Comparative Phosphoproteomic Analysis of the Developing Seeds in

Subscriber access provided by MT ROYAL COLLEGE

Article

Comparative phosphoproteomic analysis of the developing seeds in two indica rice (Oryza sativa L.) cultivars with different starch quality Yuehan Pang, Xin Zhou, Yaling Chen, and Jinsong Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00074 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

Comparative phosphoproteomic analysis of the developing seeds in

2

two indica rice (Oryza sativa L.) cultivars with different starch quality

3

Yuehan Pang1, Xin Zhou1, Yaling Chen2, Jinsong Bao1*

4 5 6

1

7

Zhejiang University, Huajiachi Campus, Hangzhou, 310029, China.

Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology,

8 9

2

College of Life Sciences, Jiangxi Normal University, Nanchang, 330022, China

10 11

*Corresponding authors: [email protected]

12

1

ACS Paragon Plus Environment


13

ABSTRACT: Protein phosphorylation plays important roles in regulation of various

14

molecular events such as plant growth and seed development. However, its

15

involvement in starch biosynthesisis less understood. Here, a comparative

16

phosphoproteomic analysis of two indica rice cultivars during grain development was

17

performed. A total of 2079 and 2434 phosphopeptides from 1273 and 1442

18

phosphoproteins were identified, covering 2441 and 2808 phosphosites in indica rice

19

9311 and Guangluai4 (GLA4), respectively. Comparative analysis identified 303

20

differentially phosphorylated peptides, and 120 and 258 specifically phosphorylated

21

peptides in 9311 and GLA4, respectively. Phosphopeptides in starch biosynthesis

22

related enzymes such as AGPase, SSIIa, SSIIIa, BEI, BEIIb, PUL and Pho1were

23

identified. GLA4 and 9311 had different amylose content, pasting viscosities and

24

gelatinization temperature, suggesting subtle difference in starch biosynthesis and

25

regulation between GLA4 and 9311. Our study will give added impetus to further

26

understanding the regulatory mechanism of starch biosynthesis at the phosphorylation

27

level.

28

KEYWORDS:Rice (Oryza sativa L.); Phosphorylation; Grain development; Starch

29

biosynthesis; Starch properties

30

2


Page 2 of 32

Page 3 of 32


31

INTRODUCTION

32

Rice is an important cereal crop in developing countries which feeds more than half of

33

the world population.1–4 Rice grain is largely composed of starch (approximately

34

80%-90%).2,5,6 Starch properties such as apparent amylose content (AAC), gel

35

consistency, gelatinization temperature, and pasting viscosity determine rice eating

36

and cooking quality (ECQ) which has become the primary consideration of rice

37

customers and breeders.1,3,4,6 Starch biosynthesis is a complex network of multiple

38

isozymes including at least four enzyme classes: ADP-glucose pyrophosphorylase

39

(AGPase), starch synthase (SS), starch branching enzyme (BE or SBE), and starch

40

debranching enzyme (DBE).1,5,7 At present, the functional characterizations of starch

41

synthesis related enzymes have been well documented. However, interactions among

42

these enzymes have not been well determined. Furthermore, post-translational

43

modifications (PTMs) of enzymes related to starch synthesis in rice have rarely been

44

studied. A better understanding of starch biosynthesis and its regulation will aid

45

breeders to design new rice varieties with desirable ECQ.1,3–5

46

Protein phosphorylation is one of the most abundant and important PTMs, which

47

mainly affects the hydroxyl group in serine, threonine and tyrosine.8 Protein

48

phosphorylation plays important biological roles in regulation of various molecular

49

events including plant growth and seed development.9–11 However, information to

50

understand the regulatory roles of protein phosphorylation related to starch synthesis

51

during seed development remains unclear. In vitro experiments from wheat

52

endosperm have shown that protein phosphorylation modified the catalytic activities

53

of SBEs in amyloplasts and chloroplasts, and the integrity of a protein complex

54

between SBEI, SBEIIb and starch phosphorylase was dependent on protein

55

phosphorylation.12 Further research indicated that SSII, SBEIIa and SBEIIb were 3



Page 4 of 32

56

phosphorylated and the formation of a 260kD SS-SBEII protein complex was

57

phosphorylation dependent.13 Similar research has been carried out in maize. On the

58

one hand, Grimaud et al. found that GBSS, BEIIb, and starch phosphorylase were all

59

phosphorylated as they occurred in the granule, suggesting protein phosphorylation

60

may play a role in their association with starch granules.14 On the other hand, a larger

61

protein complex of approximately 670 kDa whose formation is phosphorylation

62

dependent was found.15 Further analysis of this protein complex in maize endosperm

63

showed that the SBEIIb in the complex was phosphorylated.16 Makhmoudova et al.

64

demonstrated that SBEIIb of maize was phosphorylated at three sites, and the Ser 286

65

and Ser 297 phosphorylation sites are conserved in all plant branching enzymes, but

66

Ser 649 is cereal specific.17 In barley, Ahmed et al. found that GBSS, SSI, SSIIa,

67

SBEIIb and starch phosphorylase (SP) are phosphorylated in their granule bound state

68

and the formation of protein complexes was regulated by protein phosphorylation.18

69

However, less is known about the phosphorylation of starch synthesis related enzymes

70

in rice as compared to wheat, maize and barley. In an effort to study the molecular

71

mechanism of poor grain filling of rice inferior spikelet, high resolution

72

two-dimensional gel electrophoresis in combination with Pro-Q

73

phosphoprotein fluorescence stain revealed five phosphoproteins (PGM, UDPase,

74

phosphorylase, AGPase and GBSS) related to starch synthesis,19 but the exact

75

phosphorylation sequences and phosphorylation sites are still unknown. Qiu et al.

76

identified some phosphorylated proteins (SSIIIa, sucrose synthases, AGPase, etc.)

77

related to starch synthesis,10 however this study only focused on the phosphorylation

78

events in early seed development. Detailed investigations of protein phosphorylation

79

involved in starch synthesis during late rice seed development tmay provide insights

80

into the mechanisms behind starch biosynthesis in rice varieties with different ECQ. It 4


Diamond

Page 5 of 32


81

should be mentioned that all the previous studies on protein phosphorylation except

82

for the study of Zhang et al.19used the japonica rice as material, characterization of

83

protein phosphorylation in indica rice has not been comprehensively reported.

