Accumulation of Anthocyanin and its Associated Gene Expression in

Subscriber access provided by YORK UNIV

Food and Beverage Chemistry/Biochemistry

Accumulation of Anthocyanin and its Associated Gene Expression in Purple Tumorous Stem Mustard (Brassica juncea var. tumida Tsen et Lee) Sprouts when Exposed to Light, Dark, Sugar and Methyl Jasmonate Qiaoli Xie, Fei Yan, Guoping Chen, Zongli Hu, Shuguang Wei, and Jianghua Lai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04706 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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

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 35

Journal of Agricultural and Food Chemistry

1

Accumulation of Anthocyanin and its Associated Gene Expression in

2

Purple Tumorous Stem Mustard (Brassica juncea var. tumida Tsen et

3

Lee) Sprouts when Exposed to Light, Dark, Sugar and Methyl

4

Jasmonate

5

6

Qiaoli Xie1,Fei Yan3, Zongli Hu2, Shuguang Wei1, Jianghua Lai1*,Guoping Chen2*

7 8

1 Key Laboratory of the Education Ministry for Environment and Genes Related to Diseases,

9

Xi’an, Shaanxi, 710061 People’s Republic of China; 2 Bioengineering College, Campus B,

10

Chongqing University, 174 Shapingba Main Street, Chongqing 400030, People’s Republic of

11

China; 3 School of Energy and Power Engineering, Chongqing University, 174 Shapingba

12

Main Street, Chongqing 400030, People’s Republic of China.

13

*Corresponding author: Jianghua Lai & Guoping Chen.

14

E-mail: [email protected] & [email protected]

15

16

17

18

19 1

ACS Paragon Plus Environment


ABSTRACT

20

21

Tumorous stem mustard is a characteristic vegetable in Southeast Asia, as are its

22

sprouts. The purple color of the purple variety "Zi Ying" leaves is because of

23

anthocyanin accumulation. The ways in which this anthocyanin accumulation is

24

affected by the environment and hormones has remained unclear. Here, the impacts of

25

sucrose, methyl jasmonate (MeJA), light and dark on the growth and anthocyanin

26

production of “Zi Ying” sprouts were explored. The results showed that Anthocyanins

27

can be enhanced by sucrose in sprouts under light condition, and MeJA can promote

28

anthocyanins production under light and dark conditions in sprouts. The anthocyanin

29

biosynthetic regulatory genes BjTT8, BjMYB1, BjMYB2 and BjMYB4, and the EBGs

30

and LBGs were upregulated under light conditions while BjTT8, BjMYB1 and BjMYB2

31

and anthocyanin biosynthetic genes BjF3H and BjF3’H were upregulated under DM

32

condition. These results indicate that sucrose and methyl jasmonate can stimulate the

33

expression of genes encoding components of the MBW complex (MYB, bHLH and

34

WD40) and that they transcriptional activated the expression of LBGs and EBGs to

35

promote the accumulation of anthocyanins in “Zi Ying” sprouts. Our findings enhance

36

our understanding of anthocyanin accumulation regulated by sucrose and MeJA in “Zi

37

Ying”, which will help growers to produce anthocyanin-rich foods with benefits to

38

human health.

39

KEYWORDS Anthocyanin, tumorous stem mustard, sprouts, transcriptional

40

regulation, light, sucrose, methyl jasmonate 2


Page 2 of 35

Page 3 of 35


INTRODUCTION

41

42

Anthocyanins, which are a subclass of flavonoids, the primary water-soluble

43

pigments, are important visible plant pigments that are responsible for purple, blue,

44

orange, red colors in leaves, flowers, seeds and fruits, and they can attract insects for

45

pollination and seed dispersal1. They are glycosides of polyhydroxy or flavylium salts2.

46

Anthocyanins are powerful antioxidants that can be stimulated under stresses or

47

infection by pathogens3. Many studies indicate that anthocyanin-rich foods are good for

48

human health, and they can against inflammation, cardiovascular diseases, cancers, and

49

other chronic disorders4-5.

50

The anthocyanin biosynthesis pathway has been studied extensively in plants6-7.

51

Anthocyanin synthesis is a branch of the phenylpropanoid pathway. The structural

52

genes, which participate directly in anthocyanin biosynthesis, have been reported in

53

many plants8-11. These structural genes are divided into three categories of genes,

54

namely beginning, early and late biosynthetic genes12. The three beginning genes are

55

phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-

56

coumaryol CoA ligase (4CL), and they are in the phenylpropanoid pathway. The early

57

biosynthetic genes (EBGs) of anthocyanin synthesis include CHS (chalcone synthase),

58

CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), and F3’H (flavanone 3’-

59

hydroxylase). The late biosynthetic genes (LBGs) are DFR (dihydroflavonol 4-

60

reductase), ANS (anthocyanidin synthase) and UFGT (UDP-glucose: flavonoid 3-O-

61

glucosyl transferase), which form the main pathway of anthocyanin biosynthesis.

62

Glucoside can be transfered to the 3-O position of flavonoids by UFGT in regulating 3



63

anthocyanin biosynthesis (Figure 1).

64

A major regulatory mechanism of the anthocyanin biosynthesis pathway is at the

65

transcriptional level of the structural genes13-14. The structural genes are known to be

66

regulated by the MBW (R2R3-MYB, bHLH and WD40) ternary protein complex,

67

which is formed by MYB transcription factors, bHLH (helix-loop-helix) and WD40

68

proteins. They can transcriptional activate the structural genes in the anthocyanin

69

biosynthetic pathway by binding to their promoters15. The expression of the EBGs is

70

regulated by R2R3-MYB transcription factors, and the LBGs are modulated by the

71

MBW complex12. Importantly, the MBW complex is also necessary to activate EBGs

72

in maize16. In Arabidopsis, the R2R3-MYB genes Production of anthocyanin pigment

73

1 (PAP1), PAP2, MYB113, and MYB114; the bHLH genes TT8; and the WD40 gene

74

TTG1 are the key genes that encode the components of the MBW complex17. Moreover,

75

it has been reported that the other transcription factors and proteins MAPK Cascade18,

76

NAC19, MdERF1B20, and CRY1a15 could stimulate anthocyanin biosynthesis as well.

