Integrated Proteome Analysis of the Wheat Embryo ... - ACS Publications

Subscriber access provided by UPSTATE Medical University Health Sciences Library

Article

Integrated proteome analysis of the wheat embryo and endosperm reveals central metabolic changes involved in water deficit response during the grain development Aiqin Gu, Pengchao Hao, Dongwen Lv, Shoumin Zhen, Yanwei Bian, Chaoying Ma, Yanhao Xu, Wenying Zhang, and Yue-Ming Yan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00575 • Publication Date (Web): 02 Sep 2015 Downloaded from http://pubs.acs.org on September 8, 2015

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 free 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 accessible to all readers and 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 43

Journal of Agricultural and Food Chemistry

Integrated proteome analysis of the wheat embryo and endosperm reveals central metabolic changes involved in water deficit response during the grain development Aiqin Gu1, Pengchao Hao1, Dongwen Lv1, Shoumin Zhen1, Yanwei Bian1, Chaoying Ma1, Yanhao Xu2, Wenying Zhang2, Yueming Yan1, 2* 1

College of Life Sciences, Capital Normal University, Beijing 100048, China

2

Hubei Collaborative Innovation Center for Grain Industry; Yangtze University,

434025 Jingzhou, China AiqinGu: [email protected]; PengchaoHao: [email protected]; DongwenLv: [email protected]; Shoumin Zhen: [email protected]; YanweiBian: [email protected]; Chaoying Ma: [email protected]; Yanhao Xu: [email protected] ; WY Zhang: [email protected] *Corresponding author: Prof. Dr. Yueming Yan, College of Life Science, Capital Normal University, Xi San Huan Bei Lu No. 105, 100048 Beijing, P. R. of China. Tel. and Fax: 0086-10-68902777 E-mail: [email protected] 1 ACS Paragon Plus Environment


1

ABSTRACT

2

Embryo and endosperm of wheat have different physiological functions and big

3

differences in protein level. In this study, we performed the first integrated proteome

4

analysis of wheat embryo and endosperm in response to water deficit during grain

5

development. In total, 155 and 130 differentially expressed protein (DEP) spots in

6

embryo and endosperm were identified respectively by nonlinear two-dimensional

7

electrophoresis (2-DE) and tandem mass spectrometry. These DEPs in embryo were

8

mainly involved in stress/defense responses such as heat shock related proteins (HSP)

9

and peroxidase, whereas those in endosperm were mainly related to starch and

10

storage protein synthesis such as alpha-amylase inhibitor and globulin-1 S allele.

11

Particularly, some storage proteins such as avenin-like proteins and high molecular

12

weight (HMW) glutenin subunit Dy12 displayed higher expression levels in the

13

mature endosperm under water deficit, which might contribute to the improvement

14

of breadmaking quality.

15

KEYWORDS: wheat, embryo, endosperm, water deficit, proteome

16 17 18 19 20 21

2 ACS Paragon Plus Environment

Page 2 of 43

Page 3 of 43


22

INTRODUCTION

23

Wheat is one of the three most important grain crops, and is cultivated worldwide.

24

Wheat flour can be used to make various specific products, such as noodles, bread,

25

biscuits, and pasta, and serves as one of the most important food for humans and

26

livestock. Wheat protein can provide essential amino acids required by humans,

27

although protein content is relative low (usually 8–15%) 1. Along with the constantly

28

increasing world population, the global demand for wheat has also risen. 2 However,

29

the growth and development of wheat are readily influenced by various biotic and

30

abiotic stresses, such as drought, flooding, salt, and high and low temperatures.

31

Water stress, as one of the most important abiotic stresses, has a great impact on

32

wheat grain yield, especially at the flowering and early grain-filling stages. 3

33

Under drought stress, plants often suffer oxidative damage due to disruption of

34

the balance between the production and elimination of reactive oxygen species

35

(ROS). The accumulation of ROS can damage lipids, proteins, and DNA, finally

36

leading to cell death.

37

evolved efficient signaling networks to eliminate or reduce this damage. Proteins

38

related to antioxidant and stress/defense responses, such as superoxide dismutase,

39

peroxidase, and glutathione play important roles in the process. 5 Along with the

40

great progress in plant genomics, bioinformatics, and proteomics, considerable work

41

has been done on the plant drought response at different levels, such as the genome, 6

42

transcriptome,

43

improvements in wheat genome sequencing 10-12 are expected to facilitate wheat

7

4

To survive under these unfriendly conditions, cells have

and proteome

8-9

under drought stress. Notably, continuous



44

Page 4 of 43

proteome studies.

45

The embryo and endosperm are the most important organs of wheat grains, and

46

both develop from double fertilization. The embryo comprises embryonic axis and a

47

single cotyledon, and plays important roles in organ regeneration during grain

48

germination. Endosperm mainly accumulates storage starch and proteins. The

49

biosynthesis of starch majors in determinant of wheat yield. 13 Under drought stress,

50

the size, yield, and breadmaking quality of wheat grains are significantly affected.

51

Although a number of proteomic studies on wheat grain development and adverse

52

stress have been carried out, most of these investigations have only focused on a

53

single tissue or organ, such as the embryo 14 or on whole grains 8-9, 15-16. Several

54

proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme

55

metabolism were identified in embryo

56

alpha-amylase inhibitors identified upregulation in the grain of drought resistant

57

varieties 9, 16. However, comparative proteome analysis of the wheat embryo and

58

endosperm in response to drought stress has not been reported. It is necessary to

59

further investigate how these two organs respond to water stress differently.

14

and ascorbate peroxidases

8, 15

, LEA,

60

In this study, we performed the first integrated comparative proteome analysis of

61

the wheat embryo and endosperm under field water deficit during grain development.

62

We identified considerable differentially expressed proteins to reveal the central

63

metabolic changes in the embryo and endosperm development under water deficit,

64

which provide new insights into the biochemical mechanism of wheat grain

65

development in response to drought stress. 4 ACS Paragon Plus Environment

Page 5 of 43


66

MATERIALS AND METHODS

67

Wheat Materials, Water Deficit Treatment, and Sampling. The elite

68

Chinese winter wheat cultivar Jing 411 (Triticum aestivum L, 2n = 6x = 42,

69

AABBDD) was used as material in this study, which has been widely cultivated in

70

Beijing and north wheat production area of China since 1990s. It has many desirable

71

features such as high yield and good yield stability, strong cold hardiness, wide

72

adaptability and commendable resistance

73

experimental station of Agricultural University of China during 2013-2014 growing

74

season. The station is located in Hebei province (latitude 37º41´02˝N, longitude

75

116º37´23˝E) with average annual sunshine of 2690 h, highest temperature of 32°C

76

and about 125 mm rainfall during the wheat growing season. Plants were grown at

77

three biological replicates and each plot was 50 m2. Water stress treatment was

78

conducted as follows: in the control group as local field cultivation management,

79

wheat was watered approximately 750 m3/ha at sowing, jointing and flowering

80

stages, respectively. While in the stress group, no watering was used after sowing.

