Transcriptomic Analyses of Ascorbic Acid and Carotenoid Metabolites

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Transcriptomic analyses of Ascorbic acid and Carotenoid metabolites influenced by root restriction during grape berry development and ripening Feng Leng, Dan Dan Tang, Qiong Lin, Jin Ping Cao, Di Wu, Shiping Wang, and Chongde Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05322 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Agricultural and Food Chemistry

1

Transcriptomic analyses of Ascorbic acid and Carotenoid metabolites

2

influenced by root restriction during grape berry development and

3

ripening

4

Feng Lenga, Dandan Tanga, Qiong Lina,b, Jinping Caoa,d, Di Wua, Shiping Wangc, Chongde Suna*

5

a

Laboratory of Fruit Quality Biology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth,

6

Development and Quality Improvement, Zhejiang University, Zijingang Campus, Hangzhou 310058, P. R.

7

China

8

b

9

Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Opening Laboratory of Agricultural Products Processing and Quality Control, Ministry of Agriculture, Beijing 100193, P. R. China

10

c

Shanghai Jiao Tong University, School of Agriculture and Biology, Shanghai 200240, P. R. China

11

d

Taizhou Academy of Agricultural Sciences, Linhai 317000, PRChina

12 13

Abstract Ascorbic acid (AsA) and carotenoids are recognized as crucial metabolites

14

for various biological processes in plants. The contents of AsA and carotenoids in

15

fruits are influenced by external environmental stimuli, such as water, temperature,

16

light and hormones. However, it is still not clear whether it can be affected by root

17

restriction (RR) treatment. In this study, ‘Summer Black’ grape berry (Vitis vinifera ×

18

V. labrusca) under RR and control treatments during development and ripening were

19

used as materials. The results showed that RR significantly increased the contents of

20

AsA, and the transcript VIT_08s0040g03150 related to AsA recycling pathways may

21

be the main regulator for AsA. Similarly, the contents of most of the carotenoids at

22

the earlier stages significantly increased by RR, the enzyme crtB encoded by

23

VIT_12s0028g00960, the enzyme crtZ encoded by VIT_02s0025g00240 and

ACS Paragon Plus Environment


24

VIT_16s0050g01090 were inferred to play major roles in the carotenoid metabolic

25

pathways.

26 27

Keywords: grape berry, RNA-Seq, ascorbic acid, carotenoid, root restriction

28 29

Introduction

30

Root restriction (RR) is a type of cultivation technique to improve the utilizing

31

efficiency of agricultural resources by restricting the root growth in a certain volume1.

32

It has been adopted in many fruit species, such as tomato2, 3, mangoe4, 5, peach6,

33

mandarin7, sweet cherry8, persimmon9, and grape1, 10-15. RR treatment was proved to

34

be responsible in increasing the sugars contents, total and individual anthocyanin

35

concentrations1, 10, 11, 13, 15, but no research has been conducted on the investigation of

36

other important quality attributes, such as ascorbic acid (AsA) and carotenoids.

37

AsA, commonly known as vitamin C, is an important biological molecule

38

involved in many biochemical processes and is an important antioxidant responsible

39

for main processes in the human body16. Also, it is usually used as an index of the

40

health-related quality of fruits17. AsA is active growth and development in plants, and

41

is also influenced by many factors, such as light18, pathogens19, chemical exposures19

42

and temperature20,

43

important roles as provitamin A and antioxidant compounds, which are important for

44

photosynthesis, regulating growth and development in plant22. They are recognized as

45

crucial to the human diets because of their valuable and beneficial health effects23.

21

. Carotenoids are the secondary metabolites known to play


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Carotenoids are influenced by several factors, such as water24, 25, temperature26, and

47

phytohormones27, 28.

48

‘Summer Black’ is one of the most widely grown seedless table grapes in China.

49

During maturation, berries undergo a series of physical and biochemical changes.

50

Because of the importance of AsA and carotenoids to the human health and the fruit

51

quality, it is important to understand their physiology and biochemical processes in

52

fruits. To the best of our knowledge, there are no previous studies of ascorbic acid and

53

carotenoid metabolism influenced under RR treatment throughout the growing season.

54

For this purpose, we carried out a global analysis of the grape transcriptome under RR

55

and control treatments during the grape development using the RNA-Seq method. The

56

aim of this study was to investigate the regulatory role of RR treatment towards AsA

57

and carotenoid metabolism in grape berry.

58 59

Materials and Methods

60

Plant Materials. This study was carried out in a greenhouse in an orchard with three

61

years old table grape ‘Summer Black’ (V. vinifera × V. labrusca) during the fruiting

62

season of 2013-2014 in Jinhua Academy of Agricultural Sciences (Zhejiang,

63

China).The grapes of the first group were planted in 40 cm depth and 100 cm wide

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ridges isolated with the plastic film from the outside ground as the RR treatment,

65

whereas those of the other group were planted in a raise bed (40 cm deep) with the

66

same soil at the open ground as the control. The same watering and fertilizer strategy

67

were applied to the RR and the control to avoid different environmental conditions.



