein in wheat and its homologues in other 553

species. 554

Supplemental Figure S6. Phylogenetic tree of GRP2 proteins based on the alignment analysis of 555

TaGRP2 and its homologues in these species. 556

Supplemental Figure S7. Sequencing identification of mutated nucleotide to cause an encoding amino 557

acid change (T17 to A17, S87 to A87 and S152 to A152) in GRP2 used in the RNA-EMSA. 558

Supplemental Figure S8. TaGRP2 regulated vernalization inhibited flowering transition in winter 559

wheat. 560

Supplemental Figure S9. KEGG analysis of O-GlcNAcylated proteins in metabolism processing. 561

Supplemental Table S1. Details of O-GlcNAcylated peptides in non-vernalized wheat. 562

Supplemental Table S2. Details of O-GlcNAcylated peptides in vernalized wheat. 563

Supplemental Table S3. 124 phosphoproteins which are significant changes in phosphorylation level 564

(SCPL) between vernalization and non-vernalization. 565

Supplemental Table S4. Details of O-GlcNAcylated proteins homologous in rice and Arabidopsis. 566

Supplemental Table S5. The O-GlcNAc modified proteins are consistent with the potential interactors 567

of SEC (O-GlcNActransferase) detected in Arabidopsis. 568

Supplemental Table S6. The list of the primers used in RT-qPCR and RNA-EMSA. 569



21

Acknowledgements 570

We gratefully acknowledge funding from the National Key Research and Development Program of 571

China (2016YFD0101004) and the China Postdoctoral Science Foundation. We also thank Dr. Zhuang 572

Lu for her MS analysis. 573

Tables 574

Table 1 The proteins with O-GlcNAcylation and phophorylation modification 575

No. GeneBank ID Protein descriptions

Metabolic process(32.3%)

1 525291 ATP synthase beta subunit [Triticumaestivum]

2 474210338 Fructose-bisphosphatealdolase cytoplasmic isozyme [Triticumurartu]

3 148508784 Glyceraldehyde-3-phosphate dehydrogenase [Triticumaestivum]

4 474433294 Peptidyl-prolylisomerase PASTICCINO1 [Triticumurartu]

5 473990310 Peroxidase 64 [Triticumurartu]

6 474111415 Polyadenylate-binding protein 2 [Triticumurartu]

7 474305843 DEAD-box ATP-dependent RNA helicase 24 [Triticumurartu]

8 473923422 Elongation factor 1-alpha [Triticumurartu]

9 461744056 Enolase [Triticumaestivum]

10 474137978 RuBisCO large subunit-binding protein subunit beta, chloroplastic

[Triticumurartu]

Response to stress(29%)

11 473949239 E3 ubiquitin-protein ligase TRIM33 [Triticumurartu]

12 474267869 Ethylene insensitive 3-like 5 protein [Triticumurartu]

13 474173714 Heat shock 70 kDa protein, mitochondrial [Triticumurartu]

14 474378056 Heat shock protein 83 [Triticumurartu]

15 294717808 Heat shock protein 90 [Triticumaestivum]

16 25989705 LEA1 protein [Triticumaestivum]

17 300681479 bZIP transcription factor domain containing protein, expressed

[Triticumaestivum]

18 474425093 Zinc finger protein VAR3, chloroplastic [Triticumurartu]

19 474302864 Putative calcium-binding protein CML7 [Triticumurartu]

Kinase and phosphatase(12.9%)

20 262192761 LRR receptor-like kinase [Triticumaestivum]

21 474016289 Serine/threonine-protein kinase PBS1 [Triticumurartu]

22 473996388 Serine/threonine protein phosphatase 2A 57 kDa regulatory subunit B~

iota isoform [Triticumurartu]

23 114145394 Glycine-rich RNA-binding protein [Triticumaestivum]

Others(25.8%)



22

24 4098272 Alpha-tubulin [Triticumaestivum]

25 215398470 Globulin 3 [Triticumaestivum]

26 2980891 Histone H1 [Triticumaestivum]

27 474360395 Nuclear-pore anchor [Triticumurartu]

