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Transcriptomic Analysis of Red-fleshed Apples Reveals the Novel Role of MdWRKY11 in Flavonoid and Anthocyanin Biosynthesis Nan Wang, Wenjun Liu, Tianliang Zhang, Shenghui Jiang, Haifeng Xu, Yicheng Wang, Zongying Zhang, Chuanzeng Wang, and Xuesen Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01273 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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Journal of Agricultural and Food Chemistry

Transcriptomic Analysis of Red-fleshed Apples Reveals the Novel Role of MdWRKY11 in Flavonoid and Anthocyanin Biosynthesis

Nan Wang, † ‡ Wenjun Liu, † ‡ Tianliang Zhang, † Shenghui Jiang, † Haifeng Xu, † Yicheng Wang,† Zongying Zhang,† Chuanzeng Wang,§ Xuesen Chen,*,†

†

State Key Laboratory of Crop Biology, College of Horticulture Science and

Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, China §

‡

Shandong Institute of Pomology, Tai’an, Shandong 271000, China

Nan Wang and Wenjun Liu are co-first authors.

*Corresponding author: Tel: +86-538-8249338. E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: In plants, flavonoids are important secondary metabolites that

2

contribute to the nutritional quality of many foods. Apple is a popular and frequently

3

consumed food because of its high flavonoid content. In this study, flavonoid

4

composition and content were detected and compared between the red- and

5

white-fleshed apples in a BC1 hybrid populations using ultra-performance liquid

6

chromatography/quadrupole

7

analysis of the red- and white-fleshed apples was then performed using RNA-seq

8

technology. By screening differentially expressed genes encoding transcription factors

9

we unearthed a WRKY family transcription factor designated MdWRKY11.

10

Overexpression of MdWRKY11 promoted the expression of F3H, FLS, DFR, ANS and

11

UFGT and increased the accumulation of flavonoids and anthocyanin in apple calli.

12

Our findings explored novel role of MdWRKY11 in flavonoid biosynthesis and

13

suggest several other genes that may be also potentially involved. This provides

14

valuable information on flavonoid synthesis for the breeding of elite red-fleshed

15

apples.

time-of-flight

mass

spectrometry.

Transcriptomic

16 17

KEYWORDS: plant secondary metabolites, flavonoids,

18

transcriptome analysis, WRKY

red-fleshed

apple,

19 20 21 22 2


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INTRODUCTION

24

In plants, flavonoids are important secondary metabolites that provide protection

25

against ultraviolet radiation, prevent pathogenic microorganism invasion, regulate

26

auxin transport, act as signaling molecules in plant–bacteria interactions, and promote

27

pollen germination and fertility.1–4 Moreover, flavonoids are natural antioxidants that

28

reduce myocardial oxygen consumption, prevent vascular sclerosis, enhance

29

immunity, and have anti-aging and anti-cancer effects.5–8 Evidence suggests that

30

flavonoids taken from various fruits and vegetables play a key role in reducing

31

disease risk.9,10

32

Apple (Malus domestica Borkh.), one of the most widely produced and

33

economically important fruit crops in temperate regions,11 is a significant source of

34

flavonoids in people's diet and is one of the top nutritionally rated and consumed fruit

35

worldwide.12–14 Boyer and Liu found that apples had the second highest level of

36

antioxidant activity when compared to many other commonly consumed fruits in the

37

United States.15 More importantly, apple contains high levels of free flavonoid, which

38

are not bound to other compounds and may be more available for eventual absorption

39

into the human bloodstream.15,16 For example, apples rich in flavonoids can

40

independently enhance endothelial function and acutely lower blood pressure,

41

benefitting cardiovascular health.17 The effects of apples on specific enzymes related

42

to cancer etiology have also been examined, with studies showing that apple juice

43

extracts rich in flavonoids reduced the growth of cancer cells and increased the

44

expression of several genes, including phase 2 enzymes associated with 3



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45

chemoprevention.18 Studies have also shown that proanthocyanidins have significant

46

effects on plasma antioxidant activity, whereas quercetin has slight effects on plasma

47

antioxidant biomarkers in vivo but can affect some carcinogenesis markers.19

48

Eberhardt found that 100 g of fresh apples had the same antioxidant activity as 1500

49

mg VC and that apple extracts inhibit the growth of colon and liver cancer cells in

50

vitro in a dose-dependent manner.20 According to these epidemiological studies,

51

apples appear to play a significant role in the intake of flavonoids and the

52

maintenance of human health.