81

The soil water content ranged from 13.3% to 9.18% in water-deficit conditions and

82

from 18.1% to 13.3% in control conditions at the jointing stage, flowering stage, and

83

mature stage. This soil water content was about 20 − 30% lower in stressed than in

84

control groups. The treatment belonged to medium-to-severe drought stress

85

according to Guttieri 18. Wheat spikes were immediately marked once flowering, and

86

collected at 15, 21, 26, 30, and 45 days after flowering (DAF), respectively. The

87

middle 4-6 grains of each ear were used as experimental materials. Husks were first

17

. The cultivar was grown at Wuqiao



88

removed from seeds, then separated embryo and aleurone layer from the remainder

89

of the seed at low temperatures, respectively 13. Embryo and endosperm samples

90

were stored at -80°C prior to use.

91

Measurement of Physiological Indicators, Agronomic Traits, and

92

Quality Parameters. Flag leaves of five stages (12, 15, 18, 21, and 26 DAF) were

93

sampled. Five biology replications of each period were measured. Physiological

94

indicator testing consisted of the contents of leaf relative water (RWC),

95

malondialdehyde (MDA), soluble sugar, free proline, and chlorophyll. The

96

measurement of RWC was followed by the method of Smart 19, MDA and soluble

97

sugar contents were based on the procedure of Zhao et al. 20, free proline content was

98

referred to the progress of Bates et al. 21, and chlorophyll content was according to

99

Arnon 22 with minor modifications.

100

After grain matured, the main agronomic traits were measured and the plant

101

samples were collected. The grain yield was measured according to three plot

102

replications, and theoretical and practical yields were obtained. Different grain

103

quality parameters were tested, including total protein content (14% moisture basis),

104

falling number, gluten content, Farinograph, Extensograph, Mixograph, loaf volume,

105

appearance score and C-cell properties. Flour protein content was determined

106

according to American Association of Cereal Chemists Approved Methods (AACC)

107

39-10A with a near-infrared (NIR) analyzer (Instalab610, Dickey-john Co. Ltd,

108

USA). Falling number (Perten, Sweden) were determined with the methods of

109

AACC 56-81B. Mixograph index (Nationalmfg, USA) including water absorption 6 ACS Paragon Plus Environment

Page 6 of 43

Page 7 of 43


110

rate, peak time, and time×width, was determined according to the method of AACC

111

54-40A. Gluten content was measured according to the method of Czuchajowska 23.

112

Farinograph and extensograph (Brabender, German) were followed by the methods

113

of AACC 54-21 and 54-10. And bread-making was followed with the method of

114

AACC 10-10B. The internal structure of bread was tested and evaluated by the

115

means of C-CELL (UK CCFRA Company). Data analysis was performed using

116

independent Student’s T-test with SPSS statistical software (version 17.0).

117

Protein Extraction and Nonlinear 2-DE. Protein was extracted from embryo

118

and endosperm according to the process of Guo et al. 24 with minor modifications. A

119

total of 500 mg samples were quickly ground into a fine power in liquid nitrogen.

120

Protein extractions were dissolved with lysis buffer (7 M urea, 2 M thiourea, 4%

121

CHAPS) for about 5 h. Protein concentrations were evaluated with a 2-D Quant Kit

122

(Amersham Bioscience, USA) using bovine serum albumin (BSA, 2 mg/ml) as the

123

standard.

124

Nonlinear two-dimensional electrophoresis (2-DE) isolation was performed

125

according to the steps of Jiang et al, which can significantly improve the resolution

126

of protein separation compared to traditional linear 2-DE. 8 In total, 500 µg protein

127

samples were mixed with 1 mg DTT, 0.5% IPG buffer (GE Healthcare/Amersham

128

Biosciences), 1 µl 1% bromophenol blue solution and moderate rehydration buffer (7

129

M urea, 2 M thiourea, 2% w/v CHAPS), and 350 µl mixed solution was loaded onto

130

an ImmobilineDryStrip (pH 3-10, 18 cm, nonlinear strip, GE Healthcare, USA) for

131

isoelectric focusing (IEF). After IEF, the strips were treaded with equilibration buffer 7 ACS Paragon Plus Environment


132

for the second SDS-PAGE. At least two technical replicates for each sample were

133

performed to obtain statistically reliable results. Protein spot images were visualized

134

by Coomassie Brilliant blue staining (R-250/G-250 = 4:1; Sigma, USA) and scanned

135

at 600 dpi with a UMAX Power Look 2100XL scanner (Maxium Tech Inc., Taiwan,

136

China). The image analysis was carried out with ImageMaster™ 2D Platinum

137

Software Version 7.0 (Amersham Bioscience). The abundance of protein spots from

138

biological and technical replicates was estimated by percentage volumes (Vol. %).

139

Data analysis was conducted using independent Student’s T-test with SPSS statistical

140

software (version 17.0). Only those with significant and reproducible changes (at

141

least 2-fold increase/decrease and p-value < 0.05) were considered as differentially

142

expressed protein (DEP) spots.

143

MALDI-TOF/TOF-MS. DEP spots were manually excised from the gels,

144

digested with trypsin and identified by matrix-assisted laser desorption/ionization

145

time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). After

146

bleaching, enzymatic and drying, the peptides were analyzed by using an ABI 4800

147

Proteomic Analyzer MALDI-TOF/TOF-MS (Applied Biosystems/MDS Sciex, USA).

148

The parameters were set as follows: laser intensity 4,500 and mass range 900-3,500

149

Da. The MS/MS spectra was searched against Viridiplantae (green plant) sequences

150

in the non-redundant National Center for Biotechnology Information (NCBI)

151

database (Nov 28, 2012; including 1,154,529 protein sequences) by using software

152

MASCOT (version: 2.1.0) with the following parameter settings: trypsin cleavage,

153

one missed cleavages allowed, peptide mass tolerance set to ± 0.3 Da, fragment 8 ACS Paragon Plus Environment

Page 8 of 43

Page 9 of 43


154

tolerance set to ± 0.4 Da. Only the proteins with both Protein Score C.I. % and Total

155

Ion Score C.I. % above 95% were identified as credible results.

156

Multivariate and Cluster Analysis. The multivariate method was carried out

157

by Principal Component Analysis (PCA) in the R language with the average relative

158

abundance of all the identified DEP spots 25-26. PCA is a way of identifying patterns

159

in data, and expressing the data in such a way as to emphasis their similarities and

160

differences. In our study, identified DEP spots of control and stressed embryo and

161

endosperm during the five development stages were analyzed by PCA. Cluster

162

analysis of the differentially abundant proteins data was performed by using cluster

163

software (version 3.0). Euclidean distances and Ward’s criteria were used for the

164

analysis. Cluster results were displayed by Java Tree view software.

165

Bioinformatics and Statistical Analysis. The possible functions of

166

identified proteins were classified according to the information from uniprot

167

database

168

(www.geneontology.org). The sequences of all identified proteins were used for

169

BLAST analysis with NCBI cluster of eukaryotic Orthologous Groups (KOG)

170

database to obtain the KOG numbers of those proteins. Information about the

171

protein-protein interaction was used these KOG numbers with the Search Tool for

172

Retrieval of Interacting Genes/Proteins (STRING) database (version 9.1.

173

www.string-db.org) to construct an interaction network graph 27 with a confidence

174

score of potential substrates at least 0.9. The network was displayed using the

175

cytoscape software (version 3.0). 28 When an unknown named protein was identified,

(www.uniprot.org)

and

Gene

Ontology


(GO)

database


176

homologous protein of known function with the highest query cover (above 90%)

177

and lowest E value were blast in NCBI database. Subcellular localizations of

178

identified proteins were forecasted by UniprotKB (http://www.uniprot.org/) database,

179

WoLF PSORT (http://wolfpsort.org/) and Predotar (http://urgi.versailles.inra.fr/

180

predotar/predotar.html) program. 29-30 The most likely location of a given protein was

181

determined by similar results from at least two programs.