28 473956884 Patatin group A-3 [Triticumurartu]

29 474241959 Tetratricopeptide repeat protein 7B [Triticumurartu]

30 473889537 40S ribosomal protein S3-3 [Triticumurartu]

31 474323352 60S ribosomal protein L22-2 [Triticumurartu]

576

Figure Legends 577

Figure 1. O-GlcNAc signaling accelerates vernalization promoted flowering transition in winter 578

wheat. 579

(A) Shoot apex morphology of winter wheat JD1 at V0, V14 and V21 with non-treatment (control) and 580

PUGNAc (OGA inhibition) treatment respectively, bar=0.5mm. The diagram on the left shows 581

different stages of wheat apex, double ridge is a clear marker to indicate the initiation of flowering. -582

(B,C) Quantification of the heading rate (the percentage of the wheat reaches double ridge when 583

observation) (B) and heading time (C) of wheat with different treatmen, the stars above two of the bars 584

emphasize that winter wheat with PUGNAc addition under V14 flowers at similar time as the control 585

plant with V21. Data are means ± SD of 20 plants for each line. Different letters indicate the significant 586

treatment difference at P<0.05, and one-way ANOVA was used for statistical analysis; (D) Relative 587

expression of key flowering genes TaVRN1, TaVRN2 and TaFT1 in JD1 wheat with non-treatment 588

(control) and PUGNAc treatment (Data was normalized to housekeeping gene Actin first, then 589

normalized to non-treated V0 plant). Data shown are means ± SD, n=3. 590

Figure 2. Experiments design to enrich and identify proteins with O-GlcNAcylation or 591

Phosphorylation at different stage of vernalization and overview of identified proteins. 592

(A) Diagram of tissue sampling at different time point during vernalization and the corresponding 593

developmental stages of shoot apex. (B-C) Strategies used for isolation, enrichment and identification 594

of O-GlcNAcylated peptide/protein (B) and phosphorylated peptide/protein with two biologic 595

replications (C). (D) Venn-diagram showing general and unique O-GlcNAcylated proteins identified at 596

V0 and V21. (E) Venn-diagram showing alternatively changed phosphorylated proteins identified in 597

response to vernalization. V21/V0, protein of significantly changed phosphorylation level (SCPL) 598

between V21 and V0; V21/V0_V2/V0, SCPL protein between V21 and V0 deduct that of V2 and V0; 599

V21/V0_V2/V0_V21+5/V0, SCPL protein between V21 and V0 subtract that of V2/V0 and V21+5/V0. 600

V0: no cold exposure, V2: vernalization for 2 days, V21: vernalization for 21days, and V21*: 601

vernalization for 21 days followed by high temperature (35℃) growth for 5 days (V21+5). 602

Figure 3. Enrichment of biological process of the identified O-GlcNAcylated proteins 603

(A-C) Enriched GO terms of all identified O-GlcNAcylated proteins in either vernalized or 604

non-vernalized wheat (A); specifically in non-vernalized (V0) wheat (B); or in vernalized (V21) wheat 605



23

(C). (D) Four major processes with phosphorylation or O-GlcNAcylation modification involved in 606

vernalization response, the red small dots mean O-GlcNAc modification and the blue dots mean 607

phosphorylation modification. 608

Figure 4. Occupancy patterns between phosphorylation and O-GlcNAcylation modification in 609

response to vernalization and SEC O-GlcNAcylates GAPD, Enolase and FBA in vitro. 610

(A) Three-dimensional structures of the proteins (such as Enolase, glyceraldehydes-3-phosphate 611

dehydrogenase (GAPD) and fructose-bisphosphatealdolase (FBA)) with O-GlcNAcylation and 612

phosphorylation modification predicted by Swiss-model (https://swissmodel.expasy.org/) and the 613

sequences of identified peptides. The amino acid with O-GlcNAc modification (g) was orange, and the 614

one with phosphorylation modification (p) was blue. There were two states between the 615

