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Anthocyanin accumulation and transcriptional regulation of anthocyanin biosynthesis in purple bok choy (Brassica rapa var chinensis) Yanjie Zhang, Guoping Chen, tingting dong, yu pan, Zhiping Zhao, shibing tian, and Zongli Hu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on December 5, 2014

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Journal of Agricultural and Food Chemistry

Anthocyanin accumulation and transcriptional regulation of anthocyanin biosynthesis in purple bok choy (Brassica rapa var chinensis) Yanjie Zhang1, Guoping Chen1, Tingting Dong1, Yu Pan1, Zhiping Zhao1, Shibing Tian2, Zongli Hu 1* 1

Bioengineering College, Chongqing University, Campus A, 174 Shapingba Main

Street, Chongqing 400044, P.R. China; 2

The Institute of Vegetable Research，Chongqing Academy of Agricultural Sciences,

401329 Chongqing , People’s Republic of China *

Corresponding author. Zongli Hu, Tel: 00862365112674; Fax: 0086 23 65112674;

E-mail: [email protected].

ACS Paragon Plus Environment


1

ABSTRACT: Bok choy (Brassica rapa var chinensis) is an important dietary

2

vegetable cultivated and consumed worldwide for its edible leaves. The purple

3

cultivars rich in health-promoting anthocyanins are usually more eye-catching and

4

valuable. Fifteen kinds of anthocyanins were separated and identified from a purple

5

bok choy cultivar (Zi He) by high-performance liquid chromatography−electrospray

6

ionization tandem mass spectrometry. To investigate the molecular mechanisms

7

underlying anthocyanin accumulation in bok choy, the expression profiles of

8

anthocyanin biosynthetic and regulatory genes were analyzed in seedlings and leaves

9

of the purple cultivar and the green cultivar (Su Zhouqing). Compared with the other

10

tissues, BrTT8 and most of anthocyanin biosynthetic genes were significantly up

11

regulated in the leaves and light-grown seedlings of Zi He. The results that

12

heterologous expression of BrTT8 promotes the transcription of partial anthocyanin

13

biosynthetic genes in regeneration shoots of tomato indicate that BrTT8 plays an

14

important role in the regulation of anthocyanin biosynthesis.

15

KEYWORDS: Anthocyanin, Purple bok choy, Brassica rapa var chinensis,

16

HPLC-ESI-MS/MS, Transcriptional regulation

17


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INTRODUCTION

19

Anthocyanins are naturally occurring flavonoids widely spread in higher plants and

20

are responsible for the wide range of colors including red, purple, and blue found in

21

lots of flowers, fruits, grains, and leaves. They are implicated to play important roles

22

in reducing the damage arising from coldness, drought and UV irradiation in plant

23

tissues (1). In addition, the brilliant colors in flowers and fruits as a result of

24

anthocyanins biosynthesis predominantly contribute to the completion of pollination

25

and seed dispersal by attracting insects and animals (2). Besides, more and more

26

evidences show that high dietary intake of foods rich in anthocyanins is closely

27

associated with the reduced risk of suffering from cardiovascular disease,

28

degenerative diseases and cancers (3-7). For the favorite color and health-promoting

29

effects, vegetables and fruits containing high amount of anthocyanins are more

30

eye-catching and valuable than the other cultivars (8).

31

The biosynthesis of anthocyanins has been widely studied in maize (Zea mays),

32

snapdragon (Antirrhinum majus), petunia (Petunia hybrida), grape (Vitis vinifera L.)

