Ectopic overexpression of bol-miR171b increases chlorophyll content

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Agricultural and Environmental Chemistry

Ectopic overexpression of bol-miR171b increases chlorophyll content and results in sterility in broccoli (Brassica oleracea L var. italic) Hui Li, Qingli Zhang, Lihong Li, Jiye Yuan, Yu Wang, Mei Wu, Zhanpin Han, Min Liu, Chengbin Chen, Wenqin Song, and Chun-guo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01531 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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Journal of Agricultural and Food Chemistry

Ectopic overexpression of bol-miR171b increases chlorophyll content and results in sterility in broccoli (Brassica oleracea L var. italic)

Hui Li 2, Qingli Zhang1, Lihong Li1, Jiye Yuan1, Yu Wang1, Mei Wu1, Zhanpin Han2, Min Liu3 Chengbin Chen1, Wenqin Song1, Chunguo Wang1** 1

College of Life Sciences, Nankai University, Tianjin, China;

2

College of Horticulture and Landscape, Tianjin Agricultural University, Tianjin,

China 3

College of Life Sciences, Shandong Normal University, Jinan, Shandong, China

**Correspondence: email: [email protected]

Hui Li ： [email protected]; Qingli Zhang: [email protected]; Lihong Li: [email protected];

Jiye

Yuan:

[email protected];

Yu

Wang:

[email protected]; Mei Wu: [email protected]; Zhanpin Han: [email protected]; Min Liu: [email protected]; Chengbin Chen: [email protected]; Wenqin Song: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

2

MiR171 plays pleiotropic roles in the growth and development of several plant

3

species. However, the mechanism underlying the miR171-mediated regulation of

4

organ development in broccoli remains unknown. In this study, bol-miR171b was

5

characterized and found to be differentially expressed in various broccoli organs.

6

The ectopic overexpression of bol-miR171b in Arabidopsis affected the leaf and

7

silique development of transgenic lines. In particular, the chlorophyll content of

8

leaves from overexpressed bol-miR171b transgenic Arabidopsis was higher than that

9

of the vector controls. The fertility and seed yield of Arabidopsis with overexpressed

10

bol-miR171b were markedly lower than those of the vector controls. Similarly,

11

overexpressed bol-miR171b transgenic broccoli exhibited dark green leaves with

12

high chlorophyll content, and nearly all the flowers were sterile. These results

13

demonstrated that overexpression of bol-miR171b could increase the chlorophyll

14

content of transgenic plants. Degradome sequencing was conducted to identify the

15

targets of bol-miR171b. Two members of the GRAS gene family, BolSCL6 and

16

BolSCL27, were cleaved by bol-miR171b-3p in broccoli. Besides the genes targeted

17

by bol-miR171b-3p, adenylylsulfate reductase 3 (APSR3), which played important

18

roles in plant sulfate assimilation and reduction, was speculated to be cleaved by

19

bol-miR171b-5p, suggesting that the star sequence of bol-miR171b may also have

20

functions in broccoli. Comparative transcriptome analysis further revealed that the

21

genes involved in chloroplast development and sulfate homeostasis should

22

participate in the bol-miR171b-mediated regulatory network. Taken together, these 2


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findings provided new insights into the function and regulation of bol-miR171b in

24

broccoli and indicated the potential of bol-miR171b as a small RNA molecule that

25

increased leaf chlorophyll in plants by genetic engineering.

26

KEY WORDS: bol-miR171b, chlorophyll, broccoli, leaf development, silique

27

development

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

3



45

1. INTRODUCTION

46

MicroRNAs (miRNAs) are endogenous noncoding RNAs that are 21-24 nucleotides

47

(nts) in length. These RNAs are widely distributed in animals and plants. In plants,

48

primary miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase II.

49

Pri-miRNAs are then processed to produce stem-loop precursors (pre-miRNAs),

50

which are further processed to produce miRNA/miRNA* duplexes. Mature miRNAs

51

are released from the duplexes and incorporated into RNA-induced silencing

52

complexes to regulate target genes through transcript cleavage or translational

53

inhibition.1-2 Considerable evidence has demonstrated that miRNA-mediated gene

54

regulation has critical roles in plant growth and development. For example, MiR156