53

In apple, studies on flavonoid biosynthesis and regulation first focused on

54

anthocyanin. Members of three TF families (MYB, bHLH, and WD40) function

55

together in a ternary MYB-bHLH-WD40 (MBW) protein complex to participate in

56

the anthocyanidin pathway, a role that is widely conserved among plant species.21–24

57

Among these, the MdMYB1 and MdMYBA genes encoding TFs were initially isolated

58

and observed to regulate anthocyanin biosynthesis in apple skin.25,26 MdMYB10, an

59

allele of MdMYB1/MYBA, determines the red pigmentation of red-fleshed apple. The

60

red-fleshed apple has a minisatellite-like structure comprising six tandem repeats in

61

the promoter of MdMYB10 (R6:MdMYB10), whereas the white-fleshed apple has only

62

one (R1:MdMYB10).27 Subsequently, MdMYB3, MdMYB9, MdMYB11, MdMYB12,

63

MdMYB22, and other genes regulating flavanol and flavanol biosynthesis have also

64

been cloned and identified.28–30 In addition to MYB TFs, MdbHLH3 reportedly binds

65

to the promoters of MdDFR and MdUFGT to promote the anthocyanin synthesis.31

66

Conversely, MdTTG1 of class WD40 cannot bind to the promoters of MdDFR and 4


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MdUFGT nor interact with MdMYB1. The regulation of anthocyanin by MdTTG1

68

may be achieved by interaction with MdbHLH3 and MdbHLH33.32

69

Many studies have examined flavonoid synthesis in apples. However,

70

considerable inbreeding has narrowed the hereditary basis of cultivated apple varieties

71

and resulted in fruit with poor nutritional quality and low flavonoid content. Thus, the

72

breeding of red-fleshed apples with a high flavonoid content is of great interest to

73

apple breeder worldwide.33–35 Since 2006, we have used wild red-fleshed apple

74

germplasm resources, Malus. sieversii f. niedzwetzkyana, to breed red-fleshed apple

75

and expand the genetic basis and diversity of cultivated apple. Although M. sieversii f.

76

niedzwetzkyana (R6R1) has red-fleshed phenotype, its flesh is soft and sour.

77

Therefore, M. domestica (R6R1) with crisp and sweet flesh was chosen to cross with

78

it. In the crossed F1 hybrid populations, we identified a red-fleshed mutant was

79

homozygous for the R6R6 genotype, in conflict with the parent R6R1 and R1R1

80

genotypes.30 In the BC1 hybrid populations of the R6R6 homozygous mutant crossed

81

with M. domestica, all progeny had the R6R1 genotype, but the flavonoid and

82

anthocyanin content of their flesh differed significantly. This genetic diversity reflects

83

the combined effect of the R6:MdMYB10 and other unknown regulation factors that

84

have not previously been reported. Therefore, RNA-seq was used in this study to

85

compare the transcriptomes of red- and white-fleshed apple strains in the BC1

86

populations. Focusing on screening TFs differentially expressed between the red- and

87

white-fleshed apples unearthed a WRKY family TF designated MdWRKY11. Its role

88

in flavonoid biosynthesis was identified through overexpression studies in ‘Orin’ calli. 5



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We aimed to identify flavonoid-related functional genes useful for providing a

90

valuable perspective on flavonoid synthesis and breeding elite red-fleshed apples with

91

a high flavonoid content.

92 93

MATERIAL AND METHODS

94

Plant materials. The BC1 hybrid populations obtained by crossing the R6R6

95

red-fleshed mutant and M. domestica were grown in the Guanxian Fruit Tree

96

Breeding Base (36°580’N, 115°420’E). Five strain lines with different degree red

97

flesh were selected. Three individual strains were selected from each line for

98

biological repetition and their fruits were harvested at the ripe stage, and then frozen

99

in liquid nitrogen and stored at −80°C. Among them, three individual strains with

100

extremely red flesh (RF) and three individual strains with white flesh (WF) fruit were

101

selected for UPLC-ESI-TOF/MS analysis and RNA-seq.