33

blood Orange (Citrus sinensis L. Osbeck) and Arabidopsis (Arabidopsis thaliana)

34

(9-11). This kind of pigments is synthesized via the flavonoid pathway which is

35

classified as a branch of the phenylpropanoid pathway. Briefly, the biosynthesis of

36

anthocyanins begins with the lysis of phenylalanine ammonia catalyzed by the

37

enzyme phenylalanine ammonia lyase (PAL). Then, the following enzymes that each

38

catalyzes a sequential reaction for anthocyanin synthesis are listed as: cinnamate

39

4-hydroxylase (C4H), 4-coumarateCoA ligase (4CL), chalcone synthase (CHS),



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chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol

41

4-reductase

42

flavonoid-3-O-glucosyltransferase (UFGT). In addition, the final enzyme in

43

anthocyanin biosynthetic pathway, UGFT which catalyzes the transfer of glucosyl

44

moiety is essential for the conversation of colorful anthocyanidins to stable status (12,

45

13). Generally, dihydrokaempferol (DHK) will be catalyzed by DFR, ANS, UFGT

46

and other enzymes sequentially with the production of brick–red pelargonins.

47

However, DHK can also be further hydroxylated by flavonoid 3′-hydroxylase (F3′

48

H) or flavonoid 3′5′-hydroxylase (F3′5′H) to produce dihydroquercetin and

49

dihydromyricetin, separately. Then dihydroquercetin and dihydromyricetin will be

50

catalyzed by DFR and following enzymes of anthocyanin pathway leading to the

51

production of the red cyanidin- and violet delphinidin-based pigments, respectively

52

(14). Therefore, the hydroxylation patterns of DHK are fundamental determinants for

53

coloration of plant tissues.

(DFR),

anthocyanidin

synthase

(ANS),

and

54

The enhanced expression of partial or all structural genes of anthocyanin

55

biosynthesis always accounts for increased levels of anthocyanin accumulation in

56

plants directly (15). Furthermore, these structural genes are regulated mainly at the

57

transcriptional level (16). Recent studies show that a serial of regulatory genes

58

including basic helix-loop-helix (bHLH) transcription factors, R2R3 MYB

59

transcription factors, and WD40 proteins are responsible for the activation of

60

anthocyanin biosynthetic genes at mRNA level (17). In addition, a ternary

61

transcriptional activation MYB–bHLH–WD40 complex (MBW) combined with


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R2R3 MYB, bHLH and WD40 proteins has been proved able to target the promoters

63

of structural genes and initiate the transcription of the corresponding genes

64

immediately (18). Extensive studies showed that abundant anthocyanin accumulation

65

in certain tissues of some purple fruits and vegetables arise from the spatiotemporal

66

transcriptional activation of R2R3 MYB or bHLH transcription factors (8, 19-23). On

67

the other hand, light which acts as an essential environmental stimulus also modulates

68

the intensity of the pigment through the regulation of anthocyanin structural genes

69

(24). It is known that a basic leucine zipper (bZIP) type of transcription factor LONG

70

HYPOCOTYL 5 (HY5) plays a central role in the biological process of the

71

coordination of light signals and the adjustment of appropriate gene expression.

72

Furthermore, HY5 has also been demonstrated that it can positively regulate

73

anthocyanin biosynthesis by directly binding to the promoters of CHS and F3H in

74

Arabidopsis (25, 26).

75

As a variant cultivar of the turnip, bok choy (Brassica rapa var chinensis) is

76

originally cultivated in China and distinct from another leaf vegetable Chinese

77

cabbage which usually forms a compact head in winter. Being winter-hardy, bok choy

78

is widely cultivated and consumed in China and northeast Asia, and increasingly

79

grown in Northern Europe and North America. Furthermore, the purple bok choy

80

receives more attention from the public than green cultivars for high levels of

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anthocyanin accumulation in mature leaves besides the well known health benefits of

82

cruciferous vegetables (27, 28). However, being an economically important crop, the

83

ingredients of anthocyanins and molecular mechanisms of anthocyanin biosynthesis



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in purple bok choy remain unknown. In this article, we characterized the components

85

of anthocyanin production in the purple cultivar (Zi He) with high-performance liquid

86

chromatography−electrospray

87

(HPLC-ESI-MS/MS). For the purpose of investigating the molecular mechanisms

88

underlying anthocyanin accumulation in the purple edible leaves, the expression level

89

of anthocyanin biosynthetic and regulatory genes was analyzed by quantitative

90

real-time polymerase chain reaction (qRT-PCR) in corresponding tissues of the purple