77

Anthocyanins can be induced by various developmental signals, sugar, plant

78

hormones, and environmental stresses such as high-intensity light, UV light,

79

temperature, pathogen infection, drought, wounding, and nutrient deficiency21. Light is

80

a key environmental factor for anthocyanin biosynthesis, and it acts by control the genes

81

in the pathway22. Blue light can quickly stimulate the accumulation of anthocyanins in

82

strawberry fruits23. In juvenile, anthocyanin accumulation is response to irradiance

83

according to the leaf sensitivity to light24. The functions of R2R3-MYB genes are seems

84

conserved between monocots and eudicots, and the transcriptional regulation is a major 4


Page 4 of 35

Page 5 of 35


85

mechanism25. Green light controls the anthocyanin production in microgreens26. In

86

Arabidopsis, light-induced anthocyanin accumulation requires MYB75 phosphorylated

87

by MPK427. Sucrose is a crucial messengers for plant growth and development, and the

88

regulation of multiple genes depends on the amount of soluble sugars present28. The

89

enhancement of sucrose on anthocyanin biosynthesis in many plant species have been

90

reported. For example, AtMyb56 regulates anthocyanin levels by modulating AtGPT2

91

expression in response to sucrose in Arabidopsis29. A ubiquitin-like protein, AtSDE2,

92

inhibits sucrose induced anthocyanin accumulation in Arabidopsis30. In apples,

93

MdJAZ18 interacts with MdSnRK1 to regulate sucrose induced anthocyanin

94

biosynthesis31. Methyl jasmonate (MeJA) has been identified as a key regulator that

95

participates in many developmental processes such as root growth, seed germination,

96

fertility, senescence, fruit ripening, anthocyanin accumulation and trichome

97

formation32 as well as defense responses against stresses such as UV damage, pathogen

98

infection, insect attack and wounding33. Many previous studies have reported that

99

MeJA can promote anthocyanin accumulation in different plants such as red

100

raspberries34, grapevines35, apples36, blueberries37 and radish sprouts38.

101

Tumorous stem mustard (B. juncea var. tumida Tsen et Lee) is a cruciferous

102

vegetable in Asian countries. They are popular for their special nutritional value and

103

flavors. Its succulent and swollen stem are always pickled for products. The purple

104

tumorous stem mustard is a mutation of Cruciferae that displays abundant anthocyanin

105

accumulation in their leaves39. Therefore, purple tumorous stem mustard could be used

106

in pharmaceutical, health protection and cosmetic industries. The cotyledons of its 5



107

sprouts contain prolific anthocyanins are also popular. Moreover, we have found that

108

the purple tumorous stem mustard predominantly contains cyanidin as its aglycone, and

109

the anthocyanin biosynthesis in leaves of “Zi Ying” is induced by regulatory genes40.

110

However, the way in which this process is affected by the environment and hormones

111

has remained unclear.

112

In our study, the impacts of sucrose, methyl jasmonate (MeJA), light and dark on

113

the growth and anthocyanin accumulation of purple tumorous stem mustard “Zi Ying”

114

sprouts were explored. The fresh weight, hypocotyl length, cotyledon diameter, the

115

major anthocyanin contents, and the transcriptional level of regulatory and structural

116

genes related to anthocyanin synthesis were examed in the sprouts of “Zi Ying” under

117

light (L), light+sucrose (LS), light+MeJA (LM), dark (D), dark+sucrose (DS) and

118

dark+MeJA (DM) from 0 to 12 days after sowing (DAS).

119

MATERIALS AND METHODS

120

1. Plant materials and growth conditions

121

The purple tumorous stem mustard cultivar “Zi Ying” was used as the experimental

122

material. Its seeds were first surface-sterilized used 70% ethanol for 1 minute, and then

123

washed 3 times using sterile water, then rinsed with 1% sodium hypochlorite solution

124

for 15 minutes, and finally washed eight times using sterile water. The sterilized seeds

125

were simultaneously sown in three different mediums. Some sterilized seeds were

126

germinated on ½ MS medium, some were germinated on ½ MS medium with 150 mM

127

sucrose and the others were germinated on ½ MS medium containing 100 μM MeJA. 6


Page 6 of 35

Page 7 of 35


128

For biological replicates, each medium was dispensed into ten bottles. There were

129

approximately 200 to 300 seeds per bottle. These seeds were all cultivated in one

130

growth chamber under light condition (16 h light/8 h dark) or dark condition (24 h dark),

131

440 μmol/m2/s light intensity at 25°C, 60% humidity. Uniformly sized sprouts were

132

sampled with liquid nitrogen from each bottle at 3, 6, 9, and 12DAS, and then stored at

133

-80°C for anthocyanin extraction and RNA isolation. Twenty to 30 sprouts were used

134

to measure the lengths and weights of the sprouts every replication.

135

2. RNA extraction and Quantitative Real-time PCR

136

We employed three bunches of mustard sprouts with the same growth status as three

137

groups (n=20 to 30 per group). These three groups were sampled separately for RNA

138

extraction and reverse transcription experiments. The total RNA was isolated from the

139

sprout samples with RNAiso (TaKaRa). And 1 to 2 µg of total RNA was used to

140

synthesize the cDNA with Reverse Transcriptase (TaKaRa) and Oligo-dT primers.

141

Then, the cDNAs were diluted 1 time with RNase-free water. qPCR analysis was

142

performed in the CFX96™ Real-Time System (C1000™ Thermal Cycler, Bio-Rad).

143

All the reactions were performed using a SYBR® Premix Taq II kit (TaKaRa) in a 10

144

μl total sample volume (3.5 μl of ddH2O, 5.0 μl of 2×SYBR Premix Taq, 1.0 μl of

145

cDNA and 0.5 μl of each primer). There were three technical repetitions for each

146

individual sample. NTCs (no template control) and NRTs (no reverse transcription

147

control) were used to remove the influence of the DNA and the environment. The melt

148

curve analysis of each gene primer and standard curves were run simultaneously. To 7



Page 8 of 35

149

confirm the specific amplification, the PCR products were sequenced. The

150

housekeeping gene BjEF-1-α41 was used as an internal standard. The expression of the

151

genes in sprouts from 3DAS without treatment were all set to 1. The primers are listed

152

in Table 1S.

153

3. Chemicals

154

We purchased anthocyanin cyanidin-3-glucoside chloride from Phytolab (Germany),

155

use as the external standards. HPLC-grade water was prepared from Milli-Q system

156

(Millipore Laboratory, Bedford, MA). HPLC-grade formic acid and methanol (MeOH)

157

were purchased from Sigma. All of the other solvents were provided by Aldrich (St.

158

Louis, MO).

159

4. Extraction of Anthocyanin and HPLC Analysis

160

The anthocyanins were extracted and separated by HPLC according to the modified

161

procedure reported by Prior and Wu40, 42. The cotyledons of fresh mustard seedlings, up

162

to approximately 5-6 grams (n=100 to 120), were freeze-dried. One hundred mg of the

163

dried samples was extracted in 1 mL (85:15:0.5) methanol/H2O/acetic acid at room

164

temperature for 10 min and centrifuged for 10 min at 12000g. Then the supernatants

165

were filtered by a 0.2 μm polytetrafluoroethylene (PTFE) syringe filter to remove the

166

cell debris. After that, the samples were analyzed using a HPLC (Waters, 2795, MA)

167

equipped with a variable wavelength detector. The results were analyzed with Waters

168

2795 HPLC ChemStation software. The loading volume was 10 μL.

8


The

Page 9 of 35


169

chromatographic separation was carried out on a Zorbax Stablebond Analytical SB-

170

C18 column (4.6 × 150 mm, 5 μm, Waters). An elution was carried out using mobile

171

phase A (aqueous 2% formic acid solution) and mobile phase B (methanol). The

172

detection was performed at 520 nm, and the column oven temperature was set to 40°C.