84

The objectives of this study were: (1) to identify phosphoproteins, phosphopeptides

85

and phosphosites of two indica-type rice with different starch properties during rice

86

grain development; (2) to characterize the functions of all phosphorylated peptides

87

including differentially and specifically phosphorylated peptides by Motif-X

88

algorithm and Blast2GO software; (3) to compare phosphopeptides of proteins related

89

to starch synthesis in two indica rice cultivars with other research results on japonica

90

rice, wheat, and maize; and (4) to determine and compare the rice ECQ of two indica

91

rice. The results of this study will give an impetus to further understanding of the

92

regulatory mechanism of starch biosynthesis at the phosphorylation level.

93 94

MATERIALS AND METHODS

95

Plant materials and sample collection. Indica rice varieties, 9311 and Guangluai4

96

(GLA4), were grown at the Zhejiang University farm in 2016, Hangzhou, China. On

97

the day of rice flowering, each panicle was labeled to facilitate collecting grains at

98

defined developmental stages. Three independent biological replicates of developing

99

seeds from 10 days after flowering were harvested, and stored at -80 oC prior to

100

analysis. Full matured rice grains of two varieties were harvested and milled rice and

101

flour was obtained.

102

Determination of starch quality. The apparent amylose content (AAC) and pasting

103

viscosity parameters were determined according to Bao et al.,20 and the thermal

104

(gelatinization) properties were determined by Differential Scanning Calorimeter Q20

105

(TA Instruments, New Castle, DE, USA) according to Bao et al.21 5



106

Extraction of proteins. Extraction of proteins was accomplished referring to the

107

procedures of Crofts et al. with minor modifications.22 Briefly, rice endosperm was

108

extracted with extraction buffer A (50 mM imidazole at pH 7.4, 8 mM MgCl2, 10%

109

glycerol, and 500 mM β-mercaptoethanol), and subsequently centrifuged at 12000×g

110

for 10 min at 4 °C. This procedure was repeated with extraction buffer B (55 mM

111

Tris-HCl at pH 6.8, 2.3% SDS, 10% glycerol, and 500 mM β-mercaptoethanol). All

112

supernatants were gathered and combined, and every replicate was adjusted to the

113

same concentration for the subsequent analysis.

114

Protein digestion and phosphopeptide enrichment. The procedures of protein

115

digestion and phosphopeptide enrichment were performed as described by Rappsilber

116

et al.23 with minor modifications. Extracted protein mixtures were directly reduced

117

with dithiothreitol (DTT), alkylated with iodoacetamide (IAA) in the dark, and

118

subsequently digested with trypsin overnight. The digested peptides were resolved

119

with DHB buffer (0.6% DHB, 16% CAN, 0.02% TFA), then incubated with TiO2

120

beads (5020-75000, Shimadzu, Japan) for 40 min and centrifuged. After TiO2 beads

121

were transferred into a homemade StageTip and rinsed three times in washing buffer 1

122

(30% ACN, 3% TFA) and washing buffer 2 (80% ACN, 0.3% TFA), the enriched

123

phosphopeptides were subsequently eluted with elution buffer (40% ACN, 15%

124

NH4OH). The combined eluates were concentrated by vacuum evaporation and

125

reconstituted in 0.1% formic acid for LC–MS/MS analysis.

126

LC-MS/MS and data analysis. LC was performed on an Easy nLC System (Thermo

127

Scientific, Bremen, Germany). The mobile phases consisted of 0.1% formic acid (A)

128

and 0.1% formic acid in 84% v/v acetonitrile (B). The column was equilibrated with

129

95% A solution. A 20 µL of phosphopeptide solution was loaded onto Thermo

130

scientific EASY column (2cm*100µm 5µm-C18), and separated by Thermo scientific 6


Page 6 of 32

Page 7 of 32


131

EASY column (75µm*100mm 3µm-C18) at a flow rate of 300 nl/min. Over the period

132

0–220 min, the concentration of B rose linearly from 0% to 55%; from 220–228 min,

133

it was increased from 55% to 100%; from 228–240 min, it was maintained at 100%.

134

The Q-Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany) was

135

operated in the positive ion mode over 240 min. Full-scan mass spectra were acquired

136

over a mass range of 300–1800 m/z. Survey scans were acquired at a resolution of

137

70000 at 200 m/z, and the resolution set for the HCD spectra was 17500 at 200 m/z.

138

Three technical replicates were performed independently for each sample.

139

Raw mass spectrometric data were analyzed with the MaxQuant software (version

140

1.3.0.5) and were compared with the rice database. Maximally, two missed cleavages

141

were allowed. For MS and MS/MS, the tolerances of the main search for peptides

142

were set at 6 and 20 ppm, respectively. For the search, trypsin allowing for cleavage

143

N-terminal to proline (trypsin/P) was chosen as enzyme specificity. Cysteine

144

carbamidomethylation was selected as a fixed modification, while protein N-terminal

145

acetylation, methionine oxidation, and phosphorylation on serine/threonine/tyrosine

146

were selected as variable modifications. A false discovery rate (FDR) of 0.01 for

147

proteins and peptides and a minimum peptide length of 7 amino acids were required.