182

RESULTS

183

Changes in Leaf physiological Indexes, Agronomic Traits and

184

Breadmaking Quality Properties in Response to Water Deficit. Results for

185

leaf physiological indexes are shown in Figure1. RWC displayed a decreasing trend

186

during the five development periods and a significantly lower content in water stress

187

samples. In contrast, the contents of free proline, MDA, and soluble sugar were all

188

significantly higher in the water stress samples. MDA, which can react with proteins

189

and nucleic acids and inhibit protein synthesis, is the final decomposition product of

190

lipid peroxidation and reflects the degree of cell damage. While free proline and

191

soluble sugars, help to protect the structure of proteins and adjust osmotic balance

192

and protect the proteins from stress damages 31. Chlorophyll content increased in the

193

first three stages, and then decreased during the subsequent stages and displayed

194

significant changes under water stress. These physiological index changes might

195

reflect the general wheat response to water deficit during grain development.

196

The results for agronomic traits, yield and quality performance showed a

197

significant difference between control and water stress samples (Table 1 and S1). 10 ACS Paragon Plus Environment

Page 10 of 43

Page 11 of 43


198

Under water stress, the seeds had significant lower spikelet numbers, spike grain

199

numbers and grain weights, and had an approximate 12% reduction in yield

200

compared to the control (Table S1). Water stress shortened the time of wheat grain

201

filling, which explains the lower weight and yield under water deficit. Grain quality

202

analysis showed that total protein and gluten contents were higher in control samples.

203

However, some breadmaking related parameters of Mixograph, Extensograph, loaf

204

volume, loaf appearance score and C-cell testing showed significant increase under

205

water-stressed treatment comparing with the control (Table 1).

206

Proteome Characterization of the Embryo and Endosperm in

207

Response to Water Deficit during Grain Development. Protein spots for the

208

embryo and endosperm were detected by nonlinear 2-DE during grain development

209

under water deficit (Figure 2A–B). In total, 876 ± 8 and 752 ± 5 spots on all gels of

210

embryo and endosperm were detected, respectively. Of these, 155 DEP spots in the

211

embryo corresponded to 112 unique proteins, and 130 DEP spots in endosperm

212

corresponded

213

MALDI-TOF/TOF-MS (Figure 2A–B, Figure 3, Table S2–3). Among these DEP

214

spots, 60 representing 47 unique DEPs were found in both embryo and endosperm

215

samples, while 95 representing 76 unique DEPs and 70 representing 60 unique DEPs

216

were identified particularly in embryo and endosperm, respectively (Figure 3).

217

Detailed peptide information for all of the identified proteins is shown in Table S4.

218

To obtain more information about the unknown/predicted proteins, their sequences

219

were used to search for homologues by BLASTP. Predicted proteins with sequence

to

96

unique

proteins,

were

successfully


identified

by


Page 12 of 43

220

similarity > 90% were considered homologues. 14 BLASTP revealed homologues for

221

20 spots (Table S5).

222

PCA showed major differences between the two organs during the grain

223

development stages under drought stress. DEP spots of different stages in endosperm

224

were located in the same plot, and were in a different plot of those in embryo (Figure

225

4A-B). These results indicated that the difference between organs was dominant over

226

differences during grain development under water stress. Samples from five different

227

development stages were grouped into different plots in these two organs, but

228

differences between control and water deficit samples were modest (Figure 4C-D).

229

Our moderate treatment might explain this phenomenon. Cluster results showed a

230

differential protein expression trends in the embryo and endosperm during grain

231

development under water deficit (Figure S1). Protein expression profiles at 15-21

232

DAF and 30-45 DAF generally showed similar patterns of accumulation in control

233

embryo, respectively, and proteins expression profiles at 26 DAF closed to those at

234

30-45 DAF (Figure S1A), whereas under water deficit, protein accumulation at 21

235

DAF similarly to those at 26 DAF and differently to those at 15 DAF (Figure S1B),

236

which might indicate proteins in embryo in response to water deficit mainly from 21

237

DAF. By comparing Figure S1C and Figure S1D, proteins in endosperm responded

238

to water deficit mainly from 26 DAF.

239

According to the protein function, these DEPs could be classified into nine

240

categories in embryo development: stress/defense response, energy metabolism,

241

storage

proteins,

nitrogen

metabolism,

protein

metabolism/folding,


starch

Page 13 of 43


242

metabolism, transport, lipid metabolism, and other important functions (Table S2).

243

The DEP spots identified in endosperm development under water deficit showed

244

similarly functional classifications (Table S3), except for the absence of lipid

245

metabolism-related proteins. Prediction of protein-protein interactions was displayed

246

in Figure 5. Proteins identified in both embryo and endosperm samples were located

247

in the middle. Proteins identified specifically in the embryo and endosperm were

248

located

249

dehydrogenase (GAPDH, KOG0657), 70 kDa heat shock-related protein (HSP70,

250

KOG0101), and ubiquitin (KOG0001) had the most connections with other proteins,

251

and were located in the center, suggesting their important roles in the embryo and/or

252

endosperm development in response to water deficit. Proteins with different

253

functions were closely connected with each other and formed a big network to play

254

center roles in response to water deficit. Subcellular localizations of the identified

255

proteins were predicted. They were mainly located in the cytoplasm, mitochondria,

256

vacuole, and nucleus (Tables S2–3).

to

the

left

and

right,

respectively.

Glyceraldehyde-3-phosphate

257

Comparative Proteome Analysis of the Embryo and Endosperm in

258

Grain Development under Water Deficit. Embryo was identified to have more

259

stress/defense response related DEP spots and less starch metabolism and storage

260

related proteins than endosperm in this study. About 40 and 34 DEP spots involving

261

in stress/defense response were identified in embryo and endosperm, respectively

262

(Table S2-3). Whereas 10 and 19 DEP spots taking part in starch metabolism were

263

identified respectively in these two organs. Aldose reductase-related protein (spot 13 ACS Paragon Plus Environment


264

30), cold regulated protein (spot 54), peroxidase 1 (spot 151), and stromal 70 kDa

265

heat shock-related protein (spot 164) involving in stress/defense response were only

266

identified in embryo, while beta-amylase (spot 45) with important roles in starch

267

metabolism, storage proteins such as putative avenin-like α precursor (spot 169) and

268

HMW gultenin subunit Dy 12 (spot 115) were only identified in endosperm (Table

269

2).

270

DISCUSSION

271

Stress/Defense-Related Proteins. Cells suffer harmful damage during grain

272

development due to various biotic and abiotic stresses, especially under water deficit.

273

Large amounts of ROS accumulate in cells under stress, leading to lipid peroxidation,

274

protein (enzyme) instability, and loss of osmotic balance.