173

The flow rate was 1 mL/min. The gradient program is described as follows: 0−2 min,

174

10−20% B; 2−40 min, 20−55% B; 40−45 min, 55−60% B; 45−60 min, 90% B; and

175

60−65 min, 10% B. This process was repeated 3 separate times. Finally, statistical

176

analyses were performed. The quantification of the total anthocyanin was calculated as

177

equivalents of the external standards and based on the peak areas. The results were

178

showed as milligrams per gram of dry weight (DW).

179

5. Statistical analysis

180

The mean values of data were taken from the measurements of the replicates, and the

181

‘Standard Errors’ of the means were calculated. The data were analyzed with Origin

182

9.0 using Student’s t-test to assess the significant differences between the means.

183

184

RESULTS AND DISCUSSION 1. The effects of light, dark, sucrose and MeJA on sprout development

185

The sprouts under the light (L) and dark (D) conditions were examined every 3

186

days until the 12th day after sowing (DAS). The sprouts under the L condition were

187

strong and short. The cotyledons were purple. Compared with the sprouts grown under

188

L, the sprouts grown in D were thinner and longer, and their cotyledons were yellow 9



189

(Figure 2A). The length of the sprouts increased with age (Figure 2B). Furthermore, the

190

cotyledons grown under the LS and LM conditions displayed smaller sizes and a deeper

191

purple color than those grown only under the L condition. Small cotyledons appear

192

yellow under dark conditions (Figure 2C). The sprouts grown under LS, LM, DS and

193

DM conditions were all apparently shorter than they were under the L and D conditions,

194

respectively. The lengths of almost the hypocotyls grown under L were lower than those

195

grown in D. The lengths of the sprouts were approximately equal to one another under

196

the DM and L conditions. Moreover, the sprouts under the D condition were the longest

197

and they grew the fastest. The sprouts were the shortest under the LM condition (Figure

198

2B). These results indicate that darkness induced hypocotyl elongation in “Zi Ying”.

199

This finding is similar to the results of a recent study in Arabidopsis43. Furthermore,

200

150 mM sucrose and 100 μM MeJA inhibits hypocotyl elongation, and the effect of

201

100 μM MeJA is stronger than that of sucrose. The fresh weight increased along with

202

the growth of the sprouts (Figure 2D). Under the L condition, the fresh weight of the

203

sprouts maintained a relatively stable growth trend and the growth rate was almost

204

unchanged. After 6DAS, the fresh weight of sprouts grown under LS was lower than

205

that under the L condition and the growth rate of the sprouts was also decreased. The

206

fresh weight and growth rate of sprouts grown under LM were always lower than those

207

grown under the L condition. The fresh weight and growth rate of sprouts grown under

208

DS were much higher than they were under the other conditions. By contrast, under the

209

DM condition, the fresh weight and growth rate of the sprouts were obviously lower

210

than they were under the D and DS conditions, and they were even lower than they 10


Page 10 of 35

Page 11 of 35


211

were under the LM condition after 6DAS. In addition, compared with the sprouts grown

212

under the L condition, the fresh weights of sprouts grown under D and DS were greater

213

(Figure 2D). The growth rate was gradually reduced after 9DAS. Therefore, dark and

214

sucrose can promote the production of “Zi Ying” sprouts, consistent with the findings

215

in corn44. Except under DM, the growth rate of the sprouts was fastest before 9DAS.

216

These results indicate that the maximum biomass of “Zi Ying” sprouts can be reached

217

under darkness within approximately 8 to 9 days, which is concordant with the findings

218

in buckwheat45.

219

In addition, figure 2D shows that the fresh weight of the sprouts under the LS

220

condition exceed that under the L condition before 6DAS, and then they become

221

gradually lower than they are under the L condition. One reasonable explanation for

222

this result is that the pigment production in the cotyledons of the “Zi Ying” sprouts

223

reduced the effectiveness of the photosynthesis by suppressing the light capture of

224

chlorophyll46. In addition, lots of production of anthocyanin consumed a great deal of

225

nutrients and energy from the plant itself during development. Under the LM condition,

226

the weights of the sprouts were always lower than those of the sprouts under the L

227

condition, which may be because MeJA inhibits the development of sprouts, and the

228

higher anthocyanin production in the cotyledons may inhibits the development of

229

sprouts as well. Measurements of the cotyledon diameters under different treatments

230

showed that the cotyledons under the D, DS and DM conditions grow quite slowly and

231

their diameters are always notably lower than they are under the L condition at 6 or

232

9DAS. Moreover, under the LS and LM conditions, the cotyledon diameters were also 11



233

significantly smaller than they were under the L condition (Figure 2E). These results

234

illustrate that sucrose and MeJA may inhibit the growth of cotyledons to varying

235

degrees, and the inhibited effect of MeJA was more pronounced. Darkness can also

236

inhibit the growth of cotyledons.

237

Growth and development of plants is a high energy consumption process. Sugars

238

are the key components of a plants’ energy source (carbon source)47. Plant growth

239

includes cell volume increases and cell division48. These molecular processes demand

240

the energy and biomass from carbohydrates49. In the presence of sucrose, the

241

overexpression of a grapevine sucrose transporter (VvSUC27) in tobacco improves the

242

plant growth rate50. Sucrose transporter 2 contributes to maize growth, development,

243

and crop yield44. Jasmonates (JAs) have been shown to inhibit plant growth, and the

244

mechanisms are not well understood. In Arabidopsis, methyl jasmonate (MeJA)

245

controls leaf growth by repressing cell proliferation and the onset of

246

endoreduplication51, and it inhibits hypocotyl elongation52. It is worth mentioning that

247

compared with the light conditions, the fresh weights of “Zi Ying” sprouts were greater

248

under D and DS, which is similar to the previous reports on buckwheat45 and radish

249

sprouts38.