102

The ‘Orin’ calli were induced and cultured based as described previously.36 The

103

regenerated tissues were placed on a callus-induction medium consisting of

104

Murashige

105

2,4-dichlorophenoxyacetic acid, 0.5 mg·l−1 thidiazuron, 30 g·l−1 sugar, and 7.5 g·l−1

106

agar. The pH was adjusted to 5.8 ± 0.1. The calli were cultured at 24°C and

107

subcultured every 15 d.

and

Skoog

(MS)

medium

supplemented

with

2.5

mg·l−1

108

R6:MYB10 Genotype identification. For genotype identification, the total DNA

109

of each sample was isolated using the Plant Genomic DNA Kit (TianGen, Beijing,

110

China). The R6 and/or R1 repetitive sequences were amplified by the forward primer 6


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proMYB10-F (5ʹ-GGTGGTCAAAGATGTGTGTTGT-3ʹ) and the reverse primer

112

proMYB10-R (5ʹ-TTTGCCTGCTACCCACTTCA-3ʹ) that were designed from the

113

MdMYB10 promoter. Genotypes were then identified by agarose gel electrophoresis.

114

Flavonoid extraction and UPLC-ESI-TOF/MS analysis. Total flavonoids were

115

extracted from 0.3 g powdered apple flesh incubated in 5 ml 1% (v/v) HCl methanol

116

for 2 h at 4°C in darkness. Then, the extract was centrifuged at 10,000 g for 15 min.

117

The obtained flavonoid extract was used for UPLC-ESI-TOF/MS analysis. The upper

118

aqueous phase was saved and filtered using an organic filter membrane (0.2 µm). The

119

UPLC-ESI-TOF/MS (MALDI SYNAPT MS, Waters, http://www.waters.com/) was

120

performed using the ACQUITY UPLC system (Waters), and the chromatographic

121

column was a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 µm particle size,

122

25°C column temperature). Samples were eluted at a 0.4 ml·min-1 flow rate. The

123

mobile phase: solvent A (acetonitrile) and solvent B (2% formic acid aqueous solution,

124

v/v). The gradient: 0–0.1 min with 5% A, 20 min with 20% A, 22 min with 80% A, 21

125

min with 5% A, and 25 min with 5% A. MS was performed using a XEVO G2-S

126

Q-TOF (Waters) with electrospray ionization (ESI+ for anthocyanin and ESI− for

127

others). The following conditions were used: sweep range: 100–150 m/z, capillary

128

voltage: 3.5 kV, cone voltage: 30 V, source temperature: 100°C, desolvation

129

temperature: 300°C, and desolvation gas flow: 500 L·h-1.

130

RNA isolation and RNA-Seq. Total RNA was extracted using the RNAprep Pure

131

Plant kit (Tiangen, Beijing, China). The quality of the purified RNA was determined

132

using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The 7



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mRNA with poly(A) tails were enriched by magnetic beads with oligo (dT). Interrupt

134

buffer was used to fragment the obtained RNA. The random N6 primers were used for

135

reverse transcription and then double-stranded cDNA was synthesized. After

136

amplification and denaturation, a single-strand circular DNA library was obtained and

137

sequenced using the BGISEQ-500 system (The Beijing Genomics Institute, Shenzhen,

138

China).

139

RNA-Seq data analysis. Raw RNA-seq reads were filtered by removing low

140

quality reads containing the connectors and more than 10% unknown base N. The

141

filtered clean reads were compared with the apple genome reference sequence using

142

HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts).37 The

143

expression levels of genes and transcripts were calculated using RSEM.38 Raw counts

144

for each gene were derived and normalized to fragments per kilobase per million

145

mapped fragments (FPKM). The differentially expressed genes (DEGs) were screened

146

using the DEseq2 algorithm39 with adjusted P-value ≤ 0.05 and fold change ≥ 2 as the

147

significance cutoffs. The red-fleshed strains (RF) were used as control. The stratified

148

cluster analysis of gene expression was based on the Cluster software to calculate the

149

common formula with Euclidean distance as matrix.40 According to the GO

150

annotation results41, KEGG annotation results42, and the official classification, the

151

DEGs were grouped into different functional and biological pathways and the Phyper

152

function of R software was used for further enrichment and analysis. FDR correction

153

was performed on the P-value, with FDR ≤ 0.01 generally regarded as significant

154

enrichment. 8


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155

Real-time RT-PCR analysis. First-strand cDNA was synthesized from 1 µg total

156

RNA using a First-strand cDNA Synthesis kit (TianGen, Beijing, China). The

157

synthesized cDNA samples were stored at −20°C for qRT-PCR. Primers were

158

designed using the Beacon Designer 7 program and synthesized by Sangon Biotech

159

(Shanghai, China). The qRT-PCR analysis was conducted using SYBR Green PCR

160

Master Mix (TransGen Biotech, Beijing, China) and the iCycler iQ5 system (Bio-Rad,

161

Hercules, CA). The MdActin gene served as an internal control and the relative

162

quantities of mRNAs were calculated using the 2−∆∆Ct method of the IQ5 2.0

163

program.43 The primers are shown in Table 1 in the Supporting Information.