275

activated in cells in response to stress. Many proteins largely accumulated in cells

276

under stress, such as peroxidase, HSP, and LEA. 1-Cys peroxiredoxin PER1 (spot 3)

277

is involved in ROS scavenging, and has been reported to be a dormancy-related

278

protein expressed in the embryo and aleurone layer of barley. 32 This enzyme may

279

help maintain dormancy and prevent germination under unfavorable conditions. 33 It

280

displayed higher expression from 21 DAF in both embryo and endosperm under

281

water deficit, and reached a 4.56 fold accumulation level in embryo at 26 DAF

282

comparing with 15 DAF, much more than that increased in endosperm (Table S 2-3).

283

The higher accumulation improved the ability to survive a harsh environment during

284

grain development. Four peroxidase proteins (spots 150-153) that also take part in

285

ROS scavenging were specifically up-accumulated in the embryo during grain 14 ACS Paragon Plus Environment

4

Defense signals are

Page 14 of 43

Page 15 of 43


286

development in response to defense stress (Table 2, Table S2). 34 Stromal 70 kDa

287

heat shock-related protein (HSP 70), a chaperone, binds extended peptide segments

288

of polypeptides with a net hydrophobic character during translation and membrane

289

translocation, and may interact with newly imported proteins to assist in their

290

maturation by protein folding and reassembly.

291

overexpression of HSP genes in wheat in response to environmental stress. 36 Three

292

HSP DEP spots (spots 164, 191 and 211) were specifically identified in the embryo

293

(Tables S2). Spots 191 and 211 (corresponding to KOG 0101), and spot 164

294

(corresponding to KOG0102) were predicated interacting many other proteins and

295

played a key role in embryo development 14 (Figure 5). Another two proteins were

296

obtained, 16.8 kDa heat-shock protein (spot 133) and predicted 17.9 kDa class I heat

297

shock protein-like (spot 216), the homologous protein of hypothetical protein

298

TRIUR3_03549 and unnamed protein product, respectively (Table S5). The

299

abundance of small Hsps in plants functioned in binding and stabilizing non-native

300

proteins, and might play a role in plant-acquired tolerance to drought and salt. 37

301

These phenomena enhanced the embryo’s ability of drought tolerance and might

302

show the main function in stress/defense response in embryo under water deficit.

303

Alpha-amylase inhibitors (spots 1, 32, 33, 52, 66, 67, 118, 147, and 181) inhibit the

304

degradation of starch and increase starch accumulation in cells, which are related to

305

adjustment of osmotic balance and protecting the cells from the damage by insects. 38

306

It has been reported that the wheat endosperm proteome contained abundant levels

307

(about 11% of total proteins) of alpha-amylase inhibitors and alpha-amylase/trypsin

35

Related study has reported the



308

inhibitors to protect the starch reserves from degradation 39. Their expressions were

309

upregulated in drough tolerance genotypes, but were downregulated in sensitive

310

genotypes 16. Consistent with our results, nine related DEP spots were identified in

311

endosperm, seven of them showed higher expression under water deficit (Table S3).

312

Especially, spot 147 had reached 5.15, 8.15 and 7.77 fold accumulation at 21, 26, 45

313

DAF (comparing with 15 DAF), respectively. These results reveal the main function

314

of adjusting starch accumulation in endosperm under drought.

315

Energy Metabolism-Related Proteins. Proteins participating in ATP

316

synthesis, glycolysis, and the tricarboxylic acid (TCA) cycle provide plenty of

317

energy and molecular materials for grain development under water deficit. In this

318

study, many DEPs related to energy metabolism were identified under drought.

319

GAPDH (spots 57 and 110-114), a key enzyme of glycolysis, catalyzes the

320

conversion of D-glyceraldehyde 3-phosphate (G3P) to 3-phospho-D-glyceroyl

321

phosphate. In the present study, this enzyme showed higher accumulation in early

322

embryo and late endosperm development and up-regulated at 30 DAF in embryo

323

under water deficit (spot 111, Table 2). This favored to embryo cells regulating stress

324

defense mechanism. Protein-protein interaction analysis showed that many other

325

proteins related to energy metabolism and stress/defense responses were closely

326

connected with GAPDH (Figure 5) and indicated its central role in metabolism.

327

Other energy metabolism related proteins such as ATP synthase-related proteins,

328

dihydrolipoyl dehydrogenase 1, Bp2A protein, triosephosphate-isomerase and so on

329

were identified in response to water deficit in embryo and/or endosperm during the 16 ACS Paragon Plus Environment

Page 16 of 43

Page 17 of 43


330

grain development. All of these proteins closely interacted with each other and

331

provided enough carbohydrates and energy for embryo and endosperm development

332

under water deficit.

333

Starch Metabolism-Related Proteins. The embryo has been reported to be a

334

powerful sink for nutrients, and also accumulates moderate starch for germination,

335

but many starches were degraded into carbohydrates to improve the desiccation

336

tolerance during the development. 40-41 Storage starch is mainly accumulated in the

337

endosperm (approximately 70–80% dry weight), and largely determines wheat yield

338

during the development. Seven and ten related proteins were identified in embryo

339

and endosperm in this study, respectively, and most of them displayed a lower

340

accumulation under drought compared with the control, leading to a yield reduction

341

(Table S1). Sucrose synthase (SUS, spots 192–197) catalyzes the first step in the

342

conversion of sucrose to starch, which provides UDP-glucose and fructose for

343

various metabolic pathways. ADP-glucose pyrophosphorylase large subunit

344

(AGPase, spot 21) is a major rate-limiting enzyme in grain starch biosynthesis. It

345

initiates starch biosynthesis in plants, and catalyzes the formation of ADP-glucose

346

from Glc-1-P and ATP. SUS and AGPase are considered to be crucial for

347

development, carbon partitioning, and synthesis of storage starch during grain

348

development, and are active when starch accumulates in storage tissues. 42-43 In the

349

present study, these two proteins mainly displayed down-accumulated in late embryo

350

and endosperm development under water deficit. Fructokinase-1-like, a homolog of

351

the hypothetical protein F775_30384 (spot 130), which may help to maintain the 17 ACS Paragon Plus Environment


Page 18 of 43

352

flux of carbon towards starch formation 44, showed a similar decreasing expression

353

under

354

4)-alpha-D-glucosidic linkages in polysaccharides and removes successive maltose

355

units from the non-reducing ends of the chains, were specifically identified in the

356

endosperm and down-regulated in the late development under water stress (Table 2).

357

So the endosperm expressed alpha-amylase inhibitors to ease the reduced starch

358

under water deficit.

drought.

Beta-amylase

(spot

45),

which

hydrolyzes

(1-

>

359

Storage proteins. Wheat storage proteins mainly present in the endosperm,

360

especially glutenins and gliadins, have important effects on nutritional quality for

361

humans and livestock and on functional properties in food processing 45. Under water

362

deficit, many of them displayed a similar expression pattern and had fewer changes

363

than the control (Table S3). Some globulins displayed downregulation during the

364

grain development, similarly to the previous results of Laino 15. However, some

365

storage proteins showed higher expression under water deficit compared with the

366

control, such as HMW glutenin subunit Dy12 (spot 115), putative avenin-like α

367

precursor (spot 169) (Table 2), and the hypothetical protein F775_27291 (spot 124),

368

a homolog of avenin-3 (Table S5). One gliadin protein spot (spot 84) and four

369

protein spots, globulin-1 S allele (spots 87-90) also displayed more than 3 fold

370

upregulation during the grain development under water deficit (Table S3). Glutenins

371

and gliadins are two prolamine groups of gluten constituents, contain many cysteine

372

residues which involved in intramolecular and intermolecular linkages and

373

contribute to the viscoelastic properties of gluten which determine dough strength. 46 18 ACS Paragon Plus Environment

Page 19 of 43


374

Avenin-like proteins are also rich in cysteine, may contribute to wheat dough

375

viscoelastic properties via inter-chain disulfide bonds. 47 Globulins are crosslinked

376

by both disulfide and non-disulfide crosslinks to affect dough and crumb properties.