55

participates in vegetative phase change by down-regulating several SPL genes.3-5

56

MiR159 targeting MYB transcription factors is required for anther development.6

57

MiR160 functions in root development by cleaving auxin response factors.7-8

58

MiR165/166 targets REVOLUTA, PHABULOSA, PHAVOLUTA, CORONA, and

59

ATHB8, all of which function in the abaxial identity of lateral organs.9-12 MiR172

60

regulates floral organ identity and flowering time by regulating APETELA 2

61

transcription factors.13-15 MiR319 contributes to leaf development by targeting TCP

62

transcription factors.16-17 MiR828 and miR858 target MYB transcription factors and

63

play crucial roles in cotton fiber development.18 In addition, miR156 also contributes

64

to aerial axillary bud formation and shoot architecture.19 MiR159 determines fruit

65

setting and plant response to the root-knot nematode Meloidogyne incognita.20-21

66

Similar to miR156, miR172 regulates vegetative phase changes.22-23 MiR319 4


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participates in plant response to stress.24-25 The miR165/166-mediated regulatory

68

module regulates abiotic stress responses and ABA homeostasis.26 These

69

investigations indicated that the functions of some miRNAs are pleiotropic in

70

regulating plant growth and development. The same miRNA, deriving from various

71

plant species, may have different functions. Meanwhile, one biological process may

72

be regulated by different miRNAs. MiR171 is another important regulator with

73

pleiotropic functions in plant growth and development. At present, over 500

74

members of the miR171 family from diverse plant species have been reported in the

75

miRbase database (miRBase 21). MiR171 from Arabidopsis contains three members,

76

ath-miR171a to c. The GRAS gene family members scarecrow-like 6

77

(SCL6)/SCL6-IV, SCL22/SCL6-III and SCL27/SCL6-II are the target genes of

78

ath-miR171c.27-28 Ath-miR171c-targeted SCLs are involved in the proliferation of

79

meristematic cells, polar organization, and chlorophyll synthesis.28-29 In tomato,

80

SlGRAS24, a member of the GRAS gene family, is targeted by Sly-miR171. By

81

regulating gibberellin and auxin homeostasis, the Sly-miR171-SlGRAS24 module

82

affects multiple agronomical traits, including plant height, flowering time, leaf

83

architecture, lateral branch number, root length, and fruit set and development.30 In

84

barley and rice, miR171 overexpression affects phase transition and floral meristem

85

determinacy.31-32 MiR171 also has been demonstrated to function upon various

86

stresses in Arabidopsis, barley, maize and Solanum tuberosum.33-36 However, the

87

functions of miR171 in other plant species, such as the major vegetables under the

88

Cruciferae, remain unknown. 5



89

Broccoli (Brassica oleracea L var. italic) is one of the most important vegetables

90

of B. oleracea. The edible organ of broccoli is its curd, which mainly composes of

91

many flower buds and shortened inflorescence branches. The curd of broccoli

92

contains abundant nutrients and bioactive substances, such as sulforaphane that

93

shows powerful potential in anti-cancer properties.37 However, knowledge of the

94

molecular processes involved in the regulation of the growth and development of

95

broccoli is currently limited. In the present study, bol-miRNA171b was identified in

96

broccoli and its expression profiles in different organs were explored. The candidate

97

targets of bol-miRNA171b were also identified through degradome sequencing. In

98

addition, to uncover the function of bol-miRNA171b, bol-miRNA171b was

99

overexpressed in Arabidopsis and broccoli, respectively. The bol-miR171b-mediated

100

regulation network was further explored by transcriptome sequencing.

101

2. MATERIALS AND METHODS

102

2.1. Plant Materials

103

Arabidopsis thaliana (Columbia ecotype) was used in the present study. The

104

Arabidopsis seeds were planted in soil at 22 °C under a 40%-65% relative humidity

105

and a 16 h/8 h light/dark photoperiod after vernalizing in darkness at 4 °C for 3 days.

106

Meanwhile, homozygous broccoli seeds were treated with 75% ethanol for 5 min,

107

and 2% NaClO for 10 min, and then rinsed three times with sterile distilled water.

108

The sterilized seeds were planted on Murashige & Skoog (MS) medium under

109

controlled conditions with a 16 h/8 h light/dark cycle at 25 °C and 22 °C,

110

respectively. The 35S::bol-miR171b transgenic broccoli was planted in the green 6


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house at 25 °C with a 12 h/12 h light/dark cycle.