164

Transformation of apple calli with MdWRKY11. For gene transformation, the

165

coding sequences of MdWRKY11 were recombined into the pRI101-AN vector

166

containing the cauliflower mosaic virus (CaMV) 35S promoter and a GFP tag. This

167

vector was transformed into Agrobacterium tumefaciens LBA4404 that were then

168

used to infect ‘Orin’ calli for 20 min and followed by co-culturing on MS solid

169

medium in the dark for 24–48 h at 24°C. The co-cultured calli were then transferred to

170

screening medium containing 250 mg·l−1 carbenicillin (Solarbio) and 50 mg·l−1

171

kanamycin (Solarbio, Beijing, China) for selection. The amplification primers are

172

shown in Table 1 in the Supporting Information.

173

Determination of flavonoid and anthocyanin content. For spectrophotometric

174

quantification, flavonoids were extracted from 1 g powdered apple flesh and then

175

incubated in 10 ml 1% (v/v) HCl methanol for 4 h at 4°C in darkness. The extract was

176

centrifuged at 12,000 g for 15 min before 0.5 ml of the upper aqueous phase was 9



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177

removed and added to 1 ml of 5% NaNO2, 1 ml of 10% Al(NO3)3, and 4 ml of 2

178

mol·l−1 NaOH. Samples were incubated at room temperature for 15 min before

179

spectrophotometric quantification was performed at 510 nm using a UV–vis

180

spectrophotometer (Shimadzu UV-2450, Kyoto, Japan). Rutin (Sigma Chemicals,

181

Saint Louis, MI) was used as the master standard.

182

Anthocyanin was extracted from 1 g powdered apple flesh and was incubated in 10

183

mL 1% (v/v) HCl-methanol for 24 h at 4°C in darkness. Then, the extract was

184

centrifuged at 10,000 g for 10 min before 1 ml supernatant was mixed with 4 ml KCl

185

buffer (pH 1.0) and 4 ml NaAc buffer (pH 4.5). After 15 min incubation at 4°C,

186

spectrophotometric quantification was performed at 510 nm and 700 nm using a UV–

187

vis spectrophotometer. Anthocyanin content was calculated using the pH differential

188

method.44

189

RESULTS

190

Genotype determination of BC1 hybrid populations and flavonoid composition

191

and content analyses between red- and white-fleshed apples. In previous studies,

192

we obtained an R6R6 homozygous F1 hybrid plant.30 Using the R6R6 individual plant

193

as the parent, we further constructed a BC1 backcross population. In the BC1 hybrid

194

populations, the degree of redness of the fruit flesh varied widely (Figure 1A). The

195

genotypes of five strain lines with different red flesh degree were identified (Figure

196

1B) with all five strains having both a 497-bp and a 396-bp fragment, indicating the

197

R6R1 heterozygous genotype. This demonstrates that the presence of R6:MdMYB10

198

cannot completely determine the red phenotype of red-fleshed apples and that other 10


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199

factors are involved.

200

To observably determine the differences in flavonoid content among these R6R1

201

individuals, the red-fleshed (RF) and white-fleshed (WF) strains that displayed

202

extremely different degrees of red flesh were selected for UPLC-ESI-TOF/MS

203

analysis. A total of 10 flavonoids, including four flavanols, three flavonols, one

204

dihydrochalcone, and two anthocyanins were detected (Figure 1C, D). Two

205

anthocyanins were detected under the positive ion signal and the others were detected

206

under the negative ion signal. The chromatogram map showed that there were

207

significant differences in the peak areas between the two apple cultivars. The levels of

208

the four flavanols in red-fleshed apples were 2.4–3.3 times higher than in the

209

white-fleshed

210

3-O-α-L-arabinoside, and phlorizin content of red-fleshed apples was 14.5, 3.7, 5.2,

211

and 8.6 times higher than that of white-fleshed apples, respectively. Significantly, the

212

cyanidin 3-O-galactoside and cyanidin 3-arabinoside content of red-fleshed apples

213

were 128.9 and 150.5 times higher than of white-fleshed apples, respectively (Table

214

1).

apples.