377

48

378

breadmaking quality. Therefore, it is suggested that these proteins have positive

379

significance for the improvement of breadmaking quality. As shown in Table 1,

380

although the amount of gluten decreased, bread quality improved under water deficit.

381

Overview of Central Metabolic Changes during Wheat Embryo and

382

Endosperm Development in Response to Water Deficit. Here, we propose a

383

putative proteomic metabolic pathway during wheat embryo and endosperm

384

development in response to water deficit, which provides an overview of the main

385

DEP differences between the embryo and endosperm (Figure 6). The embryo mainly

386

synthesized proteins related to stress/defense responses during grain development,

387

such as peroxidase and HSP while the endosperm mainly accumulated storage starch

388

and storage proteins. Many energy metabolism related enzymes were both identified

389

in these two organs. Under water deficit, most proteins related to energy metabolism

390

and stress/defense responses were upregulated in the embryo. Proteins related to

391

starch were downregulated in endosperm, which might explain the lower grain

392

weights and reduced yields. But some storage proteins, such as HMW glutenin,

393

globulins, and avenin-like proteins, showed upregulated expression under water

394

deficit, which might benefit for bread making quality.

395

Funding

As we all know, there is a great relationship between dough strength and



396

This research was financially supported by grants from the National Natural Science

397

Foundation of China (31271703, 31471485), Natural Science Foundation of Beijing

398

City and the Key Developmental Project of Science and Technology, Beijing

399

Municipal Commission of Education (KZ201410028031), the National Key Project

400

for Transgenic Crops in China (2014ZX08009-003), and International Science

401

&Technology Cooperation Program of China (2013DFG30530).

402

Notes

403

The authors declare no competing financial interest.

404

SUPPORTING INFORMATION

405

Hierarchical clustering of identified proteins in both control and stressed embryo and

406

endosperm during the grain development (Figure S1), some agronomic and yield traits

407

of wheat cultivar Jing 411 under control and water deficit treatment (Table S1),

408

identified differentially expression proteins in embryo (Table S2) and endosperm

409

(Table S3) in water deficit response during the grain development, peptide

410

information of all the identified proteins (Table S4) and homologous proteins of some

411

unknown proteins (Table S5) all support this study. These supporting materials are

412

available free of charge via the Internet at http://pubs.acs.org.

413

ABBREVIATIONS USED

414

AGPase, ADP-glucose pyrophosphorylase large subunit; DAF, days after flowering;

415

DEP, differentially expressed protein; 2-DE, two-dimensional electrophoresis;

416

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMW, high molecular weight;

417

HSP 70, stromal 70 kDa heat shock-related protein; IEF, isoelectric focusing; KOG, 20 ACS Paragon Plus Environment

Page 20 of 43

Page 21 of 43


418

eukaryotic Orthologous Groups; LEA, late embryogenesis abundant proteins;

419

MALDI-TOF/TOF-MS, matrix-assisted laser desorption/ionization time-of-flight/

420

time-of-flight mass spectrometry; MDA, malondialdehyde; NCBI, National Center

421

for Biotechnology Information; PCA, principal component analysis; ROS, reactive

422

oxygen species; RWC, leaf relative water; SUS, sucrose synthase; TCA,

423

tricarboxylic acid cycle.

424

REFERENCES

425

(1) Shewry, P. R. Wheat. J. Exp. Bot. 2009, 60 (6), 1537 − 1553.

426

(2) Tester, M.; Langridge, P. Breeding technologies to increase crop production in a

427

changing world. Science 2010, 327 (5967), 818 – 822.

428

(3) Kobata, T.; Palta, J. A.; Turner, N. C. Rate of development of postanthesis water

429

deficits and grain filling of spring wheat. Crop Sci. 1992, 32 (5), 1238 – 1242.

430

(4) Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends in Plant

431

Sci. 2002, 7 (9), 405 – 410.

432

(5) Mittler, R. Abiotic stress, the field environment and stress combination. Trends

433

in Plant Sci. 2006, 11 (1), 15 – 19.

434

(6) Habash, D. Z.; Kehel, Z.; Nachit, M.

435

durum wheat ready for climate change with a focus on drought. J. Exp. Bot. 2009, 60

436

(10), 2805 – 2815.

437

(7) Aprile, A.; Mastrangelo, A. M.; De Leonardis, A. M.; Galiba, G.; Roncaglia, E.;

438

Ferrari, F.; De Bellis, L.; Turchi, L.; Giuliano, G.; Cattivelli, L. Transcriptional

439

profiling in response to terminal drought stress reveals differential responses along

Genomic

approaches


for

designing


440

the wheat genome. BMC Genomics 2009, 10, 279.

441

(8) Ge, P.; Ma, C.; Wang, S.; Gao, L.; Li, X.; Guo, G.; Ma, W.; Yan, Y. Comparative

442

proteomic analysis of grain development in two spring wheat varieties under drought

443

stress. Anal. Bioanal. Chem. 2012, 402 (3), 1297 – 1313.

444

(9) Jiang, S. S.; Liang, X. N.; Li, X.; Wang, S. L.; Lv, D. W.; Ma, C. Y.; Li, X. H.;

445

Ma, W. J.; Yan, Y. M. Wheat drought-responsive grain proteome analysis by linear

446

and nonlinear 2-DE and MALDI-TOF mass spectrometry. Int. J. Mol. Sci. 2012, 13

447

(12), 16065 – 16083.

448

(10) Ling, H. Q.; Zhao, S.; Liu, D.; Wang, J.; Sun, H.; Zhang, C.; Fan, H.; Li, D.;

449

Dong, L.; Tao, Y.; Gao, C.; Wu, H.; Li, Y.; Cui, Y.; Guo, X.; Zheng, S.; Wang, B.; Yu,

450

K.; Liang, Q.; Yang, W.; Lou, X.; Chen, J.; Feng, M.; Jian, J.; Zhang, X.; Luo, G.;

451

Jiang, Y.; Liu, J.; Wang, Z.; Sha, Y.; Zhang, B.; Wu, H.; Tang, D.; Shen, Q.; Xue, P.;

452

Zou, S.; Wang, X.; Liu, X.; Wang, F.; Yang, Y.; An, X.; Dong, Z.; Zhang, K.; Zhang,

453

X.; Luo, M. C.; Dvorak, J.; Tong, Y.; Wang, J.; Yang, H.; Li, Z.; Wang, D.; Zhang, A.;

454

Wang, J. Draft genome of the wheat A-genome progenitor Triticum urartu. Nature

455

2013, 496 (7443), 87 − 90.

456

(11)

457

R.; Pfeifer, M.; Tao, Y.; Zhang, X. Y.; Jing, R. L.; Zhang, C.; Ma, Y. Z.; Gao, L. F.;

458

Gao, C. A.; Spannag, M.; Mayer, K.; Li, D.; Pan, S. K.; Zheng, F. Y.; Hu, Q.; Xia, X.