68

Five different developmental stages were set as the sampling points, namely S1

69

(fruitlet, 15 days after full bloom (DAFB)), S2 (immature green, 28 DAFB), S3

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(before veraison, 42 DAFB), S4 (veraison, 53 DAFB), and S5 (fully ripe, 74 DAFB).

71

Fifty grapevines were averagely distributed in the five sampling locations at each

72

treatment. For each sampling time of all treatments, ten clusters were randomly

73

picked from at least five plants with no evidence of disease or stress symptoms. All

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samples were transported to the laboratory in Hangzhou, China within 3 hours after

75

picking. Berries were selected for uniform maturity and absence of mechanical

76

damage, then cut into small pieces and frozen in liquid nitrogen and stored at -80°C

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for the future use. All samples were performed with three biological replicates.

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Measurement of Ascorbic acid. AsA contents of the berries were determined

79

according to the previously method with some slight modifications29. Approximate

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500 mg of the fresh berry powder and 10% metaphosphoric acid (MPA) (1 mL) were

81

mixed and sonicated, centrifuged at 4°C and 10000 rpm for 10 min, the supernatant

82

was filtered through a 0.22 µm membrane and was injected into the high-performance

83

liquid chromatograph (HPLC) column. AsA analysis was carried out on Waters

84

Alliance 2695 system (Waters Corporation, USA) and a 2996 PDA detector set at 245

85

nm, equipped with Waters C18 (250×3.9 mm, i.d. 5 µm). The mobile phase consisted

86

of (A) methanol and (B) 5 mM KH2PO4 pH 2.65 was used according to the eluent

87

program: linear increment starting with 5-22% A in 6 min and return to the initial

88

conditions within next 9 min at the flow rate 1 mL /min, the injected sample volume

89

was 20 µL and the column temperature was set to 25°C. Quantification of AsA was


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90

carried out using the standard method.

91

Measurement of Carotenoids. Carotenoids were extracted and analyzed by

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HPLC-PDA according to the previously described method30 with some modifications.

93

Approximate 100 mg of the lyophilized berry powder was extracted ultrasonically

94

with 1.4 mL of a mixture of methanol/chloroform/water (1:2:1, v/v/v). After the

95

centrifugation, the residue was re-extracted twice with 700 µL of chloroform. The

96

chloroform phases were combined and dried under vacuum, using a rotary evaporator

97

at 30°C. The residue was dissolved in 20 µL of diethyl ether and 350 µL of 6% (w/v)

98

KOH in methanol, then was vortexed and incubated at 60°C for 30 min in darkness,

99

and 700 µL of chloroform and 350 µL of water were added. The chloroform phase

100

was recovered and partitioned with water at some times until the aqueous phase

101

became neutral, and was then dried under vacuum and dissolved in 100 µL of HPLC

102

grade ethyl acetate. HPLC analysis for individual carotenoid was carried out on the

103

Waters Alliance 2695 system (Waters Corporation, USA) and a 2996 PDA detector

104

equipped with a 250×4.6 mm i.d., 5 µm, YMC reverse-phase C30 column and a

105

20×4.6 mm i.d., YMC C30 guard. Chromatography was carried out at 25°C with the

106

elution program as previously described30. The flow rate was 1 mL/min. Carotenoids

107

were identified on the basis of the HPLC runs with the standards as well as by

108

recording the UV-Vis spectra and their comparison with the known carotenoid

109

spectra.

110

RNA extraction and RNA-Seq. Total RNA was extracted from the frozen powder of

111

about some whole grape berries according to our previously published method31.



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112

After removal of contaminating genomic DNA with a TURBO DNA-free kit

113

(Ambion), the total RNA was quantified using Nanophotometer Pearl (Implen), and

114

used for RNA-seq and real -time PCR. For the RNA-Seq, the raw reads were obtained

115

by the Shanghai Majorbio Bio-pharm Biotechnology Co. (Shanghai, China) using

116

Illumina HiSeqTM 2000 with 5 Gb reads per sample

117

processed to get clean reads by removing the adapter and low quality sequences using

118

the software SeqPrep (https://github.com/jstjohn/SeqPrep). The clean reads were

119

aligned

120

(http://www.genoscope.cns.fr/externe/Download/Projets/Projet_ ML/data/)33 using the

121

TopHat software (http://tophat.cbcb.umd.edu/)34 and the quality was assessed by the

122

saturation analysis, duplicate reads analysis and gene coverage analysis by using the

123

RSeQC-2.3.2 program (http://code.google.com/p/rseqc/)35. Gene expression values

124

were calculated by the read/fragments per kilobase of exon per million fragments

125

mapped

126

(http://cufflinks.cbcb.umd.edu/). Differential expression was analyzed according to

127

the count values of each transcript in two libraries using the edgeR software. Gene

128

with a false discovery rate (FDR) less than 0.05, and an estimated absolute log2 fold

129

change (FC) more than 1 were used as the thresholds for judging the significant

130

differences among the transcript expression36.