O-GlcNAcylation and phosphorylation modifications from the results. One was the competition 616

relation at the same site (3%) and the other was the coexistence relationship of the two modifications at 617

proximate sites (58%) or distantsites (39%). (B) Detection of O-GlcNAc modification of His-GAPD 618

and His-Enolase, catalyzed by His-SEC△N in vitro. His-GAPD and His-Enolase were recombinantly 619

expressed and affinity purified separately, His-SEC△N (expressing residues 801–1,062 of the 620

C-terminus) exhibited OGT activity [23]. O-GlcNAcylation of His-GAPD and His-Enolase were 621

detected by anti-CTD110.6 antibody. (C) Detection of O-GlcNAc modification of His-FBA and 622

His-FBA-m catalyzed by His-SEC△N in vitro. His-FBA-m means the mutation of the three 623

identified O-GlcNAcylated sites (T35, T320 and S350) of FBA. 624

Figure 5. The O-GlcNAcylated and phosphorylated peptides of TaGRP2 identified by MS. 625

(A) The amino acid sequences of TaGRP2 with identification of phosphorylation and 626

O-GlcNAcylation modification sites; (B) The representative MS/MS spectra of O-GlcNAcylated 627

peptide; (C) The representative MS/MS spectra of phosphorylated peptide. (D) Three-dimensional 628

structure map of TaGRP2 predicted by Phyre, the phosphorylated site is S152 and the O-GlcNAcylated 629

site is S87 (http://www.sbg.bio.ic.ac.uk/phyre2/); (E) The estimated accuracy of the predicted 3D 630

structure of TaGRP2. 631

Figure 6. Mutant of S87 or S152 effects the TaGRP2’s binding to TaVRN1-RIP3 632

(A) A RNA-EMSA assay to analyze the binding of GRP2 and GRP2 mutants to the TaVRN1-RIP3. 633

T17m, S87m and S152m means the GRP2 protein with mutation of T17, S87 and S152, respectively; 634

SSm2 means GRP2 protein with mutation of S87 and S152; TSSm3 means GRP2 protein with 635

mutation of T17, S87 and S152; numbers above indicate the average band intensity of three replicates 636

as quantified using Image J. The CBB signal was normalized by EMSA signal. (B) Coomassie brilliant 637

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835

836

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838

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840

841



Figure 1 O-GlcNAc signaling accelerates vernalization promoted flowering transition in winter wheat.

(A) Shoot apex morphology of winter wheat JD1 at V0, V14 and V21 with non-treatment (control) and PUGNAc

(OGA inhibition) treatment respectively, bar=0.5mm. The diagram on the left shows different stages of wheat apex,

double ridge is a clear marker to indicate the initiation of flowering. (B,C) Quantification of the heading rate (the

percentage of the wheat reaches double ridge when observation) (B) and heading time (C) of wheat with different

treatment. The stars above two of the bars emphasize that winter wheat with PUGNAc addition under V14 flowers

at similar time as the control plant with V21. Data are means ± SD of 20 plants for each line. Different letters indicate

the significant treatment difference at P<0.05, and one-way ANOVA was used for statistical analysis; (D) Relative

expression of key flowering genes TaVRN1, TaVRN2 and TaFT1 in JD1 wheat with non-treatment (control) and

PUGNAc treatment (Data was normalized to housekeeping gene Actin first, then normalized to non-treated V0 plant).

Data shown are means ± SD, n=3.



Figure 2 Experiments design to enrich and identify proteins with O-GlcNAcylation or Phosphorylation at different

stage of vernalization and overview of identified proteins.

(A) Diagram of tissue sampling at different time point during vernalization and the corresponding developmental

stages of shoot apex. (B-C) Strategies used for isolation, enrichment and identification of O-GlcNAcylated

peptide/protein (B) and phosphorylated peptide/protein with two biologic replications (C). (D) Venn-diagram

showing general and unique O-GlcNAcylated proteins identified at V0 and V21. (E) Venn-diagram showing

alternatively changed phosphorylated proteins identified in response to vernalization. V21/V0, protein of

significantly changed phosphorylation level (SCPL) between V21 and V0; V21/V0_V2/V0, SCPL protein between

V21 and V0 deduct that of V2 and V0; V21/V0_V2/V0_V21+5/V0, SCPL protein between V21 and V0 subtract that

of V2/V0 and V21+5/V0. V0: no cold exposure, V2: vernalization for 2 days, V21: vernalization for 21days, and

V21*: vernalization for 21 days followed by high temperature (35℃) growth for 5 days (V21+5).