250

2. Anthocyanidin contents in sprout development

251

To evaluate the influence of sucrose and MeJA on anthocyanin accumulation in

252

sprouts, the anthocyanin content of sprouts under different L and D conditions were

253

detected every 3 days until 12DAS. In Figure 3, compared with the L, LS and LM 12


Page 12 of 35

Page 13 of 35


254

conditions, anthocyanins were seldom detected in sprouts grown under the D, DS and

255

DM conditions, which is consistent with their phenotype (yellow). However, a large

256

amount of anthocyanins was accumulated and the contents increased quickly from

257

3DAS to 9DAS under the L, LS and LM conditions. At 12DAS, the anthocyanin content

258

remained higher than it was at 9DAS under the L and LM conditions. At the same stage,

259

the anthocyanin levels under the LS condition were the highest, followed by those under

260

the LM and L conditions. For example, at 9DAS, the anthocyanin content reached

261

approximately 1.6 mg g-1 DW under the LS condition, which was approximately 3 and

262

1.8-times higher than the contents under the L and LM conditions, respectively. The

263

data from 3, 6 and 12DAS also showed that the anthocyanin contents under the LS

264

condition were the highest, followed by the contents under the LM and L conditions.

265

These results suggest that light is a fundamental factor in anthocyanin accumulation in

266

“Zi Ying”. Furthermore, under L condition, 150 mM sucrose and 100 μM MeJA can

267

induce the accumulation of anthocyanins, and the sucrose is a better inducer.

268

Previous studies have shown that sucrose and MeJA can induce anthocyanin

269

accumulation. For example, sucrose can induced anthocyanin biosynthesis in the

270

vegetative tissue of Petunia plants53. A ubiquitin-like protein, AtSDE2, inhibits

271

sucrose-induced anthocyanin accumulation in Arabidopsis30. The ANGUSTIFOLIA3-

272

YODA gene in Arabidopsis induces anthocyanin accumulation according to the sucrose

273

levels54. MdJAZ18 interacts with MdSnRK1 to regulate sucrose induced anthocyanin

274

biosynthesis31. Additionally, MeJA has been shown to stimulate anthocyanin

275

accumulation in black currants55. Methyl jasmonate promotes anthocyanin production 13



276

in Prunus salicina x Prunus persica shoot cultures. These results supported our findings

277

that sucrose and MeJA treatments induce a higher production of anthocyanins during

278

“Zi Ying” sprout development.

279

3. Transcript regulation of Anthocyanin Biosynthetic Genes in Sprouts

280

To determine the relationship between expression of anthocyanin biosynthetic

281

genes and the anthocyanin accumulation in“Zi Ying” sprout, the mRNA transcript

282

levels of anthocyanin biosynthetic genes were explored by qPCR in sprouts grown

283

under L, LS, LM, D, DS and DM conditions every 3 days until 12DAS (Figure 4, 5).

284

Under the L condition, the transcript levels of the EBGs, namely BjPAL, BjCHI and

285

BjF3’H, and the LBGs, specifically BjDFR and BjANS, were upregulated at 6DAS. In

286

particular, the BjPAL and BjANS levels were approximately 2.2-folds higher than they

287

were at 3DAS. The expression of these five structural genes were significantly

288

decreased after 6DAS. From 3DAS to 12DAS, the expression of BjC4H was relatively

289

stable. The expression of BjCHS, BjF3H and BjUFGT showed a downward trend, and

290

the expression of BjUFGT increased slightly during the 12DAS. Under the LS condition,

291

from 3DAS to 12DAS, the expression levels of BjPAL and BjC4H showed an upward

292

trend; BjCHS, BjF3H, BjF3'H, BjDFR, BjANS and BjUFGT all showed the highest

293

expression at 6DAS and then gradually decreased; and the change in BjCHI was not

294

significant. In particular, BjANS was induced approximately 4 times and BjF3H, BjDFR

295

and BjUFGT were induced approximately 2 times relative to the level at 3DAS. During

296

the entire process, compared to the L condition, the expression of BjCHI, BjC4H, 14


Page 14 of 35

Page 15 of 35


297

BjF3H, BjDFR, BjANS and BjUFGT was significantly induced under the LS condition.

298

The expression of BjF3H, BjANS and BjUFGT was 2-3 times greater than it was under

299

the L condition. Under the LM condition, BjPAL and BjC4H showed a downward trend

300

from 3DAS and gradually recovered after 9DAS. The expressions of BjCHI and BjF3’H

301

were the highest at 6DAS, and then they gradually decreased. BjCHS and BjF3H did

302

not change significantly during the growth cycle. The three downstream genes, BjDFR,

303

BjANS and BjUFGT, showed an upward trend in terms of induction. In particular,

304

BjDFR and BjANS were induced from 3DAS to 12DAS. In general, under the LM

305

condition, the expression of BjPAL was inhibited and the other eight genes were

306

basically induced compared to those under the L condition. From the above results, it

307

can be concluded that both sucrose and MeJA can induce the synthesis of anthocyanins

308

in “Zi Ying” seedlings under light conditions, but their regulation mechanisms were

309

different. Sucrose primarily induces BjC4H, BjCHS, BjCHI, BjF3H, BjDFR, BjANS

310

and BjUFGT while MeJA primarily induces BjC4H, BjCHI, BjDFR, BjANS and

311

BjUFGT.

312

Under the D condition, BjPAL and BjCHS had the highest expression at 6DAS,

313

followed by a decline; BjC4H showed the highest expression at 9DAS, then decreased;

314

BjCHI, BjF3H, BjF3'H and BjDFR did not change significantly; and BjANS and

315

BjUFGT showed a gradual declining trend. Under the DS condition, BjPAL, BjC4H and

316

BjANS showed the highest expression at 6DAS, followed by a downward trend. BjCHS,

317

BjF3H, BjF3’H and BjUFGT generally showed a downward trend. BjCHI showed an

318

upward trend and BjDFR did not change significantly during the entire process. Overall, 15



319

the expression of BjC4H was inhibited compared to what it was under the D condition.

320

Under the DM condition, BjPAL, BjCHS, BjF3H, BjDFR, BjANS and BjUFGT showed

321

a gradual declining trend. The expressions of BjC4H, BjCHI and BjF3'H were highest

322

at 6DAS and then gradually decreased. In summary, the expression of BjPAL was

323

significantly inhibited under the DM compared to the D condition; BjF3H and BjF3'H

324

were obviously induced from 3 to 12DAS, and BjF3'H especially was induced

325

approximately 50-fold under the DM condition. Despite the lack of visual anthocyanins

326

in sprouts cultured in the dark, low expression levels were observed for all these

327

structural genes, which is consistent with previous reports in other plant species56. From

328

these results, we can assume that under the D condition, the effect of sucrose on the

329

anthocyanin synthesis genes in “Zi Ying” sprouts were not significant except at

330

inhibiting the expression of BjC4H. MeJA can notably induce the expression of BjCHI,

331

BjF3H and BjF3’H under the D condition.

332

Anthocyanin biosynthesis is often regulated by transcriptional control of

333

anthocyanin biosynthetic genes. Therefore, we analyzed the expression of

334

transcriptional regulatory genes BjTTG1(WD40), BjMYB1, BjMYB2, BjMYB3, BjMYB4

335

and BjTT8(bHLH) (the homologs of AtTTG1, AtMYB75(PAP1), AtMYB90(PAP2),

336

AtMYB113, AtMYB114 and AtTT8, respectively) in “Zi Ying” sprouts. Under the LS

337

condition, with the exception of BjMYB3, the expression of the other five genes in

338

sprouts were upregulated to varying degrees compared to the levels under the L

339

condition. BjTT8 was first induced and the highest expression was found at 6DAS.