131

All

132

(https://www.ncbi.nlm.nih.gov/sra).

133

SRR4408346,

of

to

the

reads

these

reference

(RPKM/FPKM)

RNA-Seq

SRX2234711/

reads The

32

. Raw reads were initially

Vitis

using

the

were

codes

in are:

SRX2234711/


genome

Cuffdiff

deposited

accession

SRR4408347,

vinifera

program

NCBI-SRA SRX2234711/ SRR4408413,

Page 7 of 29


134

SRX2234711/ SRR4408414.

135

Real-Time quantitative PCR validation of RNA-Seq data. For the real-time

136

quantitative PCR analyses, the gene-specific oligonucleotide primers were designed

137

and described as ‘Supporting Information Table S1’, this material is available free of

138

charge via the Internet at http://pubs.acs.org. The gene specificity of each pair of

139

primers was checked by melting curves and product re-sequencing twice. The

140

GAPDH gene was employed as the internal control for calculating the relative

141

expression of the mRNA37. The sequences of GAPDH primers are described in

142

Supplemental Table S1. Real-time PCR was performed by the FastStart Universal

143

SYBR Green (Roche), initiated by 10 min at 95°C and followed by 40 cycles of 95°C

144

for 30 s, 60°C for 30 s, and 72°C for 10 min, and completed with a melting curve

145

analysis program. The PCR mixture (10 µL total volume) comprised 5 µL of Roche

146

FastStart Universal SYBR Green Master (ROX), 0.75 µL of each primer (10 µM),

147

0.5 µL of diluted cDNA and 3 µL PCR-grade ddH2O. No-template controls and

148

melting curve analysis were included for each gene during each run.

149

Statistical analysis. The statistical significance of differences was calculated by

150

ANOVA (single factor variance analysis). The results are the mean ± SE of at least

151

three independent replicates and were analyzed using the data processing system

152

SPSS16.0 statistical software package. Figures were drawn by the Origin 8.0

153

(Microcal Software Inc., Northampton, MA, USA).

154 155

Results and discussion



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AsA Metabolism. To understand the changes of AsA metabolic pathway influenced

157

by RR treatment, five developmental stages in both control and RR treatments were

158

considered and the AsA contents were measured using the HPLC. Berries sampled

159

from two treatments exhibited similar patterns and the content of AsA was steadily

160

decreasing during the development. Interestingly, the contents of AsA in RR

161

treatment were significantly higher compared to those in the control at S1, S2, S3, S5

162

stages (Figure 1).

163

To understand how the control and RR treatments changes in the transcript

164

expressions during the dynamic process of the grape berry development,

165

high-throughput RNA-Seq using Illumina Hiseq 2000 sequencing technology was

166

performed. The sequence reads were matched to the Pinot Noir 40024 reference

167

genome33. A quantitative evaluation of the transcripts was used to measure the levels

168

of differential expressions between the RR and control groups during all the

169

developmental stages.

170

The expression levels of genes encoding several enzymes related to the AsA

171

biosynthesis pathways in berries were analyzed. The results revealed that total 7

172

transcripts encoded 5 enzymes include GDP-D-mannose 3',5'-epimerase [EC:5.1.3.18]

173

(VIT_05s0020g04510,

174

[EC:2.7.7.69] (VIT_14s0006g01370, VIT_19s0090g01000), L-galactose 1-phosphate

175

phosphatase

176

[EC:1.1.1.316]

177

[EC:1.3.2.3] (VIT_08s0007g05710). The FPKM values of all transcripts are also

VIT_14s0030g02180),

[EC:3.1.3.25]

GDP-L-galactose

(VIT_10s0405g00030),

(VIT_03s0088g01250),

phosphorylase

L-galactose

dehydrogenase

L-galactono-1,4-lactone

dehydrogenase


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shown in Table 1. From our data, all the transcripts exhibited similar patterns of

179

expression in the two treatments, and the earlier developmental stages of berry

180

showed higher relative expressions of transcripts compared with the later stages with

181

a few exceptions. The results of this study demonstrated a rapid accumulation of AsA

182

in the berries at the earlier stages.