112

2.2. Bol-miR171b Expression Pattern Assay

113

Total RNAs from different organs of broccoli (e.g., cotyledons, hypocotyls, leaves,

114

roots, stems and flowers) were isolated with TRIzol reagent (Invitrogen, USA) in

115

accordance with the manufacturer’s instructions. RNAs free of contaminated

116

genomic

117

bol-miR171b-5p stem-loop primers (Table S1). The expression levels of

118

bol-miR171b-3p and bol-miR171b-5p in the different organs of broccoli were

119

analyzed by real-time quantitative stem-loop RT-PCR. Small nuclear RNA U6 was

120

used as an internal reference. Faststart Universal SYBR Green Master (Roche,

121

Germany) was used in all real-time quantitative RT-PCR experiments (Table S1).

122

The relative expression levels of bol-miR171b-3p and bol-miR171b-5p were

123

calculated by the comparative 2−∆∆CT method in accordance with the manufacturer’s

124

recommendations. To ensure the reliability of quantitative analysis, three batches of

125

independently isolated RNAs from different organs of broccoli were used, and three

126

technological replicates were performed.

127

2.3. Vector Construction and Plant Transformation

128

Specific primers with Nco I and BstE II restriction sites were designed to amplify the

129

precursor sequences of bol-miR171b (Table S1). The PCR products were then cloned

130

into the pEASY-T1 vector and double-digested with Nco I and BstE II. The digested

131

products were subcloned into the pCAMBIA3301 binary vector. The recombinant

132

plasmids and empty vectors were transformed into Agrobacterium tumefaciens strain

DNAs

were

reverse-transcribed

using

7


bol-miR171b-3p

and


133

LBA4404 and then introduced into Arabidopsis thaliana via the floral dip method.

134

The T1 seeds of the transgenic plants were sowed in soil, and the 12-day-old

135

seedlings were selected by spraying 1:10, 000 dilute Basta solution. The positive

136

transgenic lines were further identified by RT-PCR with unique primers (Table S1).

137

The homozygous T3 generations of the transgenic Arabidopsis lines were used for

138

subsequent analyses. The recombinant 35S::bol-miR171b expression vector and the

139

empty vector were also transformed into the broccoli by A. tumefaciens-mediated

140

method. In brief, the hypocotyls of the 7-day-old broccoli seedlings were cut to 0.5

141

cm and pre-cultured on MS medium for 2 days. Subsequently, the hypocotyl

142

explants were dipped into Agrobacterium suspension with 35S::bol-miR171b

143

expression vector or empty vector for 1 min. The infected explants were then

144

transferred onto the co-cultivation medium, cultured for 2 days under dark condition.

145

To eliminate the Agrobacterium that was not transformed into the cells of the

146

hypocotyl explants, after co-culturing, all explants were washed three times with

147

sterile water and transferred into the callus induction medium (MS medium + 0.1

148

mg/L NAA (1-naphthlcetic acid) + 1 mg/L 6-BA (6-Benzylaminopurine) + 0.5% agar,

149

Ph = 5.8) containing 200 mg/L cefotaxime sodium for 2 weeks. Finally, the explants

150

were transferred into the root initiation medium (1/2 MS medium + 0.1 mg/L NAA +

151

1 mg/L 6-BA + 0.5% agar, pH = 5.8) containing 100 mg/L cefotaxime sodium and 3

152

mg/L Basta. The positive transgenic broccoli plants were further identified by

153

RT-PCR using unique primers (Table S1).

154

2.4. Phenotypic Data Collection and Analysis 8


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The phenotypes of 35S::bol-miR171b transgenic Arabidopsis and broccoli were both

156

observed. In brief, in the 35S::bol-miR171b transgenic Arabidopsis, the numbers of

157

leaves, branches, flower buds and siliques were statistically analyzed. The size of the

158

leaves and siliques, the height of the branches and the fertility of the flowers were

159

also measured. In the 35S::bol-miR171b transgenic broccoli, the phenotypes of the

160

leaves and the fertility of flowers were emphatically observed. In addition, the

161

chlorophyll content in the 35S::bol-miR171b transgenic Arabidopsis and the vector

162

controls were measured in accordance with a previously described protocol.38

163

2.5. Transcriptome Sequencing and Differential Gene Expression Profiling

164

Analysis

165

The equal proportion of the leaves, curds, flowers, and roots from the

166

35S::bol-miR171b transgenic broccoli was mixed and used to isolate the total RNAs

167

by TRIzol reagent (Invitrogen, CA, USA). Similarly, the total RNAs from the vector