148

Bioinformatic Analysis. Significantly enriched phosphorylation motifs were

149

extracted

150

(http://motif-x.med.harvard.edu/). The phosphopeptides were centered at the

151

phosphorylated amino acid residues and aligned, and six positions upstream and

152

downstream of the phosphorylation site were included. Thirteen amino acids (AAs)

153

sequences centered by the phosphorylation site were extracted, and the rice protein

154

database was used as the background database to normalize the scores against the

155

random distributions of AAs. As for differentially phosphorylated (DP) peptides, only

from

phosphopeptides

using

the

7


Motif-X

algorithm


156

the phosphopeptides that met the following restrictions were regarded as differentially

157

phosphorylated: (1) phosphopeptide detected in over two biological replicates, (2) P
2. Only the phosphopeptides that met the

160

following restrictions were regarded as specifically phosphorylated: (1) in one group,

161

there was no data in any of the three independent biological analyzes, and (2) in the

162

other group, there were more than two quantitative data. For cluster analysis, the

163

quantitative information of phosphorylated proteins was normalized to the (-1, 1)

164

interval, and then proteins were clustered by hierarchical clustering via average

165

linkage using Cluster 3.0, the results were visualized using Java TreeView. Kyoto

166

Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed by

167

using KEGG Automatic Annotation Server (KAAS) software. The biological

168

processes, molecular functions, and cellular components of the identified

169

phosphoproteins were examined using Blast2GO software to perform gene ontology

170

(GO) annotation (http://www.blast2go.com/b2ghome).

171 172

RESULTSAND DISCUSSION

173

Identification of phosphorylation sites, peptides and proteins.The label-free

174

phosphopeptide quantification identified 2079 and 2434 phosphopeptides from 1273

175

and 1442 phosphoproteins, covering 2441 and 2808 phosphosites in 9311 and GLA4

176

samples respectively (Figure 1). The majority of the phosphopeptides, 71.69% (9311)

177

and 74.68% (GLA4), in two rice samples were only single phosphorylated, whereas

178

12.21% (9311) and 10.79% (GLA4) peptides carried two phosphorylation groups, but

179

only around 1% peptides carried three or more phosphorylation modifications. The

180

distribution ratios of phosphosite on serine, threonine and tyrosine were counted. 8


Page 8 of 32

Page 9 of 32


181

Phosphoserine was the predominant phosphorylation type and accounted for >93% of

182

the

183

phosphotyrosine only accounted for 0.1%-0.4% of the total phosphosites (Figure 1).

184

In agreement with previous reports,10,24 our results showed that phosphorylation on

185

the three residue types are highly conservative among crop plants. The level of

186

tyrosine phosphorylation is close to that expected for human cells (1.8% - 6%),

187

suggesting that tyrosine signaling may be similarly important in plants.25

188

Conserved phosphorylation motif analysis of the phosphosites. With the aid of the

189

Motif-X algorithm, we extracted 13 amino acids (AA) sequences centered by the

190

phosphorylation site, and the over-presented motifs around the phosphosites were

191

analyzed. We detected 47 over-represented motifs for phosphoserine (Table S1). [Sxs]

192

was the most common motifs for phosphoserine as 333 matches were found in our

193

result (Figure S1). Followed were [pxSp] and [Gs] with around 200 hits detected.

194

There were also more than 150 hits of [sxS], [sPxR] and [sP] motifs. Among all

195

over-represented motifs detected, [sP], [Rxxs], [sDxE], [sDDD], [sxD] and [sxE],

196

[sDxD] were also found in the seeds of Arabidopsis, rapeseed, and soybean.26

197

Proline-directed motifs, such as [sP] and [LxRxxs], were recognized by GSK-3,

198

mitogen-activated protein kinases, and orcyclin-dependent kinase.27 [Rxxs] was a

199

basophilic motif recognized by calmodulin kinase-II (CaMK-II), protein kinase A

200

(PKA), and PKC.28 In addition, both [sxD] and [sDxE], the substrate of casein

201

kinase-II (CK-II), were acidic motifs. The motif [sF] was rarely found in our

202

investigation. However, recent studies found that it was an over-represented motif in

203

rice.10,29 This might be attributed to the different tissues and different methods for

204

protein extraction and MS identification.

205

total

phosphosites.

Phosphothreonine

accounted

for

around

6%

and

As shown in Figure S2, eight types of conserved motifs were significantly enriched 9



Page 10 of 32

206

around the phosphothreonine sites. [St] and [tP] were the over-represented motif we

207

discovered for phosphothreonine sites, the occurrence frequency was relatively high

208

as 88 and 53 matches were found in our result, respectively. No obvious conserved

209

motif could be extracted from phosphotyrosine peptides due to the small number of

210

phosphorylated tyrosine sites.

211

Differentially phosphorylated (DP) peptides and proteins. After the quantitative

212

normalization of the phosphorylation intensity of three biological replicates, a total of

213

303 peptides were found to be differentially phosphorylated between the 9311 and

214

GLA4, of which 95 peptides in GLA4 and 208 peptides in 9311 had a higher level of

215

phosphorylation. Moreover, 120 and 258 phosphorylated peptides were specifically

216

identified

217

phosphopeptides were abundant in the starch-related proteins (see below). To gain an

218

in-depth insight of their phosphorylation patterns, a clustering analysis of the DP

219

peptides based on their phosphorylation intensity were conducted, implying their

220

functions in seed development between two varieties (Figure S3). The clusters fall

221

into two major groups; the members of group I were most strongly phosphorylated in

222

GLA4, while the members of group II were most strongly phosphorylated in 9311.

223

Notably, proteins in group II had more DP proteins than group I. A differential

224

phosphorylation pattern usually indicates the important regulatory roles of the

225

phosphoprotein. KEGG pathway analysis revealed that differentially and specifically

226

phosphorylated proteins are mainly over-represented in the pathways of RNA

227

transport, mRNA surveillance pathway and spliceosome (Figure 2).

in

9311

and

GLA4,

respectively.

As

expected,

differentially

228

In order to obtain an overview of the phosphorylation events during grain

229

development, GO analysis was performed to classify DP proteins using Blast2GO in

230

the vocabulary of “biological process”, “molecular function” and “cellular component” 10


Page 11 of 32


231

(Figure 3). From the “biological process” perspective, phosphoproteins related to

232

“cellular process,” “metabolic process” accounted for over 70 % of all the

233

phosphoproteins identified, whereas “rhythmic process”, “locomotion” and “immune

234

system process” were under-represented. Regarding the “molecular function”,

235

phosphorylated proteins were preferentially cataloged into “binding” and “catalytic

236

activity”, whereas the “molecular transducer activity” and “electron carrier activity”

237

only accounted for less than 0.5 %. Considering that ‘‘binding’’ is closely related to

238

mRNA transcription, the GO analysis results also indicated that protein

239

phosphorylation might be involved in the gene transcription, which is consistent with

240

our KEGG pathway results. In terms of “cellular component,” phosphoproteins

241

related to “binding” and “metabolic process” accounted for over 85% of all the

242

phosphoproteins identified, only less than 1 % of the phosphoproteins were related to

243

“extracellular region” and “extracellular region part”.