91

and green cultivars. Based the fact that production of anthocyanins in seedlings of Zi

92

He is strictly dependent on lightness, the expression profiles of anthocyanin

93

biosynthesis associated genes were further analyzed in seedlings of different

94

developmental stages under light and dark conditions. These results certainly

95

expanded our understanding about the mechanisms of anthocyanin accumulation in

96

purple bok choy at both the metabolic and transcriptional levels.

ionization

tandem

mass

spectrometry

97 98

MATERIALS AND METHODS

99

Chemicals and Solvents. Anthocyanin (cyanidin 3,5-diglucoside) for external

100

standards was purchased from Phytolab (Germany). High-performance liquid

101

chromatography (HPLC)-grade formic acid and methanol (MeOH) were bought from

102

Sigma. All the other solvents were provided from Aldrich (St. Louis, MO).

103

Plant materials and Culture Conditions. Bok choy (Brassica rapa var chinensis)

104

seeds of green cultivar (Su Zhouqing) and purple cultivar (Zi He) were got from

105

Chongqing

Academy

of

Agriculture

Sciences.

The


samples

used

for

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HPLC-ESI-MS/MS and qRT-PCR analysis are collected from edible leaves of green

107

and purple cultivars which were grown in a greenhouse with a 16-h photoperiod at

108

25℃. The samples used for total anthocyanin and qRT-PCR analysis are collected

109

from seedlings of green and purple cultivars which were generated by follow

110

procedures. Bok choy seeds were surface-sterilized with 70% ethanol for 30 s and 4%

111

(v/v) bleach solution for 15 min, and rinsed several times in sterile water. These seeds

112

were then placed on half-strength sterilized Murashige−Skoog medium (1/2 MS)

113

solidified with 0.8% agar. The two cultivars were germinated in a growth chamber

114

under light/dark (16/8 h) or dark conditions at 25 °C and approximately 60% humidity.

115

Samples were harvested after 3, 6, 9, and 12 days, measured for their length and fresh

116

weight, frozen in liquid nitrogen, and stored at −80 °C until other analysis.

117

RNA Extraction and qRT-PCR Analysis. The samples of the two bok choy cultivars

118

were grounded into powder in liquid nitrogen. Total RNA was isolated from various

119

tissues for three biological repeats using RNAiso reagent according to the

120

manufacturer’s instruction (Takara, Dalian, PRC). RNA samples (1 µg) were reverse

121

transcripted into complementary DNA (cDNA) with an oligo(dT)20 primer and

122

M-MLV

123

manufacturer’s protocol. The synthesized cDNAs were diluted five times in H2O for

124

qRT-PCR analysis.

reverse

transcriptase

(Promega,

Madison,

WI)

following

the

125

qRT-PCR was carried out using the CFX96TM Real-Time System (C1000 thermal

126

cycler). All reactions were performed using the GoTaq qPCR Master Mix according

127

to the manufacturer’s instructions. Reactions were performed in triplicate using 5 µL



128

of Master Mix, 0.25 µM each primer, 1 µL of diluted cDNA and DNase-free water to

129

a final volume of 10 µL. The PCR amplification was as follows: 1 cycle of 3 min at

130

95℃, 40 cycles of denaturation for 15 s at 95 ℃, annealing for 30 s at 60℃, and

131

elongation for 15 s at 72℃. Amplification was followed by a melting curve analysis

132

with continual fluorescence data acquisition during the 60–95℃ melt. Melt curve

133

analysis of qPCR samples revealed that there was only one product for each gene

134

primer reaction. The primers used for qPCR analysis of bok choy were designed by

135

Primer Premier 5 and are listed in Supplementary Table 1 (Supporting Information).

136

In addition, primers used for qPCR analysis for regeneration shoots of tomato are

137

listed in Supplementary Table 2 (Supporting Information).

138

sequenced to confirm the specific amplifications. The gene expression was

139

normalized to BrApr and SlCAC as a reference gene for bok choy and tomato

140

respectively (29, 30). Values reported here were calculated from three biological

141

repeats for each sample.