183

In order to facilitate the comparison and visualization of the changed transcripts

184

in both treatments during the developmental stages, the metabolites and transcripts

185

depicted in Figure 2 were normalized to the expression level of the before veraison

186

stage in the control treatment to visualize the change among treatments and stages. In

187

relation to the AsA biosynthesis, our analysis of transcripts supports that the

188

biosynthesis of AsA might occur through the L-galactose (a key intermediate)

189

pathway during grape berry development and ripening. The GDP-D-mannose

190

3',5'-epimerase, which has been proposed to play a role in the control of the

191

L-galactose pathway38, it was mostly expressed at the fruitlet stage and then

192

down-regulated during the development39. Previous reports suggested that the

193

regulation of AsA content was determined at the biosynthetic level40, which was

194

consistent with our results in immature berries, and the AsA content was closely

195

related

196

dehydrogenase expression.

with

the

L-galactose

dehydrogenase

and


197

Our data revealed that the expression of transcripts related with the AsA contents

198

exhibited opposite trends between the control and RR treatment at the same phases

199

during the development. AsA exists in equilibrium with its oxidised form



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200

L-dehydroascorbate, and was limited to oxido-reductase reactions that alter the

201

balance of AsA to L-dehydroascorbate39. AsA was reduced to monodehydroascorbate

202

due

203

(VIT_02s0025g00340,

204

VIT_19s0014g05380), and the enzymes L-ascorbate oxidase (VIT_06s0009g01320,

205

VIT_06s0009g01340)

206

(VIT_08s0007g05710) were converted oxidation of AsA to L-dehydroascorbate. The

207

enzyme L-ascorbate peroxidase encoded by 11 transcripts was catabolized to

208

L-dehydroascorbate as well as recycled to AsA (Figure 2). From the data, the

209

expression of the transcript (VIT_08s0040g03150) was up-regulated significantly by

210

the RR treatment at the fruitlet stage, and was then decreased during the later

211

development stages (Table 1). The expression pattern was consistent with the

212

variations of the AsA contents, indicating that the transcripts related to AsA recycling

213

pathways may be the main regulators of AsA. However, the content of AsA was not

214

strictly associated with the concentration of regulatory genes, which could be due to

215

the post-translational modifications or to its regulation associated with some other

216

transcripts.

to

the

catalysis

of

the

enzyme

monodehydroascorbate

VIT_08s0007g03610,

and


reductase

VIT_14s0066g01100,

dehydrogenase

217

AsA as the precurs for the synthesis of both oxalic and tartaric acids, is not a

218

stable metabolic end-product. It can be converted to oxalic acid and L-threonic acid

219

via the intermediate 4-O-oxalyl-L-threonate, and tartaric acid converted from AsA is

220

known by the L-idonate dehydrogenase via an L-idonate intermediate (Figure 2). In

221

our data, L-idonate dehydrogenase encoded by the transcripts was not detected, which


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suggests that the tartaric acid accumulated in our studies from other pathway. Oxalic

223

acid and L-threonic acid were also not found, which might be because the contents

224

were too small to detection.

225

Carotenoids Metabolism. From the results, the main carotenoids in the grape berries

226

were lutein and β-carotene. A rapid decrease of the total carotenoids was observed

227

before veraison and a slow decrease continues during the maturation, which mainly

228

attributed to the decreases of all compositions but with some exceptions after veraison.

229

It was noticed that RR treatment significantly increased the contents of total

230

carotenoids before veraison but slightly decreased after veraison compared with the

231

control treatment. In addition, RR treatment significantly increased the contents of all

232

individual compositions at the fruitlet stage (Figure 3).

233

Expression abundance of the 23 carotenoid metabolic transcripts encoding 16

234

enzymes in the developing berries is listed in Table 1. From the data, the expression

235

of seven transcripts were down-regulated throughout the berry development stages in

236

both

237

VIT_05s0020g01240,

238

VIT_16s0050g01090

239

VIT_04s0023g00600 encoding isopentenyl-diphosphate delta-isomerase (IDI) in both

240

treatments were up-regulated dramatically from the immature green phase, and

241

reached peak values at the S3 stage then declined progressively towards the maturity

242

stage. On the other hand, the expression profiles of a number of transcripts

243

(VIT_15s0024g00850,

treatments,

including

VIT_19s0015g01010,

VIT_04s0023g01210,

VIT_19s0090g00530, and

VIT_11s0016g01880.

VIT_03s0038g03050,


VIT_04s0079g00680, The

expression

of

VIT_18s0001g12000,


VIT_14s0030g01740,

Page 12 of 29

244

VIT_09s0002g00100,

245

VIT_08s0032g00800,

246

VIT_07s0031g00620) decreased before the veraison stage and ascended to at or

247

around the veraison stage, then decreased until the S5 stage. Interestingly, almost all

248

expression values of the transcripts were down-regulated from S4 to S5 stages. In our

249

studies, RR treatment significantly increased the expressions of VIT_12s0028g00960

250

at the S4 stage, and increase VIT_02s0025g00240 and VIT_16s0050g01090 at the S1

251

and S2 stages.

VIT_04s0023g00080,

VIT_05s0062g01110, VIT_04s0043g01010

and

252

Carotenoid biosynthesis begins with the condensation of two GGPP molecules to

253

form 40-carbon phytoene, which is catalyzed by the enzyme phytoene synthase (crtB).