244

Phosphorylation regulates starchbiosynthesis. Phosphoproteomic analysis strongly

245

indicated that phosphorylation-mediated regulation is a crucial mechanism controlling

246

starch biosynthesis during rice grain development. Therefore, phosphorylation

247

peptides of enzymes and regulatory proteins related to starch synthesis were

248

summarized (Table 1). It was found that peptides from AGPase, SS, BE, DBE and

249

other proteins related to starch synthesis were phosphorylated.

250

AGPase is the starch synthesis rate-limiting enzyme, catalyzing the formation of

251

ADP-glucose and pyrophosphate in higher plants.30 It was suggested that AGPase

252

from bread wheat (Triticum aestivum L.) under well-watered and water-deficit

253

conditions was shown to be phosphorylated at Ser69.24 As for rice, a phosphorylated

254

peptide was found in each of the two AGPase (OsAGPS2b and OsAGPL2) in japonica

255

rice Nipponbare.16 Indica rice varieties were used in our study, and AGPase was 11



256

phosphorylated at Ser62, Ser381 and Thr68 (Table 1). Both japonica10 and indica rice

257

had the same phosphopeptide (CVFTSDADRDT(ph)PHLR) of AGPase. Therefore, it

258

is tempting to speculate that phosphorylation at Thr68 of AGPase is prevalent in rice.

259

SS plays particularly central roles in starch biosynthesis, and could be classified at

260

five isoforms: GBSS, SSI, SSII, SSIII and SSIV.31 GBSS is mainly responsible for the

261

synthesis of amylose, and different alleles of the Waxy (Wx) gene determines the

262

amylose content of rice.32 Teng et al. suggested a possibility of phosphorylation for

263

Wx proteins may play an important role in regulating GBSS activity at the

264

post-translational level.33 In addition, GBSS were stained with Pro-Q Diamond dye

265

and identified as a phosphoprotein in rice19 and wheat34. However, no phosphorylation

266

sites for GBSS were found in our study. This might be attributed to the different

267

samples and different methods of protein extraction and MS identification. Soluble

268

starch synthases catalyze the chain-elongation reaction of α-1,4-glucosidic linkage in

269

amylopectin synthesis.7 It was reported that wheat starch synthesis enzymes SSI,

270

SSIIa were phosphorylated.12,13,34 Consistent with a previous report,10 SSIIIa was

271

shown to be phosphorylated at the same phosphosites (Table 1). Additionally, a

272

phosphopeptide of SSIIa was found in 9311 and GLA4 with significant intensity

273

differences, indicating it may have a specific function in indica rice varieties.

274

BE, including BEI, BEIIa and BEIIb, is the only enzyme that can introduce

275

α-1,6-glucosidic linkages into α-polyglucans in plants.7Tetlow and Emes summarized

276

regulation of SBEs by protein phosphorylation in plants，indicating that SBEs with

277

conserved phosphorylation sites form protein complexes with other starch synthesis

278

related enzymes，and assembly of starch synthesis protein complexes in cereals were

279

dependent upon protein-phosphorylation.35 Experiments from wheat endosperm

280

showed formation of a protein complex of approximately 260 kDa between SSI, SSII 12


Page 12 of 32

Page 13 of 32


281

and SBEII required protein phosphorylation.12,13 Related research was also carried out

282

in maize SBEIIb in which the protein complex was phosphorylated in maize

283

endosperm.16 Further analysis demonstrated that SBEIIb of maize was phosphorylated

284

at three sites, and the Ser286 and Ser297 phosphorylation sites are conserved in all

285

plant branching enzymes, but Ser649 is cereal specific.17 We identified three and two

286

phosphopeptides of SBEI and SBEIIb in indica rice, respectively. It is noteworthy that

287

all phosphorylated peptides of SBEI were found only in indica rice GLA4.

288

Considering the endosperm starch from the sbe1 mutant had a lower gelatinization

289

temperature and better taste performance,36 we speculate that phosphorylation events

290

of SBEI in GLA4 may affect starch biosynthesis and eating quality. In addition, the

291

Ser715 of SBEIIb were phosphorylated in both indica rice, while phosphorylation of

292

Ser685 appears only in GLA4. By amino acid sequence alignments of SBEII and

293

SBEI, we found that the Ser685 phosphorylation site conserved among all plant BEs

294

(Figure 4), suggesting a general regulatory role. Moreover, phosphorylation of ser715

295

may have a more specialized function as this site appeared only in rice BEIIb.

296

DBE, classified as isoamylase and pullulanase (PUL), specifically hydrolyze

297

α-1,6-glucosic linkages of α-polyglucans.7 Recent experiments have shown that there

298

is a significant correlation between PUL and the physicochemical properties of rice

299

starch.37,38 Two serine phosphopeptides of PUL were identified; the one was found

300

only in 9311, while the other appeared only in GLA4. Therefore, it is speculated that

301

phosphorylations in PUL may affect the physicochemical properties of rice starch.

302

Additionally, there is evidence that α-glucan phosphorylase (Pho1 and Pho2) are

303

also involved in starch synthesis to catalyze the glucose extension of nonreducing end

304

of the α-glucan chain.39,40 Plastidial phosphorylase (Pho1), a temperature-dependent

305

enzyme, accounted for the vast majority of the total phosphorylase activity in 13



Page 14 of 32

306

developing riceseeds.41 As shown in Table 1, three phosphopeptides of Pho1 were

307

identified, one of which were specifically phosphorylated in GLA4, indicating that the

308

phosphorylation sites of this peptide may be closely related to the regulation of starch

309

synthesis.

310

Sehnke et al. speculated that 14-3-3 protein plays a role in starch regulation through

311

protein phosphorylation because all members of the starch synthase III family contain

312

the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif.42 We

313

identified seven phosphorylated peptides of 14-3-3-like protein, and found some

314

phosphopeptides were also identified by previous reports29,43 (Table S2), suggesting

315

those phosphorylation events may play general regulatory roles in starch biosynthesis.