The PCR products were

142

Anthocyanin Extraction and HPLC-ESI-MS/MS Analysis. Anthocyanin

143

extraction of bok choy was carried out as the same way as for radish (31). The extract

144

was filtered through a 0.2 µm PTFE syringe filter. The samples were then analyzed by

145

an Agilent Technologies 1200 Series HPLC (Agilent Technologies, Palo Alto, CA),

146

equipped with an Agilent 1200 HPLC variable wavelength detector. The results were

147

analyzed by Agilent 1200 HPLC ChemStation software. The chromatographic

148

separation was performed on a Zorbax Stablebond Analytical SB-C18 column (4.6

149

mm × 250 mm, 5µm, Agilent Technologies, Rising Sun, MD). The injection sling was


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5 µL. Elution was performed using mobile phase A (aqueous 2% formic acid solution)

151

and mobile phase B (methanol). The detection was at 520 nm, and the column oven

152

temperature was set at 40℃. The flow rate was 0.6 mL/min. The gradient program is

153

described as follows: 0–2 min, 10–20% B; 2–40 min,20–55% B; 40–41 min, 55

154

–80% B; 41–45 min, 80% B; 45–50 min, 80–10% B; 50–55 min, 10% B.

155

Quantification of the different anthocyanins was based on peak areas and calculated

156

as equivalents of the standard compounds. All contents were expressed as milligrams

157

per grams dry weight. Low-resolution electrospray mass spectrometry was performed

158

with a solariX ion trap mass spectrometer (Bruker Daltoniks, Billerica, MA). The

159

experimental conditions were as follows: ESI interface, nebulizer, 50 psi; dry gas,

160

15.0 psi; dry temperature, 320 °C; MS/MS, scan from m/z 200 to 1500; ion trap, scan

161

from m/z 200 to 1500; source accumulation, 50 ms; ion accumulation Time, 300 ms;

162

flight time to acq. cell, 1 ms; smart parameter setting (SPS), compound stability, 50%;

163

trap drive level, 60%.

164

Total Anthocyanin Analysis. Spectrophotometric differential pH method was used

165

for the total anthocyanin measurement of bok choy following the procedure of Yuan et

166

al.(15).

167

Gene cloning and Sequence Analyses. The primers used for BrMYB1、BrMYB2、

168

BrMYB3 and BrTT8 were designed from the sequences of known orthologous

169

sequences (Table S3 in Supporting Information). Then, the PCR products were cloned

170

into a T-Blunt vector (Takara, Dalian, PRC) and sequenced. Sequence similarities

171

were calculated with DNAMAN version 5.2.2. The phylogenetic and molecular



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172

evolutionary analysis was used with MEGA (Molecular Evolutionary Genetics

173

Analysis) version 3.1.

174

Statistical Analyses. SPSS, version 17.0 (SPSS Inc., Chicago, IL) was used for the

175

data analysis. One-way analysis of variance (ANOVA) followed by pairwise

176

comparisons was performed with posthoc Tukey’s honestly significant different (HSD)

177

test, with significance set at p < 0.05.

178

Vector construction and transformation of tomato. The complement DNA was

179

synthesized with total RNA with M-MLV (Promega, Madison, USA) as the

180

recommendation of manufacturer. The full-length coding sequence of BrTT8 was

181

cloned into pBI121 for the replacement of GUS to obtain the construct pBI121-BrTT8.

182

Primers

183

TCTAGATACAGGTTTTCATCTCGGG

184

GAGCTCCATTAAGGTTAGAATCTCGGAA. The binary vector pBI121- BrTT8

185

containing the BrTT8 cDNA driven by the 35S promoter was transferred into

186

Agrobacterium tumefaciens strain LBA4404 by the freeze–thaw method. Tomato

187

(solanum lycopersicum Mill. cv. Ailsa Craig) was used for the generation of

188

transgenic shoots by Agrobacterium-mediated transformation, using the protocol

189

previously reported (32).

used

for

amplification

of

BrTT8 and

are

BrTT8-con-F: BrTT8-con-R

190 191 192

RESULTS AND DISCUSSION Identification and Quantitative Analyses of Anthocyanins. By analyzing the