254

crtB is considered as the main rate limiting step for the carotenoid biosynthesis22, 41, 42

255

(Figure

256

VIT_06s0004g00820 and VIT_12s0028g00960) detected in our results, the RR

257

treatment significantly increased the expression of VIT_12s0028g00960 at the S4

258

stage. Phytoene is transformed into lycopene via a series of desaturation and

259

isomerization reactions43,

260

desaturase (PDS) and zeta-carotene desaturase (ZDS)) and two isomerase enzymes

261

(zeta-carotene isomerase (Z-ISO) and prolycopene isomerase (crtISO). All of these

262

enzymes are encoded by single transcript, and all of these transcripts down-regulated

263

at the early stages and up-regulated at the later stages by the RR treatment.

264

Phytofluene and ζ-carotene are the intermediates in this process, phytofluene was

265

increased by the RR treatment through the developmental stages, and ζ-carotene was

2).

There

are

three

crtB

transcripts

(VIT_04s0079g00680,

44

, involving two desaturase enzymes (15-cis-phytoene


Page 13 of 29


266

increased significantly at the S1 stage and decreased significantly at the S3 and S4

267

stages by the RR treatment.

268

Lycopene is the branching point of carotenoid biosynthesis, which is converted

269

to the cyclic carotenoids by the lycopene beta-cyclase (crtL1) and lycopene

270

epsilon-cyclase (crtL2). One of the branches leads to β-carotene with two β rings, in

271

the other branch leads to α-carotene with one β ring and one Ɛ ring. β-carotene is

272

further hydroxylated to the zeaxanthin by the beta-carotene 3-hydroxylase (crtZ), then

273

epoxidated twice to make the violaxanthin. α-carotene is eventually hydroxylated to

274

lutein by the crtZ45-47. In our results, the enzyme crtZ is encoded by two transcripts

275

VIT_02s0025g00240 and VIT_16s0050g01090, and the expression of these two

276

transcripts were significantly increased by the RR treatment at the young berry stage.

277

A previous research showed that the total carotenoid content of the grape berries

278

was decreasing gradually throughout the growth season with the lowest content at the

279

fully ripening stage48, which was consistent with our results. At the earlier stages, the

280

decreasing in contents was more likely due to the dilution effect by the quick berry

281

volume increment, but the transcript abundance of VIT_02s0025g00240 and

282

VIT_16s0050g01090 were also significantly increased by the RR treatment, indicating

283

important roles in regulating the contents of carotenoids. Carotenoids are precursors

284

of abscisic acid and volatiles49. At the later stages, carotenoids are converted to the

285

flavor and aroma compounds50, the transcript abundance of VIT_12s0028g00960 was

286

increased by the RR treatment, indicating that it played the important roles in the

287

abscisic acid and volatiles pathways.



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288

Root restriction might be regarded as one type of physical stress for roots of

289

grapevines, which can increase root mass and the amount of fibrous roots, reduced

290

shoot growth and photosynthetic rate1, 14. Meanwhile the allocation and partitioning of

291

assimilates between vegetative and reproductive organs were influenced by RR

292

treatment, which finally distributed more sugar into grape berries1. Glucose is the

293

precursor of AsA and carotenoids, RR treatment can significantly increase the glucose

294

and fructose concentration11, may be the main influence factor on the biosynthesis of

295

ascorbic acid and carotenoids.

296

Validation of gene expression using qRT-PCR. To confirm the accuracy and

297

reproducibility of the RNA-Seq data, real-time RT-PCR was performed on ten

298

transcripts at each stage in both treatments. These transcripts involved in

299

VIT_08s0040g03150,

300

VIT_16s0050g01090 significantly influenced by the RR treatment. Other six

301

transcripts were randomly chosen including up-regulated, down-regulated and

302

unaffected during the berry development. Correlation between the two methods was

303

measured by scatter ploting log2 fold changes (Figure 4). It was found that the

304

qRT-PCR results are generally consistent with the expression determined by the

305

RNA-Seq, suggesting the reliability of the RNA-Seq data.

VIT_12s0028g00960,

VIT_02s0025g00240

and

306 307

AUTHOR INFORMATION

308

Corresponding Author

309

*E-mail: [email protected].

Phone: +86 571 88982229. Fax: +86 571


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310

88982224.

311 312

Present Addresses

313

Chongde Sun: Laboratory of Fruit Quality Biology/The State Agriculture Ministry

314

Laboratory of Horticultural Plant Growth, Development and Quality Improvement,

315

Zhejiang University, Zijingang Campus, Hangzhou 310058, PR China

316 317

Authors Contributions

318

Feng Leng and Chongde Sun designed the experiments. Feng Leng, Dandan Tang,

319

Qiong Lin and Jinping Cao performed the experiments. Feng Leng, Di Wu and

320

Chongde Sun analyzed the data. Feng Leng, Qiong Lin, Jinping Cao, Shingping Wang

321

and Chongde Sun contributed reagents, materials and analytical tools. Feng Leng and

322

Chongde Sun composed the paper.