168

controls were isolated. The RNAs with high purity (OD260/280 = 1.8-2.2) and high

169

integrity (RNA integrity number, RIN > 8.0) were used to construct sequencing

170

libraries by TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA,

171

USA). These libraries were sequenced on the Illumina HiSeqTM 2500 sequencing

172

platform and 150 bp/125 bp paired-end raw reads were generated. To ensure the

173

reliability of the sequencing data, three batches of independently isolated RNAs

174

from the 35S::bol-miR171b transgenic broccoli and the vector controls were used to

175

construct the sequencing libraries and then sequenced with three technological

176

replicates. 9



177

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Clean reads were generated by discarding the low-quality reads, adaptors, and

178

other

contaminants

179

(http://hannonlab.cshl.edu/fastx_toolkit/license.html).

180

annotated and classified by mapping them to the reference genome of B. oleracea

181

var.

182

(http://www.ccb.jhu.edu/software/hisat/index.shtml). The count of the clean reads

183

that matched each gene denoted the expression abundance or level of this gene. Then,

184

the expression level of each gene was calculated and normalized by the fragments

185

per kilobase of transcript sequence per million base pairs sequenced (FPKM).

186

Differential expression analysis of the genes was performed by using the R package

187

DESeq (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). The

188

genes that showed significantly expression levels between the 35S::bol-miR171b

189

transgenic broccoli and vector control were identified according to the following

190

thresholds: │log2 (fold-change (35S::bol-miR171b transgenic broccoli / vector

191

control))│ > 1 and corrected p-value < 0.05. Gene ontology (GO) analysis of the

192

differentially expressed genes was performed by using the agriGO (http://

193

bioinfo.cau.edu.cn/agriGO/) platform, and hypergeometric test was conducted to

194

identify the significantly enriched GO terms (corrected p-value < 0.05). To visualize

195

the statistically overrepresented GO terms, the GO terms were analyzed by ReviGO

196

(http://revigo.irb.hr/).

197

2.6. Degradome Sequencing and MiRNA Target Identification

198

Total RNA was isolated from mixtures containing equal proportions of broccoli

oleracea

deposited

from

in

the

the

raw

NCBI

reads The

genome

10


by clean

database

fastx_toolkit reads

by

were

HISAT

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cotyledons, hypocotyls, leaves, roots, stems, and flowers. The quantity and purity of

200

RNAs were evaluated by using Bioanalyzer 2100 (Agilent, USA) and NanoDrop

201

2000C (Thermo Scientific, USA). Approximately 200 µg of the total RNA (RIN >

202

8.0) was used for degradome library construction following previously described

203

protocols.39 In brief, (1) approximately 150-250 ng of poly (A)+ RNA was annealed

204

with biotinylated random primers. (2) The streptavidin capture of the RNA

205

fragments was conducted with biotinylated random primers. (3) RNAs containing

206

5’-monophosphates were ligated by 5’ adaptors. (4) RNAs were reverse transcription

207

and RT-PCR was conducted. (5) Libraries were constructed. Then, the single-end

208

sequencing (50 bp) was performed on an Illumina Hiseq2000 sequencing platform,

209

and 50 nt raw reads were produced. After pre-processing, clean tags were generated,

210

stored, and classified by the alignment to the database to remove the ncRNAs.

211

Finally, the miRNA-mRNA pairs were identified and mapped to the reference

212

transcriptome data of broccoli. The clean tags were mapped to the broccoli

213

transcriptome by using SOAP2.20 (http://soap.genomics.org.cn/). The cleavage sites

214

of the miRNAs in the targeted genes were identified in accordance with the

215

information of the obtained miRNA-mRNA pairs and the p-value was calculated by

216

using PAREsnip (http://srna-workbench.cmp.uea.ac.uk/tools/paresnip/). Only the

217

cleavage sites with P-value < 0.05 were shown.

218

2.7. Differentially Expressed Gene Assay by qRT-PCR

219

The expression patterns of the differentially expressed genes and the targets of

220

bol-miR171b detected by transcriptome and degradome analyses were further 11



221

demonstrated by qRT-PCR. Specific primer pairs were designed for the detection of

222

corresponding genes (Table S1). The Actin gene from broccoli was selected as

223

internal control. Similarly to qRT-PCR in bol-miR171b expression pattern assay,

224

Faststart Universal SYBR Green Master (Roche, Germany) was used and the

225

comparative 2−∆∆CT method was conducted to calculate the relative expression levels

226

of genes.