316

It seems that synergism between bZIP transcription factor RISBZ110 and RPBF10,29

317

would regulate rice grain filling, and same serine phosphopeptides were found (Table

318

S2). Other proteins related to starch synthesis, such assucrose synthase,

319

glucose-6-phosphate

320

serine/threonine-protein

321

glucose-6-phosphate isomerase, pyruvate, phosphate dikinase (regulates starch and

322

fatty acid biosynthesis and accumulation), WRKY transcription factor 78 (regulates

323

internodes and seed development) and tetratricopeptide repeat domain containing

324

protein (regulates starch content and grain size) and so on were also detected and

325

compared with other studies (Table S2).10,29,43

isomerase,

glucose-1-phosphate

phosphatase,

UDP-glucose

adenylyltransferase, pyrophosphorylase,

326

To better understand the regulatory mechanism of phosphorylation in starch

327

synthesis, rice mutants with point mutation in the phosphosites of the related enzymes

328

should be generated. Changes in the enzyme activities and the formation of multiple

329

enzyme complexes will be investigated with the mutants. These mutants are also

330

useful for the study of phosphorylation functions in response to the different internal 14


Page 15 of 32


331

and external factors during seed development.

332

Starch physicochemical properties. Rice ECQ is mainly determined by its

333

physicochemical properties. The indica rice 9311 and GLA4 had an average AAC of

334

16.5% and 24.4%, respectively (Figure 5A). They also had distinctive pasting

335

viscosity profiles (Figure 5B). The 9311 had higher peak viscosity (PV), hot paste

336

viscosity (HPV), cold paste viscosity (CPV) than GLA4. The breakdown (PV-HPV)

337

viscosity of 9311 was higher than that of GLA4, but the setback (CPV-PV) of 9311

338

was much lower than that of GLA4. The characteristics of pasting viscosity profiles

339

suggested that 9311 had better ECQ than GLA4.1,20 The onset, peak and conclusion

340

temperatures of 9311 were 64.8, 70.3 and 76.1 oC, respectively, which were much

341

lower than those of GLA4 (77.0, 80.5 and 85.3 oC, respectively) (Figure 5C).

342

Genetically, amylose content and pasting viscosity profiles are controlled by Waxy

343

gene, while thermal properties are mainly controlled by SSIIa which is responsible for

344

amylopectin biosynthesis.1,3 It is well known that 9311 and GLA4 carry different

345

alleles of Waxy and SSIIa,44which are responsible for the distinct pasting and thermal

346

properties. The dramatic difference in starch physicochemical properties may reflect

347

subtle difference in starch biosynthesis in two varieties. Multiple protein complexes in

348

developing rice endosperm have been demonstrated previously.5,22 Crofts et al.

349

revealed the associations of starch biosynthetic isozymes and the formation of

350

enzymatically active protein complexes in japonica rice.22 Chen and Bao found

351

different interaction patterns of SSI−BEI and SSI−BEIIb between the two

352

zymographic forms of SSI from different rice varieties, and proposed two possible

353

protein-protein interaction models based on SSI-1 and SSI-2.5 Similarities as well as

354

differences in the multiple enzyme complex were also revealed when compared to

355

wheat and maize,22 whose complexes were catalyzed as a result of phosphorylation 15



Page 16 of 32

356

events. 12,13,16 Identification of phosphorylated enzymes in rice in this study will pave

357

the way for further validation of the biochemical role of protein phosphorylation in

358

the formation of multiple enzyme complex during starch biosynthesis.

359

In

conclusion,

this

study

systematically

carried

out

a

large

scale

360

phosphoproteome analysis of two indica rice cultivars with different grain quality

361

during seed development. A large number of phosphorylated peptides were identified

362

and analyzed, and differentially and specifically phosphorylated peptides were

363

determined as well. Since the starch physicochemical properties and ECQ are distinct

364

between two indica rice, the phosphopeptides of proteins identified in this study

365

especially those related to starch synthesis may provide insights into the regulatory

366

mechanisms of starch biosynthesis at the phosphorylation level in indica rice.

367 368

ASSOCIATED CONTENT

369

* Supporting Information

370

The Supporting Information is available free of charge on the ACS

371

Publicationswebsite at DOI: 10.1021/

372 373 374 375 376

Tables of the over-represented motifs for phosphoserine and the phosphorylated sequences of other proteins in relation to starch biosynthesis in rice. Figures of the over-represented motifs for phosphoserine and phosphothreonine, and clustering analysis image (PDF).

377

AUTHOR INFORMATION

378

Corresponding Author

379

*Telephone: +86-571-86971932. Fax: +86-571-86971421. E-mail: [email protected].

380

Notes

381

The authors declare no competing financial interests.

382 16


Page 17 of 32


383

ACKNOWLEDGEMENTS

384

This work was financially supported by the National Key R & D Program of China

385

(2016YFD0400104), and the Fundamental Research Funds for the Central

386

Universities at Zhejiang University, Hangzhou, China (Grant No. 2016XZZX001-09).

387 388

ABBREVIATIONS USED

389

AAC, apparent amylose content; AAs, amino acids; AGPase, ADP-glucose

390

pyrophosphorylase; BE, starch branching enzyme; DBE, starch debranching enzyme;

391

DP, differentially phosphorylated; DTT, dithiothreitol; ECQ, eating and cooking

392

quality; FDR, false discovery rate; GBSS, granule-bound starch synthases; GO, gene

393

ontology; GLA4, Guangluai 4; IAA, iodoacetamide; KAAS, KEGG Automatic

394

Annotation Server; KEGG, Kyoto Encyclopedia of Genes and Genomes; PGM,

395

phosphoglucomutase;

396

phosphorylase; SS, starch synthase; UDPase, UDP-glucose pyrophosphorylase;

PTMs,

post-translational

modifications;

SP,

starch

397 398

REFERENCES

399

(1)

Zhang, C.; Chen, S.; Ren, X.; Lu, Y.; Liu, D.; Cai, X.; Li, Q.; Gao, J.; Liu, Q.