323 324

Funding

325

The work was supported by the National Natural Science Foundation of China

326

(31471836), the Fundamental Research Funds for the Central Universities (2016) and

327

Agricultural Outstanding Talents and Innovation Team of State Agricultural Ministry

328

on Health and Nutrition of Fruit.

329 330

Notes

331

The authors declare no competing financial interest.



Page 16 of 29

332 333

ABBREVIATIONS USED

334

S1, fruitlet; S2, immature green; S3, before veraison; S4, veraison; S5, fully ripe; AsA,

335

ascorbic acid; RR, root restriction; HPLC, high performance liquid chromatography;

336

DAFB, days after full bloom; MPA, metaphosphoric acid; FPKM, fragments per

337

kilobase of exon per million fragments mapped reads; GME, GDP-D-mannose 3',

338

5'-epimerase; VTC2_5, GDP-L-galactose phosphorylase; VTC4, inositol-phosphate

339

phosphatase/L-galactose

340

dehydrogenase;

341

monodehydroascorbate

342

L-idonate dehydrogenase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl

343

diphosphate; IDI, isopentenyl-diphosphate delta-isomerase; GPS, geranyl diphosphate

344

synthase; FDPS, farnesyl diphosphate synthase; GGPS, geranylgeranyl diphosphate

345

synthase; crtB, phytoene synthase; PDS, 15-cis-phytoene desaturase; Z-ISO,

346

zeta-carotene isomerase; ZDS, zeta-carotene desaturase; crtISO, prolycopene

347

isomerase; crtL1, lycopene beta-cyclase; crtZ, beta-carotene 3-hydroxylase; LUT5,

348

beta-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase;

349

crtL2, lycopene epsilon-cyclase; LUT1, carotene epsilon-monooxygenase.

1-phosphate

GLDH,

phosphatase;


reductase;

E1.10.3.3,

GalDH, dehydrogenase;

L-ascorbate

oxidase;

L-galactose NADH, L-IdnDH,

350 351

Supporting Information Available: This material is available free of charge via the

352

Internet at http://pubs.acs.org.

353


Page 17 of 29


354

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529 530 531 532 533

Table 1. Ascorbic acid and carotenoids metabolisms Description

RefSeq

treatment

FPKM value S1

S2

S3

S4

S5

Ascorbic acid metabolism GME; GDP-D-mannose 3',

Control

1285.30

679.61

502.29

297.80

44.48

RR

745.17

334.08

534.28

250.00

51.65

Control

1026.70

279.32

235.59

213.77

81.20

RR

629.13

189.28

213.10

155.75

68.42

Control

0.41

0.10

0.03

0.06

0.17

RR

0.07

0.11

0.05

0.03

0.11

Control

4437.55

2689.16

156.52

192.91

47.05

RR

2666.16

1286.41

134.06

128.59

58.54

Control

55.30

26.49

43.79

46.52

29.14

VIT_05s0020g04510 5'-epimerase VIT_14s0030g02180 VTC2_5; GDP-L-galactose VIT_14s0006g01370 phosphorylase VIT_19s0090g01000 VTC4; L-galactose 1-phosphate VIT_10s0405g00030 phosphatase

RR

56.11

34.29

47.91

42.20

33.64

GalDH; L-galactose

Control

351.67

228.87

52.98

35.76

11.20

dehydrogenase

RR

194.44

157.79

37.81

20.95

9.39

GLDH; L-galactono-1,4-lactone

Control

20.36

10.47

7.64

7.81

2.49

RR

11.40

10.21

7.46

6.02

2.66

Control

30.43

17.67

26.90

32.28

14.93

VIT_03s0088g01250

VIT_08s0007g05710 dehydrogenase NADH; monodehydroascorbate VIT_02s0025g00340 reductase

RR

22.38

17.79

29.34

27.73

17.31

Control

179.14

111.67

118.00

170.15

97.74

VIT_08s0007g03610 RR

154.10

146.32

138.78

174.37

125.08

Control

15.75

12.17

9.54

9.48

7.71

RR

12.67

13.45

10.47

10.35

10.43

Control

0.65

0.40

0.39

0.46

1.06

RR

1.12

1.00

0.20

0.70

1.81

Control

49.43

14.73

35.81

49.02

17.57

RR

34.18

13.46

40.07

49.07

22.79

Control

0.08

0.17

0.20

0.13

0.00

RR

0.08

0.00

0.31

0.21

0.15

Control

0.57

0.56

0.64

0.94

0.48

RR

0.96

0.49

1.22

0.52

0.43

Control

108.12

126.07

203.44

217.23

207.08

VIT_14s0066g01100

VIT_19s0014g05380 E1.10.3.3; L-ascorbate oxidase VIT_06s0009g01320

VIT_06s0009g01340 E1.11.1.11; L-ascorbate VIT_03s0017g00140 peroxidase VIT_03s0038g02320 RR