459

C.; Li, J. W.; Liang, Q. S.; Chen, J.; Wicker, T.; Gou, C. Y.; Kuang, H. H.; He, G. Y.;

460

Luo, Y. D.; Keller, B.; Xia, Q. J.; Lu, P.; Wang, J. Y.; Zou, H. F.; Zhang, R. Z.; Xu, J.

461

Y.; Gao, J. L.; Middleton, C.; Quan, Z. W.; Liu, G. M.; Wang, J. International Wheat

Jia, J. Z.; Zhao, S. C.; Kong, X. Y.; Li, Y. R.; Zhao, G. Y.; He, W. M.; Appels,


Page 22 of 43

Page 23 of 43


462

Genome Sequencing Consortium; Yang, H. M.; Liu, X.; He, Z. H.; Mao, L.; Wang, J.

463

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat

464

adaptation. Nature 2013, 496 (7443), 91 − 95.

465

(12)

466

L.; Sourdille, P.; Couloux, A.; Paux, E.; Leroy, P.; Mangenot, S.; Guilhot, N.; Le

467

Gouis, J.; Balfourier, F.; Alaux, M.; Jamilloux, V.; Poulain, J.; Durand, C.; Bellec, A.;

468

Gaspin, C.; Safar, J.; Dolezel, J.; Rogers, J.; Vandepoele, K.; Aury, J. M.; Mayer, K.;

469

Berges, H.; Quesneville, H.; Wincker, P.; Feuillet, C. Structural and functional

470

partitioning of bread wheat chromosome 3B. Science 2014, 345 (6194), 1249721.

471

(13) Emes, M. J.; Bowsher, C. G.; Hedley, C.; Burrell, M. M.; Scrase-Field, E. S. F.;

472

Tetlow, I. J. Starch synthesis and carbon partitioning in developing endosperm. J.

473

Exp. Bot. 2003, 54 (382), 569 – 575.

474

(14)Irar, S.; Brini, F.; Goday, A.; Masmoudi, K.; Pagès, M. Proteomic analysis of

475

wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D)

476

— A wider perspective of the proteome. J. Proteomics 2010, 73 (9), 1707 – 1721.

477

(15) Laino, P.; Shelton, D.; Finnie, C.; De Leonardis, A. M.; Mastrangelo, A. M.;

478

Svensson, B.; Lafiandra, D.; Masci, S. Comparative proteome analysis of metabolic

479

proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress. Proteomics

480

2010, 10 (12), 2359 – 2368.

481

(16) Hajheidari, M.; Eivazi, A.; Buchanan, B. B.; Wong, J. H.; Majidi, I.; Salekdeh,

482

G. H. Proteomics Uncovers a Role for Redox in Drought Tolerance in Wheat. J.

483

Proteome Res.2007, 6 (4), 1451 – 1460.

Choulet, F.; Alberti, A.; Theil, S.; Glover, N.; Barbe, V.; Daron, J.; Pingault,



484

(17) Guo, G. F.; Ge, P.; Ma, C. Y.; Li, X. H.; Lv, D. W.; Wang, S. L.; Ma, W. J.; Yan,

485

Y. M. Comparative proteomic analysis of salt response proteins in seedling roots of

486

two wheat varieties. J. Proteomics 2012, 75 (6), 1867 – 1885.

487

(18) Guttieri, M. J.; Stark, J. C.; O’Brien, K.; Souza, E. Relative sensitivity of spring

488

wheat grain yield and quality parameters to moisture deficit. Crop Sci. 2001, 41 (2),

489

327 − 335.

490

(19) Smart, R. E. Rapid estimates of relative water content. Plant Physiol. 1974, 53

491

(2), 258 – 260.

492

(20) Zhao, S. J.; Xu, C. C.; Zou, Q.; Meng, Q. W. Improvements of method for

493

measurement of malondialdehvde in plant tissues. Plant Physiol. Commun. 1994, 30

494

(3), 207 – 210.

495

(21) Bates, L. S.; Waldren, R. P.; Teare, I. D. Rapid determination of free proline for

496

water-stress studies. Plant Soil 1973, 39 (1), 205 – 207.

497

(22) Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in

498

Beta vulgaris. Plant Physiol. 1949, 24 (1), 1 – 15.

499

(23) Czuchajowska, Z.; Paszczynska, B. Is wet gluten good for baking? Cereal

500

chemistry 1996, 73 (4), 483 – 489.

501

(24) Guo, G. F.; Lv, D. W.; Yan, X.; Subburaj, S.; Ge, P.; Li, X. H.; Hu, Y. K.; Yan, Y.

502

M. Proteome characterization of developing grains in bread wheat cultivars (Triticum

503

aestivum L.). BMC Plant Biol. 2012, 12, 147.

504

(25) Valledor, L.; Jorrin, J. Back to the basics: maximizing the information obtained

505

by quantitative two dimensional gel electrophoresis analyses by an appropriate 24 ACS Paragon Plus Environment

Page 24 of 43

Page 25 of 43


506

experimental design and statistical analyses. J. Proteomics 2011, 74 (1), 1 – 18.

507

(26) Kosová, K.; Vítámvás, P.; Planchon, S.; Renaut, J.; Vanková, R.; Prášil, I. T.

508

Proteome Analysis of Cold Response in Spring and Winter Wheat (Triticum aestivum)

509

Crowns Reveals Similarities in Stress Adaptation and Differences in Regulatory

510

Processes between the Growth Habits. J. Proteome Res. 2013, 12, 4830 − 4845.

511

(27) Franceschini, A.; Szklarczyk, D.; Frankild, S.; Kuhn, M.; Simonovic, M.; Roth,

512

A.; Lin, J.; Minguez, P.; Bork, P.; Mering, C.; Jensen, L. J. STRING v9.1:

513

protein-protein interaction networks, with increased coverage and integration.

514

Nucleic Acids Res. 2013, 41 (D1), D808 − 815.

515

(28) Cline, M. S.; Smoot, M.; Cermai, E.; Kuchinsky, A.; Landys, N.; Workman, C.;

516

Christmas, R.; Avila-Campilo, I.; Creech, M.; Gross, B.; Hanspers, K.; Isserlin, R.;

517

Kelley, R.; Killcoyne, S.; Lotia, S.; Maere, S.; Morris, J.; Ono, K.; Pavlovic, V.; Pico,

518

A. R.; Vailaya, A.; Wang, P. L.; Adler, A.; Conklin, B. R.; Hood, L.; Kuiper, M.;

519

Sander, C.; Schmulevich, I.; Schwikowski, B.; Warner, G. J.; Ideker, T.; Bader, G. D.

520

Integration of biological networks and gene expression data using Cytoscape. Nat.

521

Protoc. 2007, 2 (10), 2366 − 2382.

522

(29)Horton, P.; Park, K. J.; Obayashi, T.; Fujita, N.; Harada, H.; Adams-Collier, C. J.;

523

Nakai, K. Protein subcellular localization prediction with WOLF PSORT. Nucleic

524

Acids Res. 2007, 35, W585 − 587.

525

(30) Small, I.; Peeters, N.; Legeai, F.; Lurin, C. Predotar: A tool for rapidly screening

526

proteomes for N-terminal targeting sequences. Proteomics 2004, 4 (6), 1581 − 1590.