227

3. RESULTS

228

3.1. Bol-miR171b Precursor was Identified in Broccoli

229

On the basis of the small RNA high-throughput sequencing data in broccoli and

230

cauliflower, bol-miR171b-3p, which is also called mature bol-miR171b, was

231

detected in the hypocotyls of seedlings, whereas the star sequence of bol-miR171b

232

(bol-miR171b-5p or bol-miR171b*) was detected only in the cotyledons. To reveal

233

the function of bol-miR171b, the precursor sequence of bol-miR171b was cloned in

234

broccoli. A 103 bp sequence containing mature bol-miR171b and bol-miR171b* was

235

identified. This sequence can form a steady secondary structure and represent the

236

bol-miR171b precursor (Figure 1a). Sequence alignment analysis results indicated

237

that the bol-miR171b precursor is similar to the precursor sequences of miR171b in

238

A. thaliana, A. lyrata, B. napus, and B. rapa but differs from other miR171b

239

precursors (Figure 1b).

240

3.2. Bol-miR171b Displayed Differential Expression Profiling in Various Organs

241

of Broccoli

242

The expression patterns of bol-miR171b-3p and bol-miR171b-5p were both further 12


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analyzed. In seedlings, bol-miR171b-3p and bol-miR171b-5p displayed higher

244

expression levels in the cotyledons than those in the hypocotyls. Strikingly, the

245

expression levels of bol-miR171b-5p were significantly higher than those of

246

bol-miR171b-3p (Figure 1c). In the adult plants, bol-miR171b-3p exhibited

247

significantly differential expression levels in leaves, roots, stems, and flowers.

248

Specifically, bol-miR171b-3p displayed the highest expression level in the sterile

249

flowers. Similarly, bol-miR171b-5p was also expressed in the leaves, roots, stems,

250

and flowers. Moreover, the expression patterns of bol-miR171b-5p were similar to

251

those of bol-miR171b-3p in different organs of broccoli (Figure 1c, d). These results

252

indicated that bol-miR171b-3p and bol-miR171b-5p both displayed organ-specific

253

expression patterns in broccoli.

254

3.3. Overexpression of bol-miR171b in Arabidopsis Increased Chlorophyll

255

Content

256

To uncover the roles of bol-miR171b, the overexpression vectors of bol-miR171b

257

precursor driven by the enhanced CaMV 35S promoter were constructed and

258

transformed into Arabidopsis. The homologous T3 generations of three independent

259

transgenic lines (at least 20 individual plants from each line) with significantly high

260

expression levels of bol-miR171b were used for the subsequent analysis (Figure S1,

261

Table S2). At 3 weeks after sowing, the phenotypes of 35S::bol-miR171b transgenic

262

Arabidopsis were similar to those of the vector controls. However, the growth of

263

35S::bol-miR171b transgenic lines appeared to be inhibited over time. The

264

phenotypes of the 6-week-old 35S::bol-miR171b transgenic lines significantly 13



265

differed from those of the vector controls (Figure S2). Briefly, the numbers of rosette

266

leaves, branches, flowers, and siliques in the 35S::bol-miR171b transgenic lines were

267

significantly lower than those in the vector controls (Figures 2a, b, d, e). The rosette

268

leaves of the overexpressed bol-miR171b transgenic lines were frizzy and dark green,

269

and contained approximately 50% more chlorophyll than those of the vector controls

270

(Figure 2c). Moreover, the fertility of the 35S::bol-miR171b transgenic Arabidopsis

271

was significantly decreased. Many flowers were sterile and unable to generate

272

normal siliques. Even if the siliques formed in the 35S::bol-miR171b transgenic lines,

273

they were smaller than those of the vector controls. Consequently, the seeds

274

produced by the individual plant of 35S::bol-miR171b transgenic Arabidopsis were

275

significantly lower than those of the vector controls (Figure S2).

276

3.4. 35S::bol-miR171b Transgenic Broccoli Exhibited Dark Green Leaf with

277

High Chlorophyll and Flower Sterility

278

The bol-miR171b precursor was also overexpressed in broccoli. A total of 18

279

independent 35::bol-miR171b transgenic broccoli lines were obtained (Figure S1).

280

Similar to the 35::bol-miR171b transgenic Arabidopsis, phenotypic data indicated

281

that the overexpression of bol-miR171b in broccoli can inhibit growth and

282

development. Under the same growth condition, the aerial organs of the

283

35::bol-miR171b transgenic broccoli were smaller than those of the vector controls.