Figure 3 Enrichment of biological process of O-GlcNAcylated proteins identified

(A-C) Enriched GO terms of all identified O-GlcNAcylated proteins in either vernalized or non-

vernalized wheat (A); specifically in non-vernalized (V0) wheat (B); or in vernalized (V21) wheat

(C). (D) Four major processes with phosphorylation or O-GlcNAcylation modification involved in



vernalization response, the red small dots mean O-GlcNAc modification and the blue dots mean

phosphorylation modification.



Figure 4 Occupancy patterns between phosphorylation and O-GlcNAcylation modification in response to

vernalization and SEC O-GlcNAcylates GAPD, Enolase and FBA in vitro.

(A) Three-dimensional structures of the proteins (such as Enolase, glyceraldehydes-3-phosphate dehydrogenase

(GAPD) and fructose-bisphosphatealdolase (FBA)) with O-GlcNAcylation and phosphorylation modification

predicted by Swiss-model (https://swissmodel.expasy.org/) and the sequences of identified peptides. The amino acid

with O-GlcNAc modification (g) was orange, and the one with phosphorylation modification (p) was blue. There

were two states between the O-GlcNAcylation and phosphorylation modifications from the results. One was the

competition relation at the same site (3%) and the other was the coexistence relationship of the two modifications at

proximate sites (58%) or distantsites (39%). (B) Detection of O-GlcNAc modification of His-GAPD and His-Enolase,

catalyzed by His-SEC△N in vitro. His-GAPD and His-Enolase were recombinantly expressed and affinity purified

separately, His-SEC△N (expressing residues 801–1,062 of the C-terminus) exhibited OGT activity [23]. O-

GlcNAcylation of His-GAPD and His-Enolase were detected by anti-CTD110.6 antibody. (C) Detection of O-

GlcNAc modification of His-FBA and His-FBA-m catalyzed by His-SEC△N in vitro. His-FBA-m means the

mutation of the three identified O-GlcNAcylated sites (T35, T320 and S350) of FBA.


https://swissmodel.expasy.org/


Figure 5 The O-GlcNAcylated and phosphorylated peptides of TaGRP2 identified by MS.

(A) The amino acid sequences of TaGRP2 with identification of phosphorylation and O-GlcNAcylation modification

sites; (B) The representative MS/MS spectra of O-GlcNAcylated peptide; (C) The representative MS/MS spectra of

phosphorylated peptide. (D) Three-dimensional structure map of TaGRP2 predicted by Phyre, the phosphorylated

site is S152 and the O-GlcNAcylated site is S87 (http://www.sbg.bio.ic.ac.uk/phyre2/); (E) The estimated accuracy

of the predicted 3D structure of TaGRP2.


http://www.sbg.bio.ic.ac.uk/phyre2/


Figure 6 Mutant of S87 or S152 effects the TaGRP2’s binding to TaVRN1-RIP3

(A) A RNA-EMSA assay to analyze the binding of GRP2 and GRP2 mutants to the TaVRN1-RIP3. T17m, S87m

and S152m means the GRP2 protein with mutation of T17, S87 and S152, respectively; SSm2 means GRP2 protein

with mutation of S87 and S152; TSSm3 means GRP2 protein with mutation of T17, S87 and S152; numbers above

indicate the average band intensity of three replicates as quantified using Image J. The CBB signal was normalized

by EMSA signal. (B) Coomassie brilliant blue (CBB) staining result of the EMSA samples in A. (C) The quantitative

data of three replicates for the binding-affinity comparisons among wild-type and mutant GRP2 proteins. Data shown

are means ± SD, n=3.