527

(31) Yang, F.; Xu, X.; Xiao, X.; Li, C. Responses to drought stress in two poplar 25 ACS Paragon Plus Environment


528

species originating from different altitudes. Biol. Plant 2009, 53 (3), 511 – 516.

529

(32) Aalenf, R. B.; Opsahl-Ferstad, H. G.; Linnestad, C.; Olsen, O. A. Transcripts

530

encoding an oleosin and a dormancy-related protein are present in both the aleurone

531

layer and the embryo of developing barley (Hordeum vulgare L.) seeds. Plant J.

532

1994, 5 (3), 385 − 396.

533

(33) Haslekås, C.; Stacy, R. A.; Nygaard, V.; Culiáñez-Macià, F. A.; Aalen, R. B. The

534

expression of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is

535

seed-specific and related to dormancy. Plant Mol. Biol. 1998, 36 (6), 833 − 845.

536

(34) Radyuk, S. N.; Klichko, V. I.; Spinola, B.; Sohal, R. S.; Orr, W. C. The

537

peroxiredoxin gene family in Drosophila melanogaster. Free Radic. Biol. Med. 2001,

538

31 (9), 1090 − 1100.

539

(35) Tsugeki, R.; Nishimura, M. Interaction of homologues of Hsp70 and Cpn60 with

540

ferredoxin-NADP+ reductase upon its import into chloroplasts. FEBS Lett. 1993, 320

541

(3), 198 − 202.

542

(36) Duan, Y. H.; Guo, J.; Ding, K.; Wang, S. J.; Zhang, H.; Dai, X. W.; Chen, Y. Y.;

543

Govers, F.; Huang, L. L.; Kang, Z. S. Characterization of a wheat HSP70 gene and

544

its expression in response to stripe rust infection and abiotic stresses. Mol. Biol. Rep.

545

2011, 38 (1), 301 – 307.

546

(37) Krasensky, J.; Jonak, C. Drought, salt, and temperature stress-induced metabolic

547

rearrangements and regulatory networks. J. Exp. Bot. 2012, 63 (4), 1593 – 1608.

548

(38) Franco, O. L.; Rigden, D. J.; Melo, F. R.; Grossi-De-Sa, M. F. Plant

549

alpha-amylase inhibitors and their interaction with insect alpha-amylases. Eur. J. 26 ACS Paragon Plus Environment

Page 26 of 43

Page 27 of 43


550

Biochem.2002, 269 (2), 397 − 412.

551

(39) Skylas, D. J.; Mackintosh, J. A.; Cordwell, S. J.; Basseal, D. J.; Walsh, B. J.;

552

Harry, J.; Blumenthal, C.; Copeland, L.; Wrigleya, C.W.; Rathmell, W. Proteome

553

approach to the characterisation of protein composition in the developing and mature

554

wheat-grain endosperm. J. Cereal Sci. 2000, 32, 169 – 188.

555

(40) Smart, M. G.; O'Brien, T. P. The development of the wheat embryo in relation to

556

the neighbouring tissues. Protoplasma 1983, 114, 1 – 13.

557

(41) Blackig, M.; Corbineau, F.; Grzesikit, M.; Guyi, P.; Côme, D. Carbohydrate

558

metabolism in the developing and maturing wheat embryo in relation to its

559

desiccation tolerance. J. Exp. Bot. 1996, 47, 161 − 169.

560

(42) Sturm, A.; Tang, G. Q. The sucrose-cleaving enzymes of plants are crucial for

561

development, growth and carbon partitioning. Trends Plant Sci. 1999, 4 (10), 401 –

562

407.

563

(43) Weschke, W.; Panitz, R.; Gubatz, S.; Wang, Q.; Radchuk, R.; Weber, H.; Wobus,

564

U. The role of invertases and hexose transporters in controlling sugar ratios in

565

maternal and filial tissues of barley caryopses during early development. Plant J.

566

2003, 33 (2), 395 – 411.

567

(44) Jiang, H.; Dian, W.; Liu, F.; Wu, P. Isolation and characterization of two

568

fructokinasec DNA clones from rice. Phytochem. 2003, 62 (1), 47 − 52.

569

(45) Shewry, P. R.; Halford, N. G. Cereal seed storage proteins: structures, properties

570

and role in grain utilization. J. Exp. Bot. 2002, 53 (370), 947 − 958.

571

(46) Branlard, G.; Dardevet, M.; Saccomano, R.; Lagoutte, F.; Gourdon, J. Genetic 27 ACS Paragon Plus Environment


572

diversity of wheat storage proteins and bread wheat quality. Wheat in a global

573

environment. Springer Netherlands, 2001, 119 (1-2), 59 – 67.

574

(47) Ma, F. Y.; Li, M.; Yu, L. L.; Li, Y.; Liu, Y. Y.; Li, T. T.; Liu, W.; Wang, H. M.;

575

Zheng, Q.; Li, K. X.; Chang, J. L.; Yang, G. X.; Wang, Y. S.; He, G. Y.

576

Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene

577

improves flour mixing properties. Mol Breed 2013, 32 (4), 853 – 865.

578

(48) Rasiah, I. A.; Sutton, K. H.; Low, F. L.; Lin, H. M.; Gerrard, J. A. Crosslinking

579

of wheat dough proteins by glucose oxidase and the resulting effects on bread and

580

croissants. Food Chem. 2005, 89 (3), 325 – 332.


Page 28 of 43

Page 29 of 43


Table 1.Comparison of different breadmaking quality parameters of wheat cultivar Jing 411 between normal cultivation conditions and water deficit treatment. Basic quality parameters Treatments

Total

Total gluten

protein

content

(%)

(%)

313

12.67

±3

±0.05

FN

Control Stress

Mixograph Peak time

Time×width

(min)

(%)

3.26

6.34

5.46

±0.03

±0.06

±0.8

Farinograph Water

Extensograph Maximum

DT

Stability

Energy area

Extensibility

(min)

(min)

(cm2)

(mm)

50.1

1.9

3.5

50

161

192

±0.1

±0.1

±0.1

±1

±1

±4

absorption (%)

resistance (B.U.)

325

12.51

2.92

6.33

6.12

50

1.7

4

57

165

208

±6*

±0.02*

±0.03**

±0.03

±0.84**

±0.1

±0.1*

±0.2*

±1*

±2*

±1*

Loaf volume and appearance Treatments

score Loaf volume (ml3)

Control Stress

C-cell parameters

Score (100)

Cell diameter /px

Cell contrast

Curvature

Non-

Number

Slice area

Slice

Total

Volume of

uniformity

of cells

(px)

brightness

concavity (%)

holes

820

59

20.03

0.705

1383.5

1.81

2436

270709

145.1

1.75

0.75

±12

±1

±0.16

±0.005

±64.5

±0.09

±15

±348

±0.3

±0.02

±0.25

883

67

21.00

0.717

1788

1.04

2512

287615

150.9

3.425

0.25

±6*

±2*

±0.32**

±0.001**

±3**

±0.17**

±37*

±176**

±0.4**

±0.08**

±0.12*

29

ACS Paragon Plus Environment


Page 30 of 43

* and**: Significance at 5% and 1% levels, respectively; FN: Falling number; DT: Dough development time.