284

The leaves of the 35::bol-miR171b transgenic broccoli were frizzy and dark green.

285

The chlorophyll content of the leaves in the 35::bol-miR171b transgenic broccoli

286

was approximately 1.1 times higher than that of the vector controls (Figure 2f). The 14


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fertility of the 35S::bol-miR171b transgenic broccoli was markedly reduced. The

288

majority of the siliques was abnormal and could not generate seeds (Figure 3).

289

3.5. Genes Targeted by bol-miR171b-3p and bol-miR171b-5p were Identified

290

Degradome sequencing is a powerful and efficient approach for validating the

291

targeted genes of miRNAs. The major principle is that if a gene is targeted by a

292

certain miRNA, the transcripts of the gene should be cleaved at the position where

293

the bases are complementarily paired with the miRNA molecules, and the 5’ end of

294

the cleaved mRNAs with poly (A) tails would be enriched at the cleavage site. Then,

295

after reverse transcription and RT-PCR, these cleaved fragments could be identified

296

by high-throughput sequencing and functionally annotated by the subsequent

297

bioinformatic analysis. Accordingly, degradome sequencing was conducted to

298

identify the potential targets of bol-miR171b. Two homologs of AtSCL6 and

299

AtSCL27, named BolSCL6 and BolSCL27, respectively, were cleaved by

300

bol-miR171b-3p in broccoli (Figure 4a). Quantitative expression assay results also

301

indicated that in the 35S::bol-miR171b transgenic broccoli lines, the expression

302

levels of BolSCL6 and BolSCL27 were significantly lower than those of the vector

303

controls (Figure 4b). In addition, bol-miR171b-5p displayed differential expression

304

patterns in the different organs of broccoli. It is interesting to know whether some

305

genes were targeted by bol-miR171b-5p. According to the plant small RNA target

306

analysis

307

reductase gene, named adenylylsulfate reductase 3 (APSR3), was speculated to be

308

cleaved by bol-miR171b-5p, although the cleaved event was not detected by the

server

(http://plantgrn.noble.org/psRNATarget/),

15


an

adenylylsulfate


309

present degradome sequencing (Figure 5a). Nevertheless, the quantitative expression

310

assay further demonstrated that the expression level of APSR3 tended to be

311

negatively correlated with that of bol-miR171b-5p in the different organs of broccoli

312

(Figure 5b). Moreover, in the 35S::bol-miR171b transgenic broccoli lines, APSR3

313

displayed significantly lower expression level than that in the vector controls (Figure

314

5c). These results suggested that APSR3 should be targeted by bol-miR171b-5p.

315

3.6. Genes Involved in bol-miR171b-mediated Regulation Network were

316

Identified

317

Comparative transcriptome analysis was conducted to understand the gene

318

regulatory networks that are associated with the roles of bol-miR171b in broccoli. A

319

total of 2,692 differentially expressed genes (DEGs) were identified between the

320

35S::bol-miR171b transgenic broccoli and vector controls. Among them, the

321

expression levels of 621 genes were down-regulated in the 35S::bol-miR171b

322

transgenic broccoli, and the other 2, 071 genes displayed higher expression levels

323

than those of the vector controls (Table S3). GO functional annotations indicated that

324

the 621 DEGs with low expression levels in the 35S::bol-miR171b transgenic

325

broccoli were mapped to 467 GO terms, among which 33, 17, and 8 GO terms were

326

significantly enriched in biological processes, molecular functions, and cellular

327

components, respectively (Table S4). The significantly enriched GO terms in the

328

biological processes were clustered by ReviGO. The results indicated that these GO

329

terms were mainly involved in the hormone response, light-mediated signaling,

330

sulfate homeostasis, and photosynthesis (Figure S3). A total of 519 GO terms were 16


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331

further identified for the 2, 071 genes with up-regulated expression patterns in the

332

35S::Bol-miR171b transgenic broccoli. Among these GO terms, 138, 40, and 15

333

were significantly enriched in biological processes, molecular functions, and cellular

334

components, respectively (Table S5). In contrast to the GO terms targeted by the

335

down-regulated genes, 138 GO terms targeted by the up-regulated genes in the

336

biological processes were mainly involved in response to stress, response to growth

337

and development, hormone response, and light-mediated signaling (Figure S3).