340

BjMYB1 and BjMYB4 were induced relatively later, with the highest expression at 16


Page 16 of 35

Page 17 of 35


341

9DAS. BjMYB2 and BjTTG1 expression showed an upward trend. The expression of

342

BjTT8 increased by 1.5 times, BjMYB1 increased by 10 times and BjMYB4 increased

343

by 12 times at 12DAS. BjTTG1 doubled and BjMYB2 increased by approximately 15

344

times. Under the LM condition, the transcript level of BjTT8 showed an induced upward

345

trend. The expression of BjMYB1 was higher than it was under the L condition, even

346

up to 12 times at 3DAS. BjMYB2, BjMYB4 and BjTTG1 were highly expressed at 3DAS,

347

and then their results were gradually consistent with those of the L condition. The

348

expression of BjMYB3 was not significantly changed over the 12-day growth cycle

349

(Figure 6). Under the DS condition, the expression of BjTT8 and BjMYB1 were induced

350

by sucrose compared with the D condition. The expression of other genes was not

351

significantly changed. Under the DM condition, BjMYB1, BjMYB2 and BjMYB4 were

352

highly expressed at 3DAS, and then they decreased gradually; the expression of BjTT8

353

began to be higher than the expression under the D condition after 6DAS. The

354

expression of BjMYB4 and BjTTG1 were inhibited by MeJA. There was no significant

355

change in BjMYB3 expression (Figure 7). These results suggest that under the L

356

condition, sucrose can induce the expression of BjTT8, BjMYB1, BjMYB2, BjMYB4 and

357

BjTTG1. Methyl jasmonate can induce the expression of BjTT8 and BjMYB1. Under the

358

D condition, sucrose can induce the expression of BjTT8. Methyl jasmonate induces

359

BjTT8, BjMYB1 and BjMYB2, and it inhibits the expression of BjMYB4 and BjTTG1.

360

Therefore, under the D condition, only the increase in BjTT8 could not induce the

361

expression of anthocyanin-synthesized structural genes. That is why there is almost no

362

accumulation of anthocyanins in “Zi Ying” sprouts treated with sucrose under the D 17



363

condition. However, under the DM condition, there was a small amount of anthocyanins

364

in the “Zi Ying” sprouts, indicating that methyl jasmonate can also induce anthocyanin

365

accumulation under darkness.

366

As we know, a major regulatory mechanism of the anthocyanin biosynthesis

367

pathway occurs at the transcriptional level of the structural genes13-14. The structural

368

genes are known to be regulated by the MBW (MYB-bHLH-WD40) protein ternary

369

complex. They activate the transcription of the structural genes in the anthocyanin

370

biosynthetic pathway by binding to promoters of the structural genes15. In this study,

371

the transcripts of BjTT8 (bHLH), BjTTG1(WD40), BjMYB1, BjMYB2, BjMYB3 and

372

BjMYB4(MYB) were detected, as shown in Figure 6 and 7. The results showed that

373

under the L condition, sucrose can induce the expression of BjTT8, BjMYB1, BjMYB2,

374

BjMYB4 and BjTTG1. Methyl jasmonate can induce BjTT8 and BjMYB1 expression.

375

The upregulation of these regulatory genes was positively correlated with the higher

376

expression of anthocyanin-synthesized structural genes BjC4H, BjCHI, BjF3H, BjF3’H,

377

BjDFR and BjANS and the accumulation of anthocyanins in the “Zi Ying” sprouts,

378

indicating, under the L condition, that sucrose and methyl jasmonate can stimulate the

379

expression of genes encoding the MBW complex, and it upregulated the expression of

380

LBGs and EBGs to promote the accumulation of anthocyanins in the “Zi Ying” sprouts.

381

As previously reported, in spinach, higher anthocyanin accumulation corresponds to

382

higher expression of anthocyanin biosynthesis genes13. AtMyb56 regulates the

383

anthocyanin levels through the modulation of AtGPT2 expression in response to

384

sucrose in Arabidopsis30. Anthocyanin accumulation induced by sucrose in the 18


Page 18 of 35

Page 19 of 35


385

vegetative tissue of Petunia needs anthocyanin regulatory transcription factors57.

386

Moreover, RsMYB was induced under the LM condition, and RsMYB overexpression

387

could upregulate the transcription of anthocyanin biosynthesis genes to promote

388

anthocyanin production58. The coronatine insensitive1 (COI1) mutant is pivotal for the

389

expression of structural gene DFR and the transcription factors PAP1, PAP2 and GL3

390

in JA-induced anthocyanin biosynthesis59. In Arabidopsis, the MBW complex is

391

participated in JA-regulated anthocyanin accumulation by regulating the ‘late’

392

anthocyanin biosynthesis genes60. There is crosstalk between the hormone signaling

393

pathways and sucrose in the regulation of the anthocyanin biosynthetic pathway61.

394

In addition, under the DM condition, a small amount of anthocyanin accumulation

395

in sprouts is positively correlated with the upregulation of the regulatory genes BjTT8,

396

BjMYB1 and BjMYB2, and the structural genes BjCHI, BjF3H and BjF3'H. As in

397

cabbage62 and Dongzao11, the upregulation of BjTT8, BjMYB1 and BjMYB2 and the

398

transcriptional activation of the structural genes likely to be the regulatory mechanism

399

of anthocyanin biosynthesis in “Zi Ying” under the DM condition. The accumulation

400

of anthocyanins was not completely dependent on light because methyl jasmonate can

401

induce anthocyanin biosynthesis under darkness. UV-B can induce anthocyanin

402

biosynthesis continues in the dark after irradiation in hypocotyls of radish sprouts63.

403

This accumulation provides evidence that it is possible to use less energy produce more

404

anthocyanin-rich sprouts. Furthermore, sucrose and MeJA promote the production of

405

anthocyanins in sprouts and yield the highest anthocyanin production at 9DAS and

406

12DAS, respectively. Judging from the fresh weights of the sprouts, sucrose not only 19



407

plays important roles in activating anthocyanin synthesis but it also promotes the yield

408

of “Zi Ying” sprouts. The best yield and the greatest anthocyanin contents in sprouts

409

can be obtained at approximately 9DAS. This result is similar to that of buckwheat45

410

and radish sprouts38.

411

In conclusion, the aim of the present work was to explore whether and how

412

anthocyanin accumulation is enhanced by MeJA and sucrose in combination with light

413

or dark in the cotyledons of the purple tumorous stem mustard “Zi Ying”. The results

414

suggested that sucrose and methyl jasmonate can stimulate the expression of genes

415

encoding the components of the MBW complex, and they upregulated the expression

416

of LBGs and EBGs to promote the accumulation of anthocyanins in “Zi Ying” sprouts.