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Chong K, Bao SL, Xu T, Tan KH, Liang TB, Zeng JZ, Huang HL, Xu J, Xu ZH (1998) Functional analysis of the ver gene using antisensetransgenic wheat. Physiologia Plantarum 102: 87-92


Copeland RJ, Bullen JW, Hart GW (2008) Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance andglucose toxicity. American Journal of Physiology-Endocrinology and Metabolism 295: E17-E28


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Gambetta MC, Oktaba K, Mueller J (2009) Essential Role of the Glycosyltransferase Sxc/Ogt in Polycomb Repression. Science 325: 93-96


Gauley A, Boden SA (2019) Genetic pathways controlling inflorescence architecture and development in wheat and barley. Journal ofIntegrative Plant Biology 61: 296-309


Guinez C, Losfeld ME, Cacan R, Michalski JC, Lefebvre T (2006) Modulation of HSP70 GlcNAc-directed lectin activity by glucoseavailability and utilization. Glycobiology 16: 22-28


Guo X, Liu D, Chong K (2018) Cold signaling in plants: Insights into mechanisms and regulation. Journal of Integrative Plant Biology 60:745-756


Gupta R, Brunak S (2002) Prediction of glycosylation across the human proteome and the correlation to protein function. Pac SympBiocomput: 310-322


Hanover JA, Krause MW, Love DC (2010) The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine. Biochimica EtBiophysica Acta-General Subjects 1800: 80-95


Hart GW, Slawson C, Ramirez-Correa G, Lagerlof O (2011) Cross talk between O-GlcNAcylation and phosphorylation: Roles insignaling, transcription, and chronic disease. Annual Review of Biochemistry 80: 825-858

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title www.plantphysiol.orgon May 27, 2020 - Published by Downloaded from

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https://www.ncbi.nlm.nih.gov/pubmed?term=Hu P%2C Shimoji S%2C Hart GW %282010%29 Site%2Dspecific interplay between O%2DGlcNAcylation and phosphorylation in cellular regulation%2E Febs Letters&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Huan Q%2C Mao Z%2C Chong K%2C Zhang J %282018%29 Global analysis of H3K4me3%2FH3K27me3 in Brachypodium distachyon reveals VRN3 as critical epigenetic regulation point in vernalization and provides insights into epigenetic memory%2E New Phytol&dopt=abstract

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https://scholar.google.com/scholar?as_q=Global analysis of H3K4me3/H3K27me3 in Brachypodium distachyon reveals VRN3 as critical epigenetic regulation point in vernalization and provides insights into epigenetic memory&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huan&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Huan Q%2C Mao Z%2C Zhang J%2C Xu Y%2C Chong K %282013%29 Transcriptome%2Dwide analysis of vernalization reveals conserved and species%2Dspecific mechanisms in Brachypodium%2E Journal of Integrative Plant Biology&dopt=abstract

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https://scholar.google.com/scholar?as_q=Transcriptome-wide analysis of vernalization reveals conserved and species-specific mechanisms in Brachypodium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huan&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kaasik K%2C Kivimaee S%2C Allen JJ%2C Chalkley RJ%2C Huang Y%2C Baer K%2C Kissel H%2C Burlingame AL%2C Shokat KM%2C Ptacek LJ%2C Fu Y%2DH %282013%29 Glucose sensor O%2DGlcNAcylation coordinates with phosphorylation to regulate circadian clock%2E Cell Metabolism&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Kim EJ %282015%29 The utilities of chemical reactions and molecular tools for O%2DGlcNAc proteomic studies%2E Chembiochem&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Kippes N%2C Debernardi JM%2C Vasquez%2DGross HA%2C Akpinar BA%2C Budak H%2C Kato K%2C Chao S%2C Akhunov E%2C Dubcovsky J %282015%29 Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia%2E Proceedings of the National Academy of Sciences of the United States of America 112%3A E5401%2DE&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Koppolu R%2C Schnurbusch T %282019%29 Developmental pathways for shaping spike inflorescence architecture in barley and wheat%2E Journal of Integrative Plant Biology&dopt=abstract