The

rutin,

quercetin

3-β-D-glucoside,

quercetin

215

Analysis of differentially expressed genes between red- and white-fleshed

216

apples by RNA-seq. To systematically study the phenotypic differences in

217

red-fleshed apples and explore the differentially expressed genes related to flavonoid

218

synthesis, transcriptome profiles of the red-fleshed (RF) and white-fleshed (WF)

219

apples were generated using RNA-seq technology and then compared. A total of 412

220

DEGs were obtained using Padj (adjusted P value) ≤ 0.05 and fold change ≥2 as the 11



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221

cutoffs. A total of 164 genes were up-regulated in WF and 248 genes were

222

down-regulated compared with RF apples (Figure 2A). Of these, the gene encoding a

223

cyclin-dependent kinase F-4 had the greatest differential expression in RF than in WF

224

apples (Table 2 in the Supporting Information). Then, based on the results of DEGs

225

detection, we used the pheatmap function for hierarchical clustering analysis. The

226

result included three biological repeats in two groups of samples and showed that

227

there was a high reproducibility between the biological repeats. In both RF and WF

228

apples, some DEGs were poorly expressed or not expressed at all, suggesting that

229

they may be associated with certain phenotypic deletions in corresponding apple

230

samples (Figure 2B). Raw reads data were deposited in the NCBI sequence read

231

archive under accession number SRP134054.

232

GO functional classification and KEGG pathway enrichment analyses.

233

According to the DEG results, we classified the Gene Ontology (GO) function and

234

analyzed its enrichment. GO terms were divided into three functional categories:

235

molecular function, cellular component, and biological process. In the molecular

236

function category, both up- and down-regulated DEGs were most enriched in

237

metabolic process. In the cellular component category, both up- and down-regulated

238

DEGs were most enriched in cell and cell part. In the biological process category, the

239

up-regulated DEGs in WF were most enriched in catalytic activity, while the

240

down-regulated DEGs were most enriched in binding activity (Figure 3). Significantly,

241

only down-regulated DEGs were enriched in developmental process, reproduction,

242

reproductive process, cytoskeleton, extracellular region, membrane-enclosed lumen, 12


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243

nucleic acid binding transcription factor activity, transporter activity, and molecular

244

transducer activity.

245

Likewise, KEGG pathway enrichment analyses were performed according to the

246

DEGs. The pathway enrichment results showed that the highest number of DEGs

247

were enriched in metabolic pathways, followed by biosynthesis of secondary

248

metabolites (Figure 4A). Furthermore, there were eight DEGs enriched in flavonoid

249

biosynthesis and two in anthocyanin biosynthesis, including cytochrome P450,

250

polyketide synthase 5, leucoanthocyanidin reductase, naringenin, 2-oxoglutarate

251

3-dioxygenase,

252

anthocyanidin 3-O-glucosyltransferase. Both up- and down-regulated DEGs in WF

253

were most enriched in metabolic process. Only down-regulated DEGs were enriched

254

in

255

monoterpenoid biosynthesis, and taurine and hypotaurine metabolism, indicating that

256

they had high metabolic activity in RF apples (Figure 4B).

citrate

chalcone-flavonone

cycle,

anthocyanin

isomerase,

biosynthesis,

UDP-glycosyltransferase,

arachidonic

acid

and

metabolism,

257

The screening of DEGs encoding transcriptional factors and qRT-PCR

258

validation. To further investigate the transcriptional pattern differences between RF

259

and WF apples, the DEGs encoding TFs were analyzed further. A total of 22 DEGs

260

that were found to encode members of 14 TF families, including 17 up-regulated and

261

five down-regulated genes, were found in RF apples compared with WF apples (Table

262

3 in the Supporting Information). Notably, the DEGs encoding MADS TF family

263

members such as MADS24 and AGL24 differed most significantly higher expression,

264

respectively, in RF than in WF apples. The expression of MYB10 in RF apples was 22 13



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265

times higher than in WF apples. Additionally, genes with similar expression patterns

266

are usually functionally related. Cluster analysis shows that DEGs encoding WLIM1,

267

WRKY11, and ETC1 had the closest cluster relationship with MYB10 (Figure 5A).