417

Methyl jasmonate can even induce anthocyanin biosynthesis in the dark. Our findings

418

not only enhance our understanding on the molecular mechanisms of anthocyanin

419

accumulation regulated by sucrose and MeJA in “Zi Ying”, but they will also help

420

growers to produce anthocyanin-rich foods with potential benefits to human health.

421

ABBREVIATIONS USED

422

PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone

423

synthase; CHI, chalcone isomerase; F3H, flavone 3-hydroxylase; F3’H, flavonoid 3’-

424

hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; UFGT,

425

UDPglucose: flavonoid 3-O-glucosyltransferase; HPLC, high-performance liquid

426

chromatography; qRT-PCR, quantitative real-time reverse transcription PCR.

427

ACKNOWLEDGMENT 20


Page 20 of 35

Page 21 of 35


428

This work was funded by National Natural Science Foundation of China (31801870),

429

China Postdoctoral Science Foundation (2016M590951), Shaanxi Natural Science

430

Foundation (2016JQ8018), the Fundamental Research Funds for the Central

431

Universities and Shaanxi Postdoctoral Science Fundation.

432

SUPPORTING INFORMATION DESCRIPTION

433

A table of primers used for real-time PCR. This material is available free of charge via

434

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

435

436

437

438

439

440

441

442

443

444 21



445

REFERENCES

446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

1.

Yang, M.; I Koo, S.; O Song, W.; K Chun, O., Food matrix affecting anthocyanin bioavailability:

review. Current medicinal chemistry 2011, 18 (2), 291-300. 2.

Kong, J.-M.; Chia, L.-S.; Goh, N.-K.; Chia, T.-F.; Brouillard, R., Analysis and biological activities of

anthocyanins. Phytochemistry 2003, 64 (5), 923-933. 3.

Sun, L. L.; Gao, W.; Zhang, M. M.; Li, C.; Wang, A. G.; Su, Y. L.; Ji, T. F., Composition and antioxidant

activity of the anthocyanins of the fruit of Berberis heteropoda Schrenk. Molecules 2014, 19 (11), 19078-96. 4.

Li, P.; Li, Y. J.; Zhang, F. J.; Zhang, G. Z.; Jiang, X. Y.; Yu, H. M.; Hou, B. K., The Arabidopsis UDP-

glycosyltransferases UGT79B2 and UGT79B3, contribute to cold, salt and drought stress tolerance via modulating anthocyanin accumulation. Plant J 2017, 89 (1), 85-103. 5.

Butelli, E.; Titta, L.; Giorgio, M.; Mock, H. P.; Matros, A.; Peterek, S.; Schijlen, E. G.; Hall, R. D.; Bovy,

A. G.; Luo, J.; Martin, C., Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 2008, 26 (11), 1301-8. 6.

Cai, H.; Zhang, M.; Chai, M.; He, Q.; Huang, X.; Zhao, L.; Qin, Y., Epigenetic regulation of

anthocyanin biosynthesis by an antagonistic interaction between H2A.Z and H3K4me3. New Phytol 2018. 7.

Feng, K.; Xu, Z. S.; Que, F.; Liu, J. X.; Wang, F.; Xiong, A. S., An R2R3-MYB transcription factor,

OjMYB1, functions in anthocyanin biosynthesis in Oenanthe javanica. Planta 2018, 247 (2), 301-315. 8.

Bajpai, A.; Khan, K.; Muthukumar, M.; Rajan, S.; Singh, N. K., Molecular analysis of anthocyanin

biosynthesis pathway genes and their differential expression in mango peel. Genome 2018, 61 (3), 157166. 9.

Cao, K.; Ding, T.; Mao, D.; Zhu, G.; Fang, W.; Chen, C.; Wang, X.; Wang, L., Transcriptome analysis

reveals novel genes involved in anthocyanin biosynthesis in the flesh of peach. Plant Physiol Biochem 2018, 123, 94-102. 10. Jiang, W.; Liu, T.; Nan, W.; Jeewani, D. C.; Niu, Y.; Li, C.; Wang, Y.; Shi, X.; Wang, C.; Wang, J.; Li, Y.; Gao, X.; Wang, Z., Two transcription factors TaPpm1 and TaPpb1 co-regulate anthocyanin biosynthesis in purple pericarps of wheat. J Exp Bot 2018, 69 (10), 2555-2567. 11. Kou, X.; He, Y.; Li, Y.; Chen, X.; Feng, Y.; Xue, Z., Effect of abscisic acid (ABA) and chitosan/nanosilica/sodium alginate composite film on the color development and quality of postharvest Chinese winter jujube (Zizyphus jujuba Mill. cv. Dongzao). Food Chem 2019, 270, 385-394. 12. Chaves-Silva, S.; Santos, A. L. D.; Chalfun-Junior, A.; Zhao, J.; Peres, L. E. P.; Benedito, V. A., Understanding the genetic regulation of anthocyanin biosynthesis in plants - Tools for breeding purple varieties of fruits and vegetables. Phytochemistry 2018, 153, 11-27. 13. Cai, X.; Lin, L.; Wang, X.; Xu, C.; Wang, Q., Higher anthocyanin accumulation associated with higher transcription levels of anthocyanin biosynthesis genes in spinach. Genome 2018, 61 (7), 487-496. 14. Kodama, M.; Brinch-Pedersen, H.; Sharma, S.; Holme, I. B.; Joernsgaard, B.; Dzhanfezova, T.; Amby, D. B.; Vieira, F. G.; Liu, S.; Gilbert, M. T. P., Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq. BMC Genomics 2018, 19 (1), 811. 15. Liu, C. C.; Chi, C.; Jin, L. J.; Zhu, J.; Yu, J. Q.; Zhou, Y. H., The bZip transcription factor HY5 mediates 22


Page 22 of 35

Page 23 of 35


486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517

CRY1a-induced anthocyanin biosynthesis in tomato. Plant Cell Environ 2018, 41 (8), 1762-1775.