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https://scholar.google.com/scholar?as_q=Developmental pathways for shaping spike inflorescence architecture in barley and wheat&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Koppolu&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Leney AC%2C El Atmioui D%2C Wu W%2C Ovaa H%2C Heck AJR %282017%29 Elucidating crosstalk mechanisms between phosphorylation and O%2DGlcNAcylation%2E Proceedings of the National Academy of Sciences of the United States of America 114%3A E7255%2De&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Xu S%2DL%2C Chalkley RJ%2C Maynard JC%2C Wang W%2C Ni W%2C Jiang X%2C Shin K%2C Cheng L%2C Savage D%2C Huhmer AFR%2C Burlingame AL%2C Wang Z%2DY %282017%29 Proteomic analysis reveals O%2DGlcNAc modification on proteins with key regulatory functions in Arabidopsis%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

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https://scholar.google.com/scholar?as_q=Cloning and characterization of vernalization-related gene (ver203F) cDNA 3 ' end&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yong&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yong WD%2C Xu YY%2C Xu WZ%2C Wang X%2C Li N%2C Wu JS%2C Liang TB%2C Chong K%2C Xu ZH%2C Tan KH%2C Zhu ZQ %282003%29 Vernalization%2Dinduced flowering in wheat is mediated by a lectin%2Dlike gene VER2%2E Planta&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Yu Q%2C An L%2C Li W %282014%29 The CBL%2DCIPK network mediates different signaling pathways in plants%2E Plant Cell Rep&dopt=abstract

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https://scholar.google.com/scholar?as_q=A cis cold memory element and a trans epigenome reader mediate Polycomb silencing of FLC by vernalization in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yuan&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yun MY%2C Wu J%2C Workman JL%2C Li B %282011%29 Readers of histone modifications%2E Cell Research&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Zentella R%2C Hu J%2C Hsieh W%2DP%2C Matsumoto PA%2C Dawdy A%2C Barnhill B%2C Oldenhof H%2C Hartweck LM%2C Maitra S%2C Thomas SG%2C Cockrell S%2C Boyce M%2C Shabanowitz J%2C Hunt DF%2C Olszewski NE%2C Sun T%2Dp %282016%29 O%2DGlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis%2E Genes %26 Development&dopt=abstract

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Google Scholar: Author Only Title Only Author and Title

Zhong K, Tan KH, Huang HL, Liang HG (1995) Molecular-cloning of a cDNA related to vernalization (VERC203) in winter-wheat. Sciencein China Series B-Chemistry Life Sciences & Earth Sciences 38: 799-806


Zhou J-X, Liu Z-W, Li Y-Q, Li L, Wang B, Chen S, He X-J (2018) Arabidopsis PWWP domain proteins mediate H3K27 trimethylation onFLC and regulate flowering time. Journal of Integrative Plant Biology 60: 362-368



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

https://scholar.google.com/scholar?as_q=Isolation of a vernalization-related cDNA clone (VRC) using mRNA differential display in winter wheat&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

https://scholar.google.com/scholar?as_q=Isolation of a vernalization-related cDNA clone (VRC) using mRNA differential display in winter wheat&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhong K%2C Tan KH%2C Huang HL%2C Liang HG %281995%29 Molecular%2Dcloning of a cDNA related to vernalization %28VERC203%29 in winter%2Dwheat%2E Science in China Series B%2DChemistry Life Sciences %26 Earth Sciences&dopt=abstract

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https://scholar.google.com/scholar?as_q=Molecular-cloning of a cDNA related to vernalization (VERC203) in winter-wheat&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhong&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhou J%2DX%2C Liu Z%2DW%2C Li Y%2DQ%2C Li L%2C Wang B%2C Chen S%2C He X%2DJ %282018%29 Arabidopsis PWWP domain proteins mediate H3K27 trimethylation on FLC and regulate flowering time%2E Journal of Integrative Plant Biology&dopt=abstract

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https://scholar.google.com/scholar?as_q=Arabidopsis PWWP domain proteins mediate H3K27 trimethylation on FLC and regulate flowering time&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

https://scholar.google.com/scholar?as_q=Arabidopsis PWWP domain proteins mediate H3K27 trimethylation on FLC and regulate flowering time&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1


The protein modifications of O-GlcNAcylation and ... · 3 49. Introduction. 50. O-linked...

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