268

Next, real-time RT-PCR was performed to validate the expression of these 22 DEGs

269

encoding TFs obtained from RNA-Seq. The qRT-PCR results showed that most of the

270

differences in expression were consistent with the expression levels obtained by

271

RNA-seq. The qRT-PCR data for TCP8 and SCL14, however, did not differ

272

significantly between RF and WF apples. Additionally, the differential expression

273

levels of WRKY11 and NAC62 were much higher in the qRT-PCR data compared with

274

the RNA-seq data, reaching 8-fold, and 7.5-fold, respectively (Figure 5B).

275

Furthermore, the expressions of MdWRKY11, MdMYB10, NAC62, WLIM1,

276

MADS24 and AGL24 with significant differences of more than five times were further

277

compared in different red-fleshed phenotypes (Figure 6A). The results showed that

278

MdWRKY11 and MdMYB10 had similar expression patterns in different red-fleshed

279

phenotype strains, which increased their expression with the increase of red flesh

280

degree. However, the expression patterns of MADS24, NAC62 and WLIM1 did not

281

appear to be related to the different red-fleshed phenotypes (Figure 6B). Thus, to

282

explore whether there was some genetic difference of MdWRKY11 in the red-fleshed

283

apple with different red degree, we compared the coding region and promoter

284

sequence of MdWRKY11 and found that there was no difference, indicating that

285

MdWRKY11 is not an additional ‘genetic’ factor for red coloration.

286

Overexpression of MdWRKY11 promotes flavonoid and anthocyanin 14


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287

accumulation in apple calli. Among the 17 up-regulated DEGs, no genes other than

288

MdMYB10 have been reported to be involved in flavonoid synthesis. A small number

289

of the WRKY TFs are, however, known to participate in flavonoid synthesis.45, 46 To

290

determine whether MdWRKY11 was involved in flavonoid synthesis, the flavonoid

291

and anthocyanin contents in the selected R6R1 hybrid individual plants were

292

measured, as well as the relative expression of MdWRKY11. Correlation analysis

293

showed that MdWRKY11 was positively correlated with the flavonoid and

294

anthocyanin content, with correlation coefficients of 0.72 and 0.835, respectively

295

(Figure 7A).

296

To further confirm the function of MdWRKY11 in flavonoid and/or anthocyanin

297

synthesis, we transferred MdWRKY11 into ‘Orin’ apple calli under the control of the

298

CaMV 35S promoter. Three independent transgenic calli lines of 35S:MdWRKY11

299

(OE1, OE2. and OE3) turned red, while the wild-type (WT) calli and control calli

300

(CK) with empty vectors did not change color (Figure 7B). The presence of the

301

transgene in OE calli was confirmed by PCR amplification (Figure 7C) and western

302

blotting (Figure 7D). Next, flavonoid and anthocyanin content were determined. The

303

results showed that OE calli produced 5.3–8.5 times more flavonoid and 2.9–5.2 times

304

more anthocyanin than the WT and CK calli (Figure 7E). Finally, the expression of

305

flavonoid pathway genes was analyzed by qRT-PCR. The transcript levels of F3H,

306

FLS, DFR, ANS and UFGT increased significantly in the OE calli, while other genes

307

remained unchanged (Figure 7F). These results demonstrate that MdWRKY11 is a

308

positive regulator for anthocyanin biosynthesis and lay a foundation for further study 15



309

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on the metabolic mechanism of flavonoids.

310 311

DISCUSSION

312

The crop and fruit tree industry is challenged by a serious inbreeding problem that

313

has narrowed the hereditary basis of the plants, resulting in lines with poor nutritional

314

quality. Using wild germplasm resources to improve the genetic diversity of cultivated

315

varieties has become an important method of counteracting this.47, 48 In apple, with the

316

completion of the apple genome sequence and the resequencing of the apple

317

germplasm resources, M. sieversii has been clarified and confirmed to be the ancestor

318

of cultivated apples.11, 49 In this study, we made full use of M. sieversii and its red

319

variety M. sieversii f. niedzwetzkyana to conduct extensive cross-breeding

320

experiments. In the F1 hybrid populations, we previously performed a transcriptomic

321

analysis comparing red- and white-fleshed apples that had the R6R1 and R1R1

322

genotypes, respectively.50 Here, in the BC1 hybrid populations, we found that while

323

the selected individual plants were all R6R1 heterozygotes, the degree of red

324

coloration of fruit flesh varied widely. Therefore, high throughput RNA-seq was

325

performed between red- and white-fleshed fruit strains to study the mechanism

326

underlying differences in flavonoid content in plants with the same genotypes in more

327

depth.