518 519 520 521 522 523 524 525 526 527 528 529

28. Rook, F.; Bevan, M. W., Genetic approaches to understanding sugar‐response pathways. Journal

16. Petroni, K.; Pilu, R.; Tonelli, C., Anthocyanins in corn: a wealth of genes for human health. Planta 2014, 240 (5), 901-11. 17. Shi, M. Z.; Xie, D. Y., Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat Biotechnol 2014, 8 (1), 47-60. 18. Meng, L. S.; Xu, M. K.; Wan, W.; Yu, F.; Li, C.; Wang, J. Y.; Wei, Z. Q.; Lv, M. J.; Cao, X. Y.; Li, Z. Y.; Jiang, J. H., Sucrose Signaling Regulates Anthocyanin Biosynthesis Through a MAPK Cascade in Arabidopsis thaliana. Genetics 2018, 210 (2), 607-619. 19. Wang, J.; Lian, W.; Cao, Y.; Wang, X.; Wang, G.; Qi, C.; Liu, L.; Qin, S.; Yuan, X.; Li, X.; Ren, S.; Guo, Y. D., Overexpression of BoNAC019, a NAC transcription factor from Brassica oleracea, negatively regulates the dehydration response and anthocyanin biosynthesis in Arabidopsis. Sci Rep 2018, 8 (1), 13349. 20. Zhang, J.; Xu, H.; Wang, N.; Jiang, S.; Fang, H.; Zhang, Z.; Yang, G.; Wang, Y.; Su, M.; Xu, L.; Chen, X., The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Mol Biol 2018, 98 (3), 205-218. 21. Gonzali, S.; Mazzucato, A.; Perata, P., Purple as a tomato: towards high anthocyanin tomatoes. Trends in plant science 2009, 14 (5), 237-241. 22. He, F.; Mu, L.; Yan, G.-L.; Liang, N.-N.; Pan, Q.-H.; Wang, J.; Reeves, M. J.; Duan, C.-Q., Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules 2010, 15 (12), 9057-9091. 23. Zhang, Y. T.; Jiang, L. Y.; Li, Y. L.; Chen, Q.; Ye, Y. T.; Zhang, Y.; Luo, Y.; Sun, B.; Wang, X. R.; Tang, H. R., Effect of Red and Blue Light on Anthocyanin Accumulation and Differential Gene Expression in Strawberry (Fragaria x ananassa). Molecules 2018, 23 (4). 24. Zhu, H.; Zhang, T. J.; Zhang, P.; Peng, C. L., Pigment patterns and photoprotection of anthocyanins in the young leaves of four dominant subtropical forest tree species in two successional stages under contrasting light conditions. Tree Physiology 2016, 36 (9), 1092-1104. 25. Yamagishi, M., A novel R2R3-MYB transcription factor regulates light-mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regale. Mol Breeding 2016, 36 (1). 26. Carvalho, S. D.; Folta, K. M., Green light control of anthocyanin production in microgreens. Acta Hortic 2016, 1134, 13-18. 27. Li, S. N.; Wang, W. Y.; Gao, J. L.; Yin, K. Q.; Wang, R.; Wang, C. C.; Petersen, M.; Mundy, J.; Qiu, J. L., MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis. Plant Cell 2016, 28 (11), 2866-2883. of Experimental Botany 2003, 54 (382), 495-501. 29. Jeong, C. Y.; Kim, J. H.; Lee, W. J.; Jin, J. Y.; Kim, J.; Hong, S. W.; Lee, H., AtMyb56 Regulates Anthocyanin Levels via the Modulation of AtGPT2 Expression in Response to Sucrose in Arabidopsis. Molecules and Cells 2018, 41 (4), 351-361. 30. Xie, Y.; Zhao, Y. L.; Tan, H. J.; Chen, Q.; Huang, J. R., An ubiquitin-like protein SDE2 negatively affects sucrose-induced anthocyanin biosynthesis in Arabidopsis. Sci Bull 2017, 62 (23), 1585-1592. 31. Liu, X. J.; An, X. H.; Liu, X.; Hu, D. G.; Wang, X. F.; You, C. X.; Hao, Y. J., MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. Journal of Experimental Botany 2017, 68 (11), 2977-2990. 32. Wasternack, C.; Hause, B., Jasmonates and octadecanoids: signals in plant stress responses and development. Progress in nucleic acid research and molecular biology 2002, 72, 165-221. 23



530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

33. Cheong, J.-J.; Choi, Y. D., Methyl jasmonate as a vital substance in plants. TRENDS in Genetics 2003, 19 (7), 409-413. 34. Moro, L.; Hassimotto, N. M. A.; Purgatto, E., Postharvest Auxin and Methyl Jasmonate Effect on Anthocyanin Biosynthesis in Red Raspberry (Rubus idaeus L.). Journal of Plant Growth Regulation 2017, 36 (3), 773-782. 35. Saw, N. M. M. T.; Moser, C.; Martens, S.; Franceschi, P., Applying generalized additive models to unravel dynamic changes in anthocyanin biosynthesis in methyl jasmonate elicited grapevine (Vitis vinifera cv. Gamay) cell cultures. Hortic Res-England 2017, 4. 36. Sun, J. J.; Wang, Y. C.; Chen, X. S.; Gong, X. J.; Wang, N.; Ma, L.; Qiu, Y. F.; Wang, Y. L.; Feng, S. Q., Effects of methyl jasmonate and abscisic acid on anthocyanin biosynthesis in callus cultures of redfleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tiss Org 2017, 130 (2), 227-237. 37. Huang, X. J.; Li, J.; Shang, H. L.; Meng, X., Effect of methyl jasmonate on the anthocyanin content and antioxidant activity of blueberries during cold storage. J Sci Food Agr 2015, 95 (2), 337-343. 38. Park, W. T.; Kim, Y. B.; Seo, J. M.; Kim, S.-J.; Chung, E.; Lee, J.-H.; Park, S. U., Accumulation of anthocyanin and associated gene expression in radish sprouts exposed to light and methyl jasmonate. Journal of agricultural and food chemistry 2013, 61 (17), 4127-4132. 39. Sun, Q.; Zhou, G.; Cai, Y.; Fan, Y.; Zhu, X.; Liu, Y.; He, X.; Shen, J.; Jiang, H.; Hu, D.; Pan, Z.; Xiang, L.; He, G.; Dong, D.; Yang, J., Transcriptome analysis of stem development in the tumourous stem mustard Brassica juncea var. tumida Tsen et Lee by RNA sequencing. BMC Plant Biol 2012, 12, 53. 40. Xie, Q.; Hu, Z.; Zhang, Y.; Tian, S.; Wang, Z.; Zhao, Z.; Yang, Y.; Chen, G., Accumulation and molecular regulation of anthocyanin in purple tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee). J Agric Food Chem 2014, 62 (31), 7813-21. 41. Zhang, B.; Hu, Z.; Zhang, Y.; Li, Y.; Zhou, S.; Chen, G., A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale (Brassica Oleracea var. acephala f. tricolor). Plant cell reports 2012, 1-9. 42. Wu, X.; Prior, R. L., Identification and characterization of anthocyanins by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains. J Agric Food Chem 2005, 53 (8), 3101-13. 43. Lian, N.; Liu, X.; Wang, X.; Zhou, Y.; Li, H.; Li, J.; Mao, T., COP1 mediates dark-specific degradation of microtubule-associated protein WDL3 in regulating Arabidopsis hypocotyl elongation. Proc Natl Acad Sci U S A 2017, 114 (46), 12321-12326. 44. Leach, K. A.; Tran, T. M.; Slewinski, T. L.; Meeley, R. B.; Braun, D. M., Sucrose transporter2 contributes to maize growth, development, and crop yield. J Integr Plant Biol 2017, 59 (6), 390-408. 45. Li, X.; Thwe, A. A.; Park, N. I.; Suzuki, T.; Kim, S. J.; Park, S. U., Accumulation of phenylpropanoids and correlated gene expression during the development of tartary buckwheat sprouts. J Agric Food Chem 2012, 60 (22), 5629-35. 46. Feng, S.; Wang, Y.; Yang, S.; Xu, Y.; Chen, X., Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta 2010, 232 (1), 245-55. 47. Smith, A. M.; Stitt, M., Coordination of carbon supply and plant growth. Plant, cell & environment 2007, 30 (9), 1126-1149. 48. Powell, A. E.; Lenhard, M., Control of organ size in plants. Current Biology 2012, 22 (9), R360-R367. 49. Lastdrager, J.; Hanson, J.; Smeekens, S., Sugar signals and the control of plant growth and development. Journal of experimental botany 2014, ert474. 50. Cai, Y.; Tu, W.; Zu, Y.; Yan, J.; Xu, Z.; Lu, J.; Zhang, Y., Corrigendum: Overexpression of a Grapevine 24