400

Molecular structure and physicochemical properties of starches from rice with

401

different amylose contents resulting from modification of OsGBSSI activity. J.

402

Agric. Food Chem.2017, 65, 2222–2232.

403

(2)

Zhu, D.; Zhang, H.; Guo, B.; Xu, K.; Dai, Q.; Wei, C.; Wei, H.; Gao, H.; Hu, Y.;

404

Cui, P.; et al. Effect of Nitrogen management on the structure and

405

physicochemical properties of rice starch. J. Agric. Food Chem. 2016, 64,

17



8019–8025.

406 407

(3)

and cooking qualities of rice. Cereal Foods World 2012, 57, 148–156.

408 409

Bao, J. S. Toward understanding the genetic and molecular bases of the eating

(4)

Zhao, C.; Xie, J.; Li, L.; Cao, C. Comparative Transcriptomic analysis in paddy

410

rice under storage and identification of differentially regulated genes in

411

response to high temperature and humidity. J. Agric. Food Chem. 2017, 65,

412

8145–8153.

413

(5)

Chen, Y.; Bao, J. Underlying mechanisms of zymographic diversity in starch

414

synthase I and pullulanase in rice-developing endosperm. J. Agric. Food

415

Chem.2016, 64, 2030–2037.

416

(6)

Zhang, C.; Zhou, L.; Zhu, Z.; Lu, H.; Zhou, X.; Qian, Y.; Li, Q.; Lu, Y.; Gu, M.;

417

Liu, Q. Characterization of grain quality and starch fine structure of two

418

japonica rice (Oryza Sativa) cultivars with good sensory properties at different

419

temperatures during the filling stage. J. Agric. Food Chem. 2016, 64, 4048–

420

4057.

421

(7)

Nakamura, Y. Towards a better understanding of the metabolic system for

422

amylopectin biosynthesis in plants: rice endosperm as a model tissue. Plant

423

Cell Physiol.2002, 43, 718–725.

424

(8)

2005, 5, 4052–4061.

425 426 427

Reinders, J.; Sickmann, A. State-of-the-art in phosphoproteomics. Proteomics

(9)

Ye, J.; Zhang, Z.; Long, H.; Zhang, Z.; Hong, Y.; Zhang, X.; You, C.; Liang, W.; Ma, H.; Lu, P. Proteomic and phosphoproteomic analyses reveal extensive 18


Page 18 of 32

Page 19 of 32


428

phosphorylation of regulatory proteins in developing rice anthers. Plant J.2015,

429

84, 527–544.

430

(10) Qiu, J.; Hou, Y.; Tong, X.; Wang, Y.; Lin, H.; Liu, Q.; Zhang, W.; Li, Z.;

431

Nallamilli, B. R.; Zhang, J. Quantitative phosphoproteomic analysis of early

432

seed development in rice (Oryza sativa L.). Plant Mol. Biol.2016, 90, 249–265.

433

(11) Han, C.; Yang, P. F.; Sakata, K.; Komatsu, S. Quantitative proteomics reveals

434

the role of protein phosphorylation in rice embryos during early stages of

435

germination. J. Proteome Res.2014, 13, 1766–1782.

436

(12) Tetlow, I. J.; Wait, R.; Lu, Z.; Akkasaeng, R.; Bowsher, C. G.; Esposito, S.;

437

Kosar-Hashemi, B.; Morell, M. K.; Emes, M. J. Protein phosphorylation in

438

amyloplasts regulates starch branching enzyme activity and protein–protein

439

interactions. Plant Cell 2004, 16, 694–708.

440

(13) Tetlow, I. J.; Beisel, K. G.; Cameron, S.; Makhmoudova, A.; Liu, F.; Bresolin,

441

N. S.; Wait, R.; Morell, M. K.; Emes, M. J. Analysis of protein complexes in

442

wheat amyloplasts reveals functional interactions among starch biosynthetic

443

enzymes. Plant Physiol.2008, 146, 1878–1891.

444

(14) Grimaud, F.; Rogniaux, H.; James, M. G.; Myers, A. M.; Planchot, V. Proteome

445

and phosphoproteome analysis of starch granule-associated proteins from

446

normal maize and mutants affected in starch biosynthesis. J. Exp. Bot.2008, 59,

447

3395–3406.

448

(15) Hennen-Bierwagen, T. A.; Lin, Q.; Grimaud, F.; Planchot, V.; Keeling, P. L.;

449

James, M. G.; Myers, A. M. Proteins from multiple metabolic pathways 19



450

associate with starch biosynthetic enzymes in high molecular weight complexes:

451

a model for regulation of carbon allocation in maize amyloplasts. Plant

452

Physiol.2009, 149, 1541–1559.

453

(16) Liu, F.; Makhmoudova, A.; Lee, E. A.; Wait, R.; Emes, M. J.; Tetlow, I. J. The

454

amylose extender mutant of maize conditions novel protein–protein interactions

455

between starch biosynthetic enzymes in amyloplasts. J. Exp. Bot.2009, 60,

456

4423–4440.

457

(17) Makhmoudova, A.; Williams, D.; Brewer, D.; Massey, S.; Patterson, J.; Silva,

458

A.; Vassall, K. A.; Liu, F.; Subedi, S.; Harauz, G.; et al. Identification of

459

multiple phosphorylation sites on maize endosperm starch branching enzyme

460

IIb, a key enzyme in amylopectin biosynthesis. J. Biol. Chem.2014, 289, 9233–

461

9246.

462

(18) Ahmed, Z.; Tetlow, I. J.; Ahmed, R.; Morell, M. K.; Emes, M. J. Protein–

463

protein interactions among enzymes of starch biosynthesis in high-amylose

464

barley genotypes reveal differential roles of heteromeric enzyme complexes in

465

the synthesis of A and B granules. Plant Sci.2015, 233, 95–106.

466

(19) Zhang, Z.; Zhao, H.; Tang, J.; Li, Z.; Li, Z.; Chen, D.; Lin, W. A proteomic

467

study on molecular mechanism of poor grain-filling of rice (Oryza sativa L.)