338

3.7. Expression Patterns of Genes Involved in Chloroplast Development and

339

Sulfate Homeostasis Significantly Changed in 35S::bol-miR171b Transgenic

340

Broccoli

341

The function of the genes displaying significantly differential expression levels

342

between the 35S::bol-miR171b transgenic broccoli and the vector controls was

343

further analyzed. The results indicated that among the 2, 692 DEGs, genes closely

344

associated with chloroplast development were overrepresented. At least 71

345

chloroplast-related genes changed their expression patterns in the 35S::bol-miR171b

346

transgenic broccoli. Among these genes, 48 displayed higher expression levels, and

347

the expression levels of the other 23 genes were lower in the 35S::bol-miR171b

348

transgenic lines than in the vector controls (Table S6). In addition, the expression

349

levels of several genes involved in sulfate homeostasis, such as APSR1 and APSR3,

350

were down-regulated in the 35S::bol-miR171b transgenic broccoli. The expression

351

patterns of several genes involved in chloroplast development and sulfate

352

homeostasis were selected for experimental confirmation. The results indicated that 17



353

the differential expression patterns of each selected gene detected by qRT-PCR were

354

consistent with the data obtained by digital expression profile analysis (Figure S4,

355

Table S6).

356

4. DISCUSSION

357

MiR171 is a conserved miRNA family in plants, which plays crucial roles not only

358

in plant growth and development but also in responses to biotic and abiotic stresses,

359

implying that miR171 performs subfunctionalization in plant evolution.28-32

360

Sequence alignment and phylogenetic analysis indicated that bol-miR171b has a

361

close genetic relationship with the three members of the miR171 family in

362

Arabidopsis. Ath-miR171b and ath-miR171c possess the same mature 3p sequences.

363

The core mature sequences of ath-miR171a differ from those of ath-miR171b and

364

ath-miR171c by one nucleotide.40 The overexpression of ath-miR171a and

365

ath-miR171c can reduce shoot branching and increase the leaf chlorophyll content of

366

the transgenic lines.28-29 However, the different phenotypes of the ath-miR171a and

367

ath-miR171c overexpression transgenic lines were also observed. For example,

368

shoot branching, which was accompanied by increased shoot height, was reduced in

369

the overexpressed ath-miR171c transgenic lines, and the fertility of these transgenic

370

lines were unaffected.29 On the contrary, in the overexpressed ath-miR171a

371

transgenic lines, the height of the shoot and the number of shoot branching decreased.

372

Moreover, the transgenic lines were dramatically abortive.41 The function of

373

ath-miR171b remains unexplored. Nevertheless, the present data confirmed that

374

bol-miR171b and ath-miR171b share the same mature 5p and 3p sequences. The 18


Page 18 of 41

Page 19 of 41


375

core sequences of these two miRNA precursors were also highly similar, implying

376

that bol-miR171b and ath-miR171b have identical functions. The overexpressed

377

bol-miR171b in Arabidopsis indicated that the transgenic lines displayed similar

378

phenotypes to those of the overexpressed ath-miR171a transgenic Arabidopsis,

379

particularly reduced shoot branching, dark green leaves with high chlorophyll

380

content, and severe sterility. Interestingly, the mature sequences of bol-miR171b and

381

ath-miR171b, as well as their precursor sequences, were similar to those of

382

ath-miR171c. However, the functions of bol-miR171b seemed to be nearly identical

383

to those of ath-miR171a and remained to be further elucidated. Bol-miR171b was

384

also overexpressed in broccoli. These results demonstrated that the overexpressed

385

bol-miR171b transgenic broccoli also displayed dark green leaves with high

386

chlorophyll content and sterility, which were observed in the overexpressed

387

ath-miR171c transgenic Arabidopsis. These findings indicated that bol-miR171b

388

mainly functions in leaf development and regulating fertility in broccoli. The

389

overexpression of bol-miR171b as well as other miR171s could increase the

390

chlorophyll content of transgenic plants. Chlorophyll is an important natural pigment

391

that has been used as a food additive and has been reported to exhibit

392

anticarcinogenic effects.42-44

393

MiRNAs are important small RNA molecules that play crucial roles in plant

394

growth and development by regulating their downstream target genes.1-2 To uncover

395

the function of miRNAs, it is vital to identify and elucidate their targeted genes.