328

The complexity of the regulation mechanism underlying flavonoid biosynthesis

329

embodies the diversity of red-fleshed apple germplasm resources. In terms of

330

flavonoid metabolism, extensive research has been conducted on the cause of 16


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331

differences in red-fleshed coloration. The overexpression of MdMYB10 in transgenic

332

‘Royal Gala’ results in a red-fleshed apple phenotype.51 Interestingly, although

333

MdMYB10 was highly expressed in type 1 red-fleshed apples, it was not expressed in

334

type 2 red-fleshed apples. Conversely, another MYB transcription factor close to

335

MdMYB10, designated MdMYB110a, is associated with the red pigmentation

336

phenotype in type 2 red-fleshed apples but is not expressed in type 1 red-fleshed

337

apples.52 In this study, the RNA-seq and qRT-PCR results showed that the expression

338

of MdMYB10 was significantly up-regulated in red-fleshed apples, proving that it was

339

associated with type 1 red-fleshed apples. No MdMYB110a was found in the screened

340

DEGs, suggesting that MdMYB110a was not the cause of the difference in red flesh

341

coloration between red- and white-fleshed apples with the same R6R1 genotype in the

342

BC1 populations. MADS family TFs have been widely reported to be closely related

343

to pollen development and flower formation.53,54 Likewise, flavonoids have also been

344

shown to be closely related to pollen development in plants.55 There are, however, no

345

reports of MADS family transcription factors being related to flavonoid or

346

anthocyanin synthesis. In this study, we found two MADS family genes that had the

347

most significant up-regulation of expression in RF. Whether MADS TFs are involved

348

in flavonoid metabolism requires further study. In terms of negative regulatory factors,

349

it has been reported that the expression of AtLOB37, AtLOB38, and AtLOB39 can be

350

induced by nitrate and can negatively regulate anthocyanin biosynthesis.56

351

Furthermore, it has been shown that overexpression of MdLOB52 can inhibit the

352

synthesis of anthocyanin in apple.57 Here, the results of our RNA-seq also showed 17



Page 18 of 36

353

that the expression of MdLOB37 was significantly up-regulated in WF to a level 11.41

354

times higher than that in RF; this may be one of the reasons why the WF apples were

355

not red.

356

Flavonoids play important roles in the protection of plants against biotic and

357

abiotic stress through their ability to inhibit reactive oxygen species formation.58

358

Under oxidative and drought stress, the overaccumulation of anthocyanin can

359

effectively mitigate the accumulation of reactive oxygen species in vivo.59 In carrot

360

cells cultivated in vitro, the ability of specific anthocyanins and other

361

phenylpropanoids to protect cells from heat stress was investigated.60 Moreover,

362

flavonoids were identified to have anti-fungal activity in the defense against Fusarium

363

species and Pyricularia oryzae.61 In this study, we screened several transcription

364

factors related to biotic and abiotic stress responses using the RNA-seq data,

365

including SPL12, AP2, NAC62, MYB44, and WRKY11. They were all found to be

366

significantly up-regulated in RF but their role in flavonoid synthesis is unknown. In

367

both Arabidopsis and tobacco, overexpression of SPL1 or SPL12 can enhance

368

thermotolerance at the reproductive stage.62 In tobacco, overexpression of an AP2 TF

369

enhanced resistance to pathogen attack and osmotic stress.63 In Arabidopsis, the

370

expression of the RD26 gene encoding a NAC TF could be induced by drought,

371

abscisic acid, and high salinity.64 The MYB family TF MYB44 has been characterized

372

as a phosphorylation-dependent positive regulator in salt stress signaling.65 Among

373

these screened TFs, the WRKY family has been widely studied in plant stress

374

resistance pathways. In rice, for example, OsWRKY11 was suggested to be induced by 18


Page 19 of 36


375

pathogens, drought, and heat stress.66 Ectopic expression of the grapevine VvWRKY11

376

in Arabidopsis seedlings resulted in higher tolerance to water stress induced by

377

mannitol than in wild-type plants.67 While sporadic, reports on the involvement of

378

WRKY family TFs in flavonoid synthesis are slowly emerging. Ectopic

379

overexpression of the Brassica napus WRKY41-1 in Arabidopsis showed its role in

380

negative regulating anthocyanin biosynthesis.45 In grape, VvWRKY26 involvement in