Page 24 of 35

Page 25 of 35


574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

Sucrose Transporter (VvSUC27) in Tobacco Improves Plant Growth Rate in the Presence of Sucrose In

605 606 607 608 609 610 611 612 613 614 615 616 617

abscisic acid modulate the sucrose ‐ induced expression of anthocyanin biosynthetic genes in

vitro. Front Plant Sci 2018, 9, 36. 51. Noir, S.; Bömer, M.; Takahashi, N.; Ishida, T.; Tsui, T.-L.; Balbi, V.; Shanahan, H.; Sugimoto, K.; Devoto, A., Jasmonate controls leaf growth by repressing cell proliferation and the onset of endoreduplication while maintaining a potential stand-by mode. Plant physiology 2013, 161 (4), 19301951. 52. Dong, T.; Hu, Z.; Deng, L.; Wang, Y.; Zhu, M.; Zhang, J.; Chen, G., A tomato MADS-box transcription factor, SlMADS1, acts as a negative regulator of fruit ripening. Plant Physiol 2013, 163 (2), 1026-36. 53. Ai, T. N.; Naing, A. H.; Arun, M.; Lim, S. H.; Kim, C. K., Sucrose-induced anthocyanin accumulation in vegetative tissue of Petunia plants requires anthocyanin regulatory transcription factors. Plant Science 2016, 252, 144-150. 54. Meng, L. S.; Li, Y. Q.; Liu, M. Q.; Jiang, J. H., The Arabidopsis ANGUSTIFOLIA3-YODA Gene Cascade Induces Anthocyanin Accumulation by Regulating Sucrose Levels. Frontiers in Plant Science 2016, 7. 55. Flores, G.; del Castillo, M. L. R., Accumulation of anthocyanins and flavonols in black currants (Ribes nigrum L.) by pre-harvest methyl jasmonate treatments. J Sci Food Agr 2016, 96 (12), 4026-4031. 56. Park, W. T.; Kim, Y. B.; Seo, J. M.; Kim, S. J.; Chung, E.; Lee, J. H.; Park, S. U., Accumulation of anthocyanin and associated gene expression in radish sprouts exposed to light and methyl jasmonate. J Agric Food Chem 2013, 61 (17), 4127-32. 57. Ai, T. N.; Naing, A. H.; Arun, M.; Lim, S. H.; Kim, C. K., Sucrose-induced anthocyanin accumulation in vegetative tissue of Petunia plants requires anthocyanin regulatory transcription factors. Plant Sci 2016, 252, 144-150. 58. Park, N. I.; Xu, H.; Li, X.; Jang, I. H.; Park, S.; Ahn, G. H.; Lim, Y. P.; Kim, S. J.; Park, S. U., Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish (Raphanus sativus). Journal of agricultural and food chemistry 2011, 59 (11), 6034-6039. 59. Shan, X.; Zhang, Y.; Peng, W.; Wang, Z.; Xie, D., Molecular mechanism for jasmonate-induction of anthocyanin accumulation in Arabidopsis. Journal of experimental botany 2009, 60 (13), 3849-3860. 60. Qi, T.; Song, S.; Ren, Q.; Wu, D.; Huang, H.; Chen, Y.; Fan, M.; Peng, W.; Ren, C.; Xie, D., The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. The Plant Cell Online 2011, 23 (5), 1795-1814. 61. Loreti, E.; Povero, G.; Novi, G.; Solfanelli, C.; Alpi, A.; Perata, P., Gibberellins, jasmonate and Arabidopsis. New Phytologist 2008, 179 (4), 1004-1016. 62. Yuan, Y.; Chiu, L. W.; Li, L., Transcriptional regulation of anthocyanin biosynthesis in red cabbage. Planta 2009, 230 (6), 1141-53. 63. Su, N. N.; Lu, Y. W.; Wu, Q.; Liu, Y. Y.; Xia, Y.; Xia, K.; Cui, J., UV-B-induced anthocyanin accumulation in hypocotyls of radish sprouts continues in the dark after irradiation. J Sci Food Agr 2016, 96 (3), 886892.

25



618 619 620 621 622 623

FIGURE CAPTIONS

624

Figure 1. Anthocyanin biosynthesis pathway. PAL, phenylalanine ammonia lyase;

625

C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase;

626

F3H, flavone 3-hydroxylase; F3’H, flavonoid 3’-hydroxylase; DFR, dihydroflavonol

627

reductase; ANS, anthocyanidin synthase; UFGT, UDPglucose: flavonoid 3-O-

628

glucosyltransferase. EBGs, early biosynthetic genes; LBGs, late biosynthetic genes.

629

Figure 2. Phenotype and growth indicators of “Zi Ying” sprouts during the

630

development under light and dark conditions. (A) Phenotype of “Zi Ying” under light

631

and dark conditions, 6 days after sowing. (B) Average length of “Zi Ying” sprouts from

632

0 to 12 days after sowing (n= 30 per treatment). (C) Phenotype of “Zi Ying” cotyledons

633

under light and dark conditions with/without SUC or MeJA trenment, 6 and 9 days after

634

sowing, Bar=5 mm. (D) Fresh weight of “Zi Ying” sprouts from 0 to 12 days after

635

sowing (n= 20 per treatment). (E) Diameter of cotyledon of “Zi Ying” sprouts under

636

light and dark conditions, 6 and 9 days after sowing (n= 20). *, indicate P

Accumulation of Anthocyanin and its Associated Gene Expression in

Recommend Documents