108.82

162.77

198.84

197.09

172.47

Control

0.84

0.56

0.35

0.39

0.29

RR

1.32

0.42

0.26

0.33

0.51

Control

6.20

3.01

0.31

0.16

0.18

RR

4.59

5.87

0.15

0.00

0.00

Control

1444.51

1004.23

1111.86

1038.52

827.90

RR

1310.63

1262.00

1241.18

815.26

859.99

VIT_04s0008g05490

VIT_04s0023g03750

VIT_06s0004g03550



Page 22 of 29

Control

245.63

221.77

168.25

56.45

44.60

VIT_08s0040g03150 RR

530.87

393.91

158.83

35.94

65.22

Control

0.99

0.38

0.24

0.84

0.71

RR

0.55

0.44

0.38

0.32

0.38

Control

128.14

171.06

345.65

292.94

171.40

RR

117.66

228.12

260.71

209.28

143.09

Control

38.16

22.72

16.78

10.70

2.83

VIT_17s0053g00180

VIT_18s0001g02470

VIT_18s0001g06370 RR

45.85

19.26

12.98

7.15

2.68

Control

0.97

0.76

0.25

0.56

0.54

RR

0.21

0.23

0.35

0.55

0.36

Control

0.49

0.34

0.56

0.81

0.45

RR

0.64

0.54

0.76

0.93

0.56

Control

112.08

126.95

581.15

490.22

438.18

RR

120.36

129.82

769.69

518.17

438.40

Control

5.95

3.70

3.59

6.05

5.38

VIT_19s0014g02400

VIT_19s0014g02410

Carotenoids metabolism IDI; isopentenyl-diphosphate VIT_04s0023g00600 delta-isomerase GPS; geranyl diphosphate VIT_15s0024g00850 synthase

RR

4.73

3.98

3.62

6.58

5.39

FDPS; farnesyl diphosphate

Control

201.82

165.94

79.19

46.18

35.27

RR

153.91

128.99

54.01

36.81

30.55

Control

28.82

23.97

43.78

39.79

29.12

RR

30.42

24.52

45.81

35.96

27.40

Control

24.77

18.85

6.08

3.38

1.64

VIT_19s0015g01010 synthase GGPS; geranylgeranyl VIT_03s0038g03050 diphosphate synthase VIT_04s0023g01210 RR

26.74

17.37

3.87

2.80

2.47

Control

14.36

9.12

2.97

0.65

0.48

RR

11.55

8.88

1.49

0.27

0.15

Control

42.92

8.10

10.60

11.17

4.00

RR

35.80

7.53

11.89

13.83

4.90

Control

22.25

20.79

4.47

2.46

1.21

VIT_05s0020g01240

VIT_18s0001g12000

VIT_19s0090g00530 crtB; phytoene synthase

RR

11.27

12.99

3.56

2.66

2.30

Control

18.54

10.57

8.82

9.89

1.70

RR

16.41

14.23

7.67

4.69

1.13

Control

0.07

0.00

0.00

0.10

0.02

RR

0.23

0.02

0.00

0.05

0.00

Control

4.16

1.51

2.11

1.76

3.62

VIT_04s0079g00680

VIT_06s0004g00820

VIT_12s0028g00960 PDS; 15-cis-phytoene

RR

2.32

1.35

4.63

6.44

5.34

Control

14.53

8.41

6.75

9.87

8.10

RR

8.65

8.23

7.02

10.48

9.66

Control

10.49

4.54

24.62

26.58

37.48

RR

9.71

9.97

36.23

62.08

43.52

Control

19.42

9.65

19.95

23.24

18.10

VIT_09s0002g00100 desaturase ZDS; zeta-carotene desaturase VIT_14s0030g01740 Z-ISO; zeta-carotene isomerase VIT_05s0062g01110 crtISO; prolycopene isomerase

RR

14.77

12.80

22.50

25.46

18.95

Control

6.17

2.99

7.84

11.39

9.62

RR

3.86

4.11

9.91

20.88

15.79

Control

26.67

18.13

25.99

22.43

17.02

VIT_08s0032g00800 crtL1; lycopene beta-cyclase

VIT_08s0007g05690


Page 23 of 29


RR crtZ; beta-carotene

22.43

27.21

22.39

23.87

18.98

Control

1.11

1.93

2.72

0.83

1.71

RR

14.06

10.29

2.98

1.81

1.29

Control

56.24

31.54

16.14

6.23

0.78

VIT_02s0025g00240 3-hydroxylase VIT_16s0050g01090 LUT5; beta-ring hydroxylase

RR

149.61

68.33

7.46

1.23

1.55

Control

22.09

6.93

9.91

15.94

8.71

RR

14.33

7.44

8.73

16.94

8.45

Control

7.77

2.45

3.14

4.50

2.09

RR

7.39

2.44

3.71

4.00

4.11

Control

10.23

2.99

3.49

5.83

3.59

VIT_04s0023g00080 VDE; violaxanthin de-epoxidase VIT_04s0043g01010 ZEP; zeaxanthin epoxidase VIT_07s0031g00620 crtL2; lycopene epsilon-cyclase