468

inferior spikelets. PloS One2014, 9, e89140.

469

(20) Bao, J. S.; Shen, S. Q.; Sun, M.; Corke, H. Analysis of genotypic diversity in

470

the starch physicochemical properties of nonwaxy rice: Apparent amylose

471

content, pasting viscosity and gel texture. Starch-Starke2006, 58, 259–267. 20


Page 20 of 32

Page 21 of 32


472

(21) Bao, J.; Sun, M.; Corke, H. Analysis of genotypic diversity in starch thermal

473

and retrogradation properties in nonwaxy rice. Carbohydr. Polym.2007, 67,

474

174–181.

475

(22) Crofts, N.; Abe, N.; Oitome, N. F.; Matsushima, R.; Hayashi, M.; Tetlow, I. J.;

476

Emes, M. J.; Nakamura, Y.; Fujita, N. Amylopectin biosynthetic enzymes from

477

developing rice seed form enzymatically active protein complexes. J. Exp.

478

Bot.2015, 66, 4469–4482.

479

(23) Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micro-purification,

480

enrichment, pre-fractionation and storage of peptides for proteomics using

481

StageTips. Nat. Protoc.2007, 2, 1896–1906.

482

(24) Zhang, M.; Ma, C.-Y.; Lv, D.-W.; Zhen, S.-M.; Li, X.-H.; Yan, Y.-M.

483

Comparative phosphoproteome analysis of the developing grains in bread

484

wheat (Triticum aestivum L.) under well-watered and water-deficit conditions. J.

485

Proteome Res. 2014, 13, 4281–4297.

486

(25) Reiland, S.; Messerli, G.; Baerenfaller, K.; Gerrits, B.; Endler, A.; Grossmann,

487

J.; Gruissem, W.; Baginsky, S. Large-scale Arabidopsis phosphoproteome

488

profiling reveals novel chloroplast kinase substrates and phosphorylation

489

networks. Plant Physiol.2009, 150, 889–903.

490

(26) Meyer, L. J.; Gao, J. J.; Xu, D.; Thelen, J. J. Phosphoproteomic analysis of seed

491

maturation in Arabidopsis, rapeseed, and soybean. Plant Physiol.2012, 159,

492

517–528.

493

(27) Lee, T. Y.; Lin, Z.Q.; Hsieh, S. J.; Bretana, N. A.; Lu, C. T. Exploiting maimal 21



494

dependence decomposition to identify conserved motifs from a group of

495

aligned signal sequences. Bioinformatics. 2011, 27, 1780-1787.

496

(28) Amanchy, R.; Periaswamy, B.; Mathivanan, S.; Reddy, R.; Tattikota, S. G.;

497

Pandey, A. A curated compendium of phosphorylation motifs. Nat.

498

Biotechnol.2007, 25, 285–286.

499

(29) Qiu, J. H.; Hou, Y. X.; Wang, Y. F.; Li, Z. Y.; Zhao, J.; Tong, X. H.; Lin, H. Y.;

500

Wei, X. J.; Ao, H. J.; Zhang, J. A comprehensive proteomic survey of

501

aba-induced protein phosphorylation in rice (Oryza sativa L.). Int. J. Mol.

502

Sci.2017, 18, 60.

503

(30) Bowsher, C. G.; Scrase-Field, E. F.; Esposito, S.; Emes, M. J.; Tetlow, I. J.

504

Characterization of ADP-glucose transport across the cereal endosperm

505

amyloplast envelope. J. Exp. Bot.2007, 58, 1321–1332.

506 507

(31) Jeon, J.-S.; Ryoo, N.; Hahn, T.-R.; Walia, H.; Nakamura, Y. Starch biosynthesis in cereal endosperm. Plant Physiol. Biochem.2010, 48, 383–392.

508

(32) Wang, Z.-Y.; Zheng, F.-Q.; Shen, G.-Z.; Gao, J.-P.; Snustad, D. P.; Li, M.-G.;

509

Zhang, J.-L.; Hong, M.-M. The amylose content in rice endosperm is related to

510

the post-transcriptional regulation of the Waxy gene. Plant J.1995, 7, 613–622.

511

(33) Teng, B.; Zeng, R.; Wang, Y.; Liu, Z.; Zhang, Z.; Zhu, H.; Ding, X.; Li, W.;

512

Zhang, G. Detection of allelic variation at the Wx locus with single-segment

513

substitution lines in rice (Oryza sativa L.). Mol. Breed.2012, 30, 583–595.

514

(34) Chen, G.-X.; Zhou, J.-W.; Liu, Y.-L.; Lu, X.-B.; Han, C.-X.; Zhang, W.-Y.; Xu,

515

Y.-H.; Yan, Y.-M. Biosynthesis and regulation of wheat amylose and 22


Page 22 of 32

Page 23 of 32


516

amylopectin from proteomic and phosphoproteomic characterization of

517

granule-binding proteins.Sci. Rep.2016, 6. 33111.

518 519

(35) Tetlow, I. J.; Emes, M. J. A review of starch-branching enzymes and their role in amylopectin biosynthesis. Iubmb Life 2014, 66, 546–558.

520

(36) Satoh, H.; Nishi, A.; Yamashita, K.; Takemoto, Y.; Tanaka, Y.; Hosaka, Y.;

521

Sakurai, A.; Fujita, N.; Nakamura, Y. Starch-branching enzyme I-deficient

522

mutation specifically affects the structure and properties of starch in rice

523

endosperm. Plant Physiol.2003, 133, 1111–1121.

524

(37) Yan, C.-J.; Tian, Z.-X.; Fang, Y.-W.; Yang, Y.-C.; Li, J.; Zeng, S.-Y.; Gu, S.-L.;

525

Xu, C.-W.; Tang, S.-Z.; Gu, M.-H. Genetic analysis of starch paste viscosity

526

parameters in glutinous rice (Oryza sativa L.). Theor. Appl. Genet.2011, 122,

527

63–76.

528

(38) Kharabian-Masouleh, A.; Waters, D. L.; Reinke, R. F.; Ward, R.; Henry, R. J.