193

extracts from edible leaves of Zi He (purple cultivar) and Su Zhouqing (green cultivar)

194

with the method of HPLC-ESI-MS/MS, a total of 15 anthocyanins were separated and


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195

characterized (Figure 1 A and B). To verify the identity of anthocyanins in the purple

196

cultivar, fragmentation patterns of MS/MS (m/z) were used to compare with those

197

radical groups emerged in previously report (33). As the results showed in table 1,

198

some new anthocyanins such as cyanidin 3-(sinapoyl) diglucoside-5-(malonyl)

199

glucoside never reported in plants were identified in purple bok choy. Moreover,

200

cyanidin was characterized as the major anthocyanidin in the purple bok choy with

201

the absence of pelargonidin (Table 1). Obviously, acylated cyanidin of anthocyanidins

202

is the most common modifications of anthocyanins except for cyanidin

203

3,5-diglucoside. Broadly, the ingredients of anthocyanins identified in purples bok

204

choy are similar with the other cruciferous vegetables except radish reported so far

205

(15, 31, 34).

206

Total anthocyanin content was found to be 3.13 mg/g of dry weight for edible

207

leaves from Zi He (Table 2). However, there were no obvious anthocyanins detected

208

in mature leaves of Su Zhouqing. Furthermore, the anthocyanins showing the highest

209

levels were cyanidin 3-(sinapoyl) diglucoside-5-(malonyl) glucoside and cyanidin

210

3-caffeoyl (sinapoyl) rutinoside-5-glucoside for 0.91 and 0.69 mg/g dry weight

211

respectively (Figure 1 C and D, Table2). Nevertheless, trace amount of petunidin

212

3,5-diglucoside was detected in HPLC profiles of anthocyanins in Zi He. This kind of

213

anthocyanin has not been reported in the species of brassica up to now. Previous

214

published reports showed that accumulation of petunidin needs further hydroxylation

215

at the 3′position and 5′ positions in the B-ring of naringenin (35). That is to say,

216

isoenzymes which share the same catalytic activity with F3'5'H may probably exist in



217

bok choy.

218

Expression Analysis of Anthocyanin Biosynthesis Associated Genes in Edible

219

Leaves of Different Cultivars. To investigate the molecular mechanisms underlying

220

the pigment accumulation in purple leaves of Zi He, the transcripts of anthocyanin

221

biosynthetic and regulatory genes were examined by qRT-PCR. The expression levels

222

of BrPAL, BrC4H, BrCHS, BrCHI, BrF3H, BrF3'H, BrANS, BrDFR and BrUFGT

223

were exhibited in Figure 2. In the edible leaves, all the genes in anthocyanin

224

biosynthetic pathway were significantly up-regulated in the purple bok choy.

225

Compared with the green cultivar, the expression levels of BrDFR and BrANS were

226

especially up regulated to at least 500 fold in the purple leaves. As the early studies

227

reported, trace amount of transcripts of all the anthocyanin biosynthetic genes were

228

always observed despite the absence of visible pigment accumulation in the

229

corresponding tissues (15). These results indicate that adequate transcripts of some

230

vital structural genes are essential for large sum of anthocyanin biosynthesis. It is

231

worth to mention that the drastic increment of F3′H should made a completely

232

conversion of DHK to dihydroquercetin because of the absence of pelargonidin-based

233

anthocyanins.

234

To investigate the mechanisms about transcriptional regulation of structural genes

235

which are responsible for pigmentation production, the transcripts of some

236

anthocyanin biosynthesis regulatory orthologous genes of Arabidopsis, BrMYB1,

237

BrMYB2, BrMYB3, BrTT8, BrEGL3 and BrTTG1 were examined by qRT-PCR. It is

238

worth mentioning that the three MYB genes (BrMYB1, BrMYB2 and BrMYB3) shares


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high similarity with AtPAP1 and AtPAP2 which regulate anthocyanin biosynthesis by

240

activating the transcription of structural genes directly in an bHLH and TTG1

241

dependent manner (18, 36). However, the up-regulation of bHLH transcription factors