Table 2. Expression characteristics of some important DEP spots in embryo and endosperm in response to water deficit during grain development. Average volume SpotIDa

GI Number

204 205

gi|13387255 0 gi|28172909 gi|46174405 8 gi|18076790 gi|47560421 7 gi|14850878 4 gi|14850878 4 gi|11124572 gi|11124572

21

gi|32812836

45

gi|32400764 gi|47403319 7 gi|1872473

48 6 73 157 175 111 113

15 61

(15:21:26:30:45)b EMCc EMSd

Protein Name Bp2A protein Cytosolic 3-phosphoglycerate kinase Enolase Phosphoglucomutase Pyrophosphate--fructose 6-phosphate Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Triosephosphate-isomerase Triosephosphate-isomerase ADP-glucose pyrophosphorylase large subunit Beta-amylase Acyl-[acyl-carrier-protein] desaturase 2, chloroplastic Delta-24-sterol methyltransferase

Protein Function ENC E

E

e

ENS E

E

f

Energy Metabolism Energy Metabolism

E

E

Energy Metabolism Energy Metabolism Energy Metabolism

E N

Energy Metabolism

E

Energy Metabolism Energy Metabolism Energy Metabolism

N E

E

E

Starch Metabolism Starch Metabolism

E N N

30


N N

Lipid Metabolism Lipid Metabolism

Page 31 of 43


23 9 49 69 182 207 3 30 50 51 54 71 145 151 164 169 87 102 115

gi|47559536 6 gi|47440751 2 gi|56606827 gi|47375387 4 gi|27735373 gi|68305063 gi|47544233 9 gi|167113 gi|47429261 0 gi|62465514 gi|26017213 gi|1169518 gi|47411003 9 gi|30008707 1 gi|47556576 0 gi|89143120 gi|47441141 9 gi|21539846 8 gi|46981764

Nitrogen Metabolism

Alanine aminotransferase 2 20 kDachaperonin, chloroplastic

E N

Calreticulin-like protein

E N

Elongation factor 1-beta Replication factor C like protein Ubiquitin

N

Protein Metabolism Protein Metabolism Protein Metabolism

N

1-Cys peroxiredoxin PER1 Aldose reductase-related protein

E

E

Stress/Defense Stress/Defense

N N

Stress/Defense Stress/Defense Stress/Defense Stress/Defense

Catalase isozyme 1 Class II chitinase Cold regulated protein Em protein H5 Late embryogenesis abundant protein Lea14-A Peroxidase 1 Stromal 70 kDa heat shock-related protein Putative avenin-like a precursor

Protein Folding Protein Metabolism

N N

Stress/Defense

E

E

E

E

Stress/Defense

E

E

Stress/Defense Storage Protein

Globulin-1 S allele

Storage Protein

Globulin 3C

Storage Protein Storage Protein

HMW glutenin subunit Dy12

E

E

a. The protein spots showed in Figure 2; b. The average relative abundanceat15, 21, 26, 30, 45 days after flower; c. Control embryo; d. Stressed

31



Page 32 of 43

embryo; e. Control endosperm; f. Stressed endosperm. E means the spot did not show significantly at least 2-fold differences. N means the spot did not exist. Green color indicates a relative lower accumulation. Red color indicates a relative higher accumulation.

32


Page 33 of 43


Figure legends: Figure 1. Physiological index changes of wheat flag leaves in the control and water stress samples during the development progress. (A) RWC, relative water content. (B) Proline content. (C) MDA, malondialdehyde content. (D) Soluble sugar content. (E) Chlorophyll content. DAF, day after flower. Control, the control samples. Stress, the water stressed samples. “**”, “*”, Significant differences between the stress and the control samples at 1% and 5% levels, respectively. Figure 2. The identified differentially expressed protein (DEP) spots in embryo (A) and endosperm (B) in response to water deficit during the grain development. The protein numbers are same as those in Table S2-S3. NL, nonlinear; EMC15, control embryo at 15 day after flower; EMC21, control embryo at 21 day after flower; EMC26, control embryo at 26 day after flower; EMC30, control embryo at 30 day after flower; EMC45, control embryo at 45 day after flower; EMS15, Stressed embryo at 15 day after flower; EMS21, Stressed embryo at 21 day after flower; EMS26, Stressed embryo at 26 day after flower; EMS30, Stressed embryo at 30 day after flower; EMS45, Stressed embryo at 45 day after flower; ENC15, control endosperm at 15 day after flower; ENC21, control endosperm at 21 day after flower; ENC26, control endosperm at 26 day after flower; ENC30, control endosperm at 30 day after flower; ENC45, control endosperm at 45 day after flower; ENS15, Stressed endosperm at 15 day after flower; ENS21, Stressed endosperm at 21 day after flower; ENS26, Stressed endosperm at 26 day after flower; ENS30, Stressed endosperm at 30 day after flower; ENS45, Stressed endosperm at 45 day after flower. Protein spot 33 ACS Paragon Plus Environment


Page 34 of 43

was numbered by the order of the first letter of the protein names. The same differential protein spot in embryo and endosperm has the same number, whereas different protein spots with the different numbers. Figure 3. Numbers of identified DEP spots in embryo and endosperm. Numbers in brackets means the corresponding numbers of unique proteins. Figure 4. Principal component analysis (PCA) of all the identified DEP spots in embryo and endosperm development under water deficit. (A) Individual stages in embryo and endosperm development. (B) Individual stages of embryo and endosperm under water deficit. (C) Individual stages of embryo development in response to water deficit. (D) Individual stages of endosperm development in response to water deficit. C, control samples, S, stressed samples. EM, Embryo. EN, Endosperm.15, 21, 26, 30, 45 mean five grain developmental stages at 15, 21, 26, 30, 45 DAF. Figure 5. Prediction of protein-protein interaction between some identified proteins with high confidence score (0.900). Proteins of the left were from embryo; Proteins of the right were from endosperm; Proteins of the middle were both shared in these two tissues; Different colors display different functions. Abbreviation: GAPDH, Cytosolic glyceraldehyde-3-phosphate dehydrogenase; HSP70, Stromal 70 kDa heat shock-related protein. Figure 6. An overview of possible metabolic pathways of embryo and endosperm development in response to water deficit. Abbreviations: AA, Amino acid; AAI, alpha-amylase

inhibitor;

ADPG,

ADP-glucose; 34

AGPase,


ADP-glucose

Page 35 of 43


pyrophosphorylase large subunit; Bp2A, Bp2A protein; DHAR, Dehydroascorbate reductase;

EF,

Elongation

factor;

GAPDH,

Glyceraldehyde-3-phosphate

dehydrogenase; G-1-P, Glucose-1-phosphate; HSP, Heat shock-related protein; LEA, Late embryogenesis abundant protein Lea14-A; MC, control embryo; MS, stressed embryo;

NC,

control

endosperm;

NS,

stressed

endosperm;

PGM,

Phosphoglucomutase; ROS, Reactive oxygen species; 24SMT, Delta-24-sterol methyltransferase; SUS, Sucrose synthase, UDPG, Uridinediphosphate glucose.



Figure 1


Page 36 of 43

Page 37 of 43


A



B

Figure 2


Page 38 of 43

Page 39 of 43


Figure 3



Figure 4


Page 40 of 43

Page 41 of 43


Figure 5



Figure 6


Page 42 of 43

Page 43 of 43


TOC Graphic


Integrated Proteome Analysis of the Wheat Embryo ... - ACS Publications

Recommend Documents