529

SNP in starch biosynthesis genes associated with nutritional and functional

530

properties of rice. Sci. Rep.2012, 2, 557.

531 532

(39) Schupp, N.; Ziegler, P. The relation of starch phosphorylases to starch metabolism in wheat. Plant Cell Physiol.2004, 45, 1471–1484.

533

(40) Dauvillée, D.; Chochois, V.; Steup, M.; Haebel, S.; Eckermann, N.; Ritte, G.;

534

Ral, J.-P.; Colleoni, C.; Hicks, G.; Wattebled, F. Plastidial phosphorylase is

535

required for normal starch synthesis in Chlamydomonas reinhardtii. Plant

536

J.2006, 48, 274–285.

537

(41) Satoh, H.; Shibahara, K.; Tokunaga, T.; Nishi, A.; Tasaki, M.; Hwang, S.-K.; 23



538

Okita, T. W.; Kaneko, N.; Fujita, N.; Yoshida, M. Mutation of the plastidial

539

α-glucan phosphorylase gene in rice affects the synthesis and structure of starch

540

in the endosperm. Plant Cell2008, 20, 1833–1849.

541

(42) Sehnke, P. C.; Chung, H.-J.; Wu, K.; Ferl, R. J. Regulation of starch

542

accumulation by granule-associated plant 14-3-3 proteins. Proc. Natl. Acad.

543

Sci.2001, 98, 765–770.

544

(43) Zhong, M.; Li, S.; Huang, F.; Qiu, J.; Zhang, J.; Sheng, Z.; Tang, S.; Wei, X.;

545

Hu, P. The phosphoproteomic response of rice seedlings to cadmium stress. Int.

546

J. Mol. Sci.2017, 18, 2055.

547

(44) Bao, J. S.; Corke, H.; Sun, M. Microsatellites, single nucleotide polymorphisms

548

and a sequence tagged site in starch-synthesizing genes in relation to starch

549

physicochemical properties in nonwaxy rice (Oryza sativa L.). Theor. Appl.

550

Genet.2006, 113, 1185–1196.

551 552

24


Page 24 of 32

Page 25 of 32


553

Legends for Figures

554

555

Figure1. (A) The number of identified phosphosites, phosphopeptides and

556

phosphoproteins; (B) The counts of phosphosites in serine (S), threonine (T) and

557

tyrosine (Y); (C) The counts of phosphopeptides carrying single (1), double (2) and

558

triple (3) phosphorylation modifications.

559

Figure 2. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment

560

analysis of the differentially phosphoproteins

561 562

Figure3. Gene ontology (GO)-based enrichment analysis of the differentially

563

phosphorylated proteins

564

565

Figure 4.Amino acid sequence alignments of SBEII and SBEI and phosphorylation

566

sites (Ser685 and Ser715) on rice SBEIIb. Ser685 shows high sequence conservation

567

among all plant BEs, whereas Ser715 is found only in rice BEIIb.

568 569

Figure 5. The difference of apparent amylose content (AAC) (A), pasting viscosities

570

(B) and thermal (gelatinization) properties (C) between 9311 and GLA4.

571

25



A

3000

Page 26 of 32

2808 2441

2500

2434

2079 2000 1500

1442

1273

1000 500 0 9311 PhosphoProteins

B

GLA4 PhosphoPeptides

PhosphoSites

3000 2627 2500

2289

2000 1500 1000 500

172

149

9

3

0 S

T 9311

Y

GLA4

C 2500 2097 2000

1750

1500 1000 500

303

298

34

31 0 1

2 9311

GLA4

Figure 1

26


≥3

Page 27 of 32


Figure 2.

27



Figure 3

28


Page 28 of 32

Page 29 of 32


Figure 4.

29



30

A AAC%

25 20 15 10 5 0 9311

GLA4

B

C

Figure 5

30


Page 30 of 32

Page 31 of 32


Table 1.The phosphorylated sequences of the starch biosynthesis related enzymes in indica rice Sequence

Annotation

Abbreviation

Phosphosite

9311 intensity

GLA4 intensity

CVFTS(ph)DADR CVFTSDADRDT(ph)PHLR TPFFTS(ph)PR YGS(ph)GGDAAR EHINS(ph)DEETFDTYNR EMYTGMSDLQPAS(ph)PTINR FVFRS(ph)SDEDCK QWS(ph)LVDTDHLR AMQS(ph)LEEK FIPGNNNS(ph)YDK LSTAS(ph)DIEQR VPS(ph)SVDVASLVK FADDEDLQS(ph)EWR ICHVLYPGDES(ph)PEGK S(ph)LEPSVVVEEK

ADP-glucose pyrophosphorylase large subunit ADP-glucose pyrophosphorylase large subunit ADP-glucose pyrophosphorylase large subunit Soluble starch synthase 2-3 Putative starch synthase DULL1 Starch branching enzyme 1 Starch branching enzyme 1 Starch branching enzyme 1 Starch branching enzyme 3 Starch branching enzyme 3 Pullulanase Pullulanase Alpha-1,4 glucan phosphorylase Alpha-1,4 glucan phosphorylase Alpha-1,4 glucan phosphorylase

AGPase AGPase AGPase SSIIa

S5 T11 S6

SSIIIa

S5

BEI

S13

BEI

S5 or S6

8.65E+06 7.54E+08 2.26E+07 2.81E+06 2.68E+07 0.00E+00 0.00E+00 0.00E+00 1.61E+06 0.00E+00 0.00E+00 3.19E+06 2.54E+07 3.82E+06 0.00E+00

1.10E+07 3.13E+09 3.84E+07 9.45E+06* 1.59E+07 9.09E+05* 2.04E+07* 1.75E+06* 2.14E+06 6.78E+06* 1.79E+06* 0.00E+00* 2.48E+07 3.39E+06 2.34E+06*

S3

BEI

S3

BEIIb BEIIb

S4

PUL

S5

PUL

S3 orS4

Pho1 Pho1 Pho1

S9 S11 S1

*indicates significant difference between 9311 and GLA4 (P

Comparative Phosphoproteomic Analysis of the Developing Seeds in

Recommend Documents