242

accounts for visible pigment accumulation in certain tissues of some species (23, 37,

243

38). As the data shown in Figure 3, BrTT8 was the only transcription factor which is

244

found to be significantly up-regulated in the leaves of purple cultivar. The deduced

245

amino acid sequences of BrTT8 were aligned with other bHLH transcription factors

246

which were known to regulate the anthocyanin biosynthesis in diverse species. BrTT8

247

exhibited highest sequence similarities with other bHLH proteins within the bHLH

248

domain and the N-terminal MYB interaction domain (Supplement Figure. 1).

249

Phylogenetic analysis divided bHLH regulators into two different clades. BrTT8 were

250

placed in the same cluster with TT8 from Arabidopsis, NtAn1a and NtAn1b from

251

Tobacco, MdbHLH3 from Apple and PhAn1 from petunia (Supplement Figure. 2).

252

Ectopic expression of NtAN1a, NtAN1b and MdbHLH3 activated the transcription of

253

anthocyanin biosynthetic genes efficiently, while ectopic expression of PhAN1 failed

254

to activate the expression of DFR (23, 39, 40). As the mechanism of anthocyanin

255

biosynthesis pathway genes regulated by MBW complex directly is widely found in

256

many species, the different background expression levels of R2R3 MYB transcription

257

factors in respective species might explicate the discrepancy observed above. These

258

results suggest that the dramatically increased expression of BrTT8 might accounts

259

for the anthocyanin accumulation in edible leaves of Zi He by activating the

260

transcription of structural genes upon the formation of MBW complex with sufficient



261 262

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amount of R2R3 MYB and WD40 proteins. The Growth of Bok Choy Seedlings under Light and Dark Conditions.

To

263

evaluate the effects of light on seedling development and anthocyanin accumulation,

264

the length, fresh weight and total pigment content of seedlings of both cultivars under

265

light and dark conditions were examined every 3 days until 12 days after sowing

266

(DAS) (Figure 4). Compared with the seedlings grown under light, the seedlings of

267

both cultivars grown in darkness were all long and yellow (Figure 4 A and C). The

268

fresh weight of seedlings grown in dark conditions of both Zi He and Su Zhouqing

269

were all obviously higher than the cultivars grown under light initially. However, the

270

fresh weight of seedlings of both cultivars grown under light exceeded the seedlings

271

grown in dark conditions gradually. Interestingly, the seedlings of Zi He grown in

272

light took about 10 days to exceed the seedlings grown in darkness at the level of

273

fresh weight, while the seedlings of Su Zhouqing took only 6 days to exceed the

274

seedlings grown in darkness (Figure 4 D). The growth rate of seedlings of purple

275

cultivar under light seems apparently lower than that of green cultivars. One

276

reasonable explanation for this phenomenon is that the pigments synthesized in

277

cotyledon of Zi He reduced the efficiency of photosynthesis by inhibiting the light

278

capture of chlorophyll (24). Moreover, large amount of anthocyanin production might

279

consume a certain amount of energy and nutrients of plant cell and interfere the

280

normal metabolic activities in plant itself. Although extensive studies showed that

281

adequate amount of anthocyanin production provides photo protective function

282

against excess visible light during early development of young leaves and facilitate


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283

nutrient recovery in autumn by shielding leaves from potentially damaging light

284

levels (41-43), the results displayed above strongly suggest that constant anthocyanin

285

accumulation in leaves might be a burden during the process of development,

286

especially in seedlings.

287

Expression profiles of anthocyanin biosynthetic and regulatory genes in

288

seedlings of different cultivars under light and dark conditions. The expression

289

profiles of anthocyanin pathway genes in the four groups of materials collected at

290

different DAS were examined by qRT-PCR. As the results shown in Figure 5, the

291

transcript levels of BrCHS, BrF3H, BrANS and BrDFR were greatly up regulated in

292

light-grown seedlings of Zi He, in contrast to the other three serials of materials. In

293

addition, the expression levels of these structural genes reached top at 6 DAS in

294

purple light-grown seedlings of different stages. The expression profiles of these

295

structural genes were highly accordance with the patterns of anthocyanin

296

accumulation in seedlings of bok choy (Figure 4 B). That is to say, it was the

297

significantly increased expression of BrCHS, BrF3H, BrANS and BrDFR activates

298

visible pigmentation production in seedlings of Zi He under light.