RR

6.08

5.11

3.07

7.01

3.85

Control

15.26

5.27

2.90

2.66

0.91

RR

11.13

5.85

2.73

2.35

1.33

Control

5.63

2.71

2.46

2.71

1.55

RR

4.56

2.55

2.57

2.31

2.44

VIT_11s0016g01880 LUT1; carotene VIT_08s0007g04530 epsilon-monooxygenase

534

Note: Expression values are shown in FPKM for each sample, and the RPKM values indicating specific

535

up-regulation or down-regulation are shown in bold (Gene with a false discovery rate (FDR) < 0.05, and the

536

estimated absolute log2 fold change (FC) > 1 were used as the thresholds for judging significant difference in

537

transcript expression). S1, fruitlet; S2, immature green; S3, before veraison; S4, veraison; S5, fully ripe; RR, root

538

restriction.

539 540 541 542 543 544

Figure 1. Contents of ascorbic acid in grape berry during different developmental stages. S1, fruitlet; S2,

545

immature green; S3, before veraison; S4, veraison; S5, fully ripe; RR, root restriction. * indicates the significant

546

differences (p < 0.05).

547 548

Figure 2. The ascorbic acid and carotenoids metabolic pathways in grape berry. GME, GDP-D-mannose 3',

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5'-epimerase; VTC2_5, GDP-L-galactose phosphorylase; VTC4, inositol-phosphate phosphatase/L-galactose

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1-phosphate phosphatase; GalDH, L-galactose dehydrogenase; GLDH, L-galactono-1,4-lactone dehydrogenase;

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NADH, monodehydroascorbate reductase; E1.10.3.3, L-ascorbate oxidase; E1.11.1.11, L-ascorbate peroxidase;

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L-IdnDH, L-idonate dehydrogenase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; IDI,

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isopentenyl-diphosphate delta-isomerase; GPS, geranyl diphosphate synthase; FDPS, farnesyl diphosphate



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synthase; GGPS, geranylgeranyl diphosphate synthase; crtB, phytoene synthase; PDS, 15-cis-phytoene desaturase;

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Z-ISO, zeta-carotene isomerase; ZDS, zeta-carotene desaturase; crtISO, prolycopene isomerase; crtL1, lycopene

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beta-cyclase; crtZ, beta-carotene 3-hydroxylase; LUT5, beta-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE,

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violaxanthin de-epoxidase; crtL2, lycopene epsilon-cyclase; LUT1, carotene epsilon-monooxygenase. Boxes from

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left to right follow the berry development. The data set was normalized to the values at the before veraison stage in

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the control treatment as log2 transformed, and the relative expression changes at the other treatment and other

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stages in the relation to the before veraison stage in the control treatment were hence expressed as log2 fold change,

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the upper set of boxes is for the control treatment and the lower set is for the RR treatment. Red arrows represent

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steps in AsA catabolism, this pathway may occur enzymatically or non-enzymatically. Dotted line represent some

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steps were omitted.

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Figure 3. Carotenoids concentration in berries during the developmental stages. S1, fruitlet; S2, immature green;

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S3, before veraison; S4, veraison; S5, fully ripe. * indicates the significant differences (p < 0.05).

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Figure 4. qRT-PCR validation of differentially expressed transcripts between two treatments of grape berries

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during the ripening. (A), Transcript relative expression level was measured by RNA-Seq and qRT-PCR. a, fruitlet

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in the control treatment; b, immature green in the control treatment; c, before veraison in the control treatment; d,

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veraison in the control treatment; e, fully ripe in the control treatment; f, fruitlet in the RR treatment; g, immature

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green in the RR treatment; h, before veraison in the RR treatment; i, veraison in the RR treatment; j, fully ripe in

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the RR treatment. The error bars represent the standard errors. (B), Correlation of fold change analyzed by

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RNA-Seq (x axis) and the data obtained using qRT-PCR (y axis).

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Figure 2

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Figure 3

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Figure 4

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TOC Graphic

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Transcriptomic analyses of Ascorbic acid and Carotenoid metabolites influenced by root restriction during

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Feng Leng, Dandan Tang, Qiong Lin, Jinping Cao, Di Wu, Shiping Wang, Chongde Sun

grape berry development and ripening

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For Table of Contents Only


Transcriptomic Analyses of Ascorbic Acid and Carotenoid Metabolites

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