381

the flavonoid pathway was shown to possibly be restricted to the control of

382

proanthocyanidin biosynthesis.46 In this study, MdWRKY11 expression was found to

383

be significantly up-regulated in RF apples. However, the promoter and coding regions

384

of MdWRKY11 are identical in all red-fleshed apples with different colors, indicating

385

that MdWRKY11 is not an additional ‘genetic’ factor for red coloration. MdWRKY11

386

may promote anthocyanin synthesis through the regulation of other factors rather than

387

the ‘genetic’ factor. The overexpression of MdWRKY11 promoted the expression of

388

F3H, FLS, DFR, ANS and UFGT genes and increased the accumulation of flavonoids

389

and anthocyanin in apple calli, confirming the role of MdWRKY11 in flavonoid

390

biosynthesis.

391 392

ASSOCIATED CONTENT

393

Supporting Information

394

Primers used for RT-qPCR and the plasmids construction, differentially expressed

395

genes between red- and white-fleshed apples, and differentially expressed genes

396

encoding transcription factors. 19



397

AUTHOR INFORMATION

398

Corresponding Author

399

*Tel: +86-538-8249338. E-mail: [email protected]

400

ORCID

401

Nan Wang: 0000-0002-8648-3941

402

Notes

403

The authors declare no competing financial interest.

404

‡

Page 20 of 36

N.W. and W.L. are co-first authors.

405

Funding

406

This work was supported by the National Key Research Project (2016YFC0501505)

407

and the National Natural Science Foundation of China (CN) (31572091, 31730080).

408

ACKNOWLEDGMENTS

409

We thank the Luntai National Fruit Germplasm Resources Garden for providing

410

germplasm resources, and the Shujing Wu and Yujin Hao Laboratories for providing

411

the vectors. We thank Emma Tacken, PhD, from Liwen Bianji, Edanz Group China

412

for editing the English text of a draft of this manuscript.

413

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FIGURE CAPTIONS Figure 1 (A) The establishment of BC1 hybrid populations. BC1 hybrid populations were derived from a cross between an R6R6 homozygous red-fleshed mutant and M. domestica. (B) Both a 497 bp and a 396 bp fragment were present in the selected red-fleshed varieties (a–e). Only a single 396 bp fragment was present in the M. domestica control variety. (C) Flavonoid composition and content analyses and comparisons between red-fleshed (RF) and white-fleshed (WF) apples using UPLC-ESI-TOF/MS. Four flavanols (a–d), three flavonols (e–g), and one dihydrochalcone (h) were detected under the negative ion signal. (D) Two anthocyanins were detected under the positive ion signal (i and j). Figure 2 Analysis of differentially expressed genes (DEGs) between RF and WF. (A) The MA-plot distribution map of all DEGs. The X-axis represents the expression level after conversion of the log2 value. The Y-axis represents the fold-change in expression after conversion of the log2 value. (B) Heatmap cluster analysis of DEGs. The amount of expression after the log10 conversion is represented by different colors. Figure 3 The GO functional classification and enrichment analysis of DEGs. GO terms were divided into three functional categories: molecular function, cellular 25



Page 26 of 36

component, and biological process. The Y-axis represents the number of DEGs enriched in each GO classification. FDR correction is performed according to P-value, and the function of FDR ≤ 0.01 is generally regarded as significant enrichment. Figure 4 KEGG pathway enrichment analyses of DEGs. (A) The different colors represent different qvalues. The bluer the color, the more significant the enrichment result is. The size of the point represents the number of DEGs enriched in the pathway. (B) According to the KEGG annotation results and the official classification, the top 30 enriched pathways are shown. FDR correction is performed on P-value, and the function of FDR ≤ 0.01 is generally regarded as significant enrichment. Figure 5 The 22 screened DEGs encoding transcription factors and real-time RT-PCR validation. (A) Heatmap cluster analysis of DEGs encoding transcription factors compared between RF and WF. Boxes represent the amount of expression after the log10 conversion. Colors represent Z-score values of normalized expression levels. (B) Real-time RT-PCR was used to validate the transcript levels of 22 selected DEGs. MdActin was used as the internal control gene. Values are means ± SD of three independent biological replicates. Statistical significance: *P

Transcriptomic Analysis of Red-Fleshed Apples ... - ACS Publications

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