299

As we have speculated above, a bHLH transcription factor, BrTT8 probably

300

regulates the anthocyanin biosynthesis by activating the transcription of structural

301

genes in purple edible leaves with MYB and WD40 proteins. To investigate the

302

molecular mechanisms of light dependent anthocyanin accumulation in purple

303

seedlings of bok choy, the transcripts of related regulatory genes were examined in the

304

four serials of materials referred above. As Figure 6 showed, only the expression



305

profile of BrTT8 agreed well with those of BrCHS, BrF3H, BrANS and BrDFR.

306

Therefore, it is reasonable to speculate that the light induced expression of BrTT8

307

plays an important role in the anthocyanin biosynthesis activation in seedlings of Zi

308

He under light. As results shows, BrHY5 is greatly up regulated in the seedlings of Zi

309

He and Su Zhouqing under dark conditions. The high-level expression of BrHY5

310

should be important for the inhibition of excessive elongation of hypocotyls of bok

311

choy seedlings grown in darkness (25, 44). Besides, the increased expression of

312

BrHY5 might account for the slightly up-regulation of BrCHS and BrF3H in early

313

stages of Su Zhouqing despite of no visible pigment accumulation in corresponding

314

tissues. Nevertheless, the functions BrHY5 play during light-induced anthocyanin

315

accumulation in seedlings of Zi He need further studies. In addition, BrMYB4 which

316

is a potential transcriptional repressor of BrC4H shows no significantly changes at

317

mRNA level under visible light, in contrast with the seedlings grown in darkness (45).

318

Heterologous expression of BrTT8 in transgenic tomato promotes the

319

expression of partial anthocyanin biosynthetic genes. Phylogenetic and sequence

320

analysis shows that BrTT8 lays in the same cluster with the anthocyanin synthesis

321

activator AmDEL 、MdbHLH1 、NtAN1a and NtAN1b (Supplement Figure 1 and

322

2).To further study the function of BrTT8 gene, full-length coding sequence of BrTT8

323

driven by 35S promoter was expressed in the callus of tomato. Compared with the

324

seedlings (the T0 generation) of tomato transformed by empty vector, the transgenic

325

seedlings which show high expression levels of BrTT8 gene display visible

326

anthocyanin accumulation as small purple spots in leaves and the lower part in stems


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327

(Figure 7). Furthermore, the expression level of anthocyanin biosynthetic genes in

328

regeneration shoots was confirmed by qRT-PCR. The heterologous expression of

329

BrTT8 was in accordance with a coordinated increase in transcript levels of SlPAL、

330

SlCHS2 、 SlF3H and Sl3GT (Figure 8). In addition, SlCHS2 and SlF3H were

331

significantly up-regulated in regeneration shoots which showed visible accumulation

332

of anthocyanins. These results suggest that the up-regulation of some vital structural

333

genes is able to enhance the anthocyanin biosynthesis in shoots of transgenic tomato.

334

As indicated before, the over-expression of NtAN1a and NtAN1b were able to

335

enhance the accumulation of anthocyanin in petals of transgenic tobacco (39).

336

Furthermore, the purple spots in transgenic leaves resembled the phenotype observed

337

in leaves of transgenic tomato which showed high expression of a heterologous gene

338

AmDEL from snapdragon (37). These results prove that heterologous expression of

339

BrTT8 in tomato is able to promote anthocyanin biosynthesis by activating the

340

transcription of some vital anthocyanin structural genes.

341

In this article, the mechanisms of anthocyanin accumulation in bok choys were

342

analyzed at both molecular and metabolic levels using the methods of

343

HPLC-ESI-MS/MS

344

diglucoside-5-(malonyl)

345

rutinoside-5-glucoside were identified as the major anthocyanins in edible leaves of

346

Zi He. Moreover, the solely up-regulated transcription factors BrTT8 in both edible

347

leaves and seedlings under lightness probably contributes to the onset of anthocyanin

348

biosynthesis by activating the transcription of structural genes in purple bok choy.

and

qRT-PCR.

glucoside

and

Cyanidin cyanidin


3-(hydroxyferuloyl) 3-caffeoyl

(sinapoyl)


Page 18 of 36

349

Similarity and phylogenetic analysis of BrTT8 with other bHLH proteins from

350

different species suggests that BrTT8 may probably be a functional anthocyanin

351

biosynthesis activator in purple bok choy. Furthermore, the anthocyanins detected in

352

the regeneration shoots of tomato transformed by pBI121-BrTT8 confirmed the

353

hypothesis we speculated above. As we have mentioned before, large amount of

354

anthocyanin accumulation in leaves inevitably counts against the growth of purple

355

cultivars. This explanation agrees well with the facts that the growth rates of plants

356

with excessive purple pigment accumulation are always significantly lower than the

357

green cultivars (24, 41). However, a large amount of anthocyanin production is an

358

important contributor to nutrition and consumer preference for bok choy. One possible

359

solution for this conflict is the breeding of new cultivars with anthocyanin

360

accumulation activators spatiotemporal specific expressed in edible parts during

361

harvesting time like the strategy used in transgenic tomato (5). Thence, the elucidation

362

of the primary transcriptional control of anthocyanin accumulation in bok choy

363

provides important basis for the breeding of new purple cultivars of bok choy with

364

more excellent agronomic characters.

365 366

Abbreviation used

367

PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL,

368

4-coumarateCoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H,

369

flavone

370

3',5'-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase;

3-hydroxylase;

F3'H,

flavonoid

3',-hydroxylase;


F3'5'H,

flavonoid

Page 19 of 36


371

UFGT,

flavonoid-3-O-glucosyltransferase;

DHK,

dihydrokaempferol;

HPLC,

372

high-performance liquid chromatography; ESI-MS/MS, elctrospray ionization tandem

373

mass spectrometry; qRT-PCR, quantitative real-time PCR.

374 375

Acknowledgment

376

This work was supported by National Natural Science Foundation of China (nos.

377

30871709, 31100089, 31101546) and Graduate Science and Technology Innovation

378

Fund (CDJXS12230001).

379 380

Supporting Information description

381

A table of primers used for qPCR analysis of anthocyanin biosynthetic genes in bok

382

choy, a table of primers used for qPCR analysis of anthocyanin biosynthetic genes in

383

tomato, a table of primers used for gene cloning, a figure showing multiple alignment

384

of deduced amino acid sequences of BrTT8 with bHLH homologues and a figure

385

depicting phylogenetic analysis of BrTT8 and other bHLHs.

386



387

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514

FIGURE CAPTIONS

515

Figure 1. Anthocyanin component analysis of bok choy. (A) Photographs of the two

516

bok choy cultivars (Zi He on the left and Su Zhouqing on the right) used in this study.

517

(B) HPLC profiles of anthocyanins of the leaves from the purple cultivar (Zi He).

518

Peak numbers refer to the anthocyanins listed in Table 1. (C) Structures and major

519

cleavage of cyanidin 3-(sinapoyl) diglucoside-5-(malonyl) glucoside in reference to

520

peak 5 of Figure 1 B. (D) Structures and major cleavage of cyanidin 3-caffeoyl

521

(sinapoyl) rutinoside-5-glucoside in reference to peak 13 of Figure 1 B.

522

Figure 2. Expression analysis of anthocyanin biosynthetic genes in edible leaves of

523

the two bok choy cultivars. P and G represent purple edible leaves and green edible

524

leaves separately. Error bars represent the standard error of the mean (n = 3).

525

Statistical significance of the differences between samples was calculated with

526

ANOVA by paired-group comparisons. Different letters indicate significance at P

Anthocyanin Accumulation and Transcriptional Regulation of

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