396

Previous investigations have indicated that the genes from the GRAS gene family, 19



397

such as SCL6, SCL22, and SCL27 in Arabidopsis, are targeted by miR171.29 In the

398

present study, two homologs of AtSCL6 and AtSCL27 were identified to be cleaved

399

by bol-miR171b in broccoli, as indicated by degradome sequencing. Moreover, the

400

comparative transcriptome data and qRT-PCR results demonstrated that the

401

expression levels of BolSCL6 and BolSCL27 in 35S::bol-miR171b transgenic

402

broccoli were significantly lower than those in the vector controls. However, neither

403

the degradome data nor the expression level assay confirmed that BolSCL22 was

404

targeted by bol-miR171b. These results indicated that BolSCL6 and BolSCL27,

405

rather than BolSCL22 and other SCL genes, were the target genes of bol-miR171b. In

406

Arabidopsis, the ath-miR171c-SCL6/22/27 module plays crucial roles in mediating

407

the GA-DELLA signaling pathway in leaf development and chlorophyll

408

biosynthesis.28 A series of genes involved in chloroplast development, including

409

chlorophyll biosynthesis, displayed significantly differential expression levels

410

between the overexpressed bol-miR171b transgenic broccoli and the vector controls.

411

Moreover, the leaves of the overexpressed bol-miR171b transgenic lines also

412

displayed darker green than those of the vector controls. It suggested that the

413

bol-miR171b-BolSCL6/27 module is also involved in the chloroplast development in

414

broccoli. However, in contrast to those in the overexpressed ath-miR171c transgenic

415

Arabidopsis,28 the vegetative growth and fertility were significantly inhibited by

416

overexpression of bol-miR171b in both Arabidopsis and broccoli. It implied that

417

besides miR171-SCLs module, other regulators may also contribute to the

418

phenotypes of 35S::bol-miR171b transgenic plants. Interestingly, the present results 20


Page 20 of 41

Page 21 of 41


419

indicated that APSR3 was the candidate target of bol-miR171b* (bol-miR171b-5p).

420

The expression pattern assay also indicated that both bol-miR171b-3p and

421

bol-miR171b-5p displayed differential expression levels in diverse organs of

422

broccoli. Moreover, in the seedlings, bol-miR171b-5p exhibited higher expression

423

level than that of bol-miR171b-3p. The comparative transcriptome data further

424

revealed that the expression levels of a few genes involved in sulfate homeostasis,

425

including APSR1, APSR3, and sulfate transporters, significantly down-regulated in

426

the overexpressed bol-miR171b transgenic broccoli, which was consistent with the

427

fact that the expression levels of miRNAs are usually negatively correlated with

428

those of their targeted genes. Sulfate homeostasis is crucial to cysteine synthesis in

429

plants. Cysteine is directly incorporated into protein or utilized for methionine and

430

glutathione synthesis, all of which are essential to plant growth and development.45 A

431

body of literature has pointed that APSRs contribute to the regulation of sulfate

432

reduction and assimilation.46-47 In B. oleracea, sulfate deprivation initiates the rapid

433

expression of APSRs and sulfate transporters, thereby enhancing sulfate uptake and

434

assimilation.48 Consequently, the down-regulated expressions of APSRs and sulfate

435

transporters should decrease the sulfate assimilation and utilization in overexpressed

436

bol-miR171b transgenic broccoli. The deficiency of sulfate affected the biosynthesis

437

of a wide range of primary and secondary S-containing metabolites, which should be

438

an important factor that resulted in abnormal leaf development and sterility in the

439

overexpressed bol-miR171b transgenic broccoli. These results implied that, different

440

from most plant miRNAs in which the star sequence released from the miRNA 21



441

precursor was unstable and rapidly degraded, the star sequence of bol-miR171b may

442

contribute to the growth and development of broccoli by targeting APRS3. The star

443

sequence of ath-miR171a targets SUVH8, and the ath-miR171a*-SUVH8 module

444

played important roles in plant growth and development.49 Ath-miR393* targeted a

445

Golgi-localized SNARE gene MEMB12, and Ath-miR393*-MEMB12 functioned in

446

innate immunity.50 These findings suggested that not only the mature sequences of

447

some plant miRNAs but also their star sequences participated in the regulation of

448

plant growth and development or stress response by targeting different genes.

449

Furthermore, the expression profiles of several genes involved in sterility in other

450

plant species were discovered. The results confirmed that some of these genes, such

451

as MS1 (male sterility 1)51, MYB8852, PSK4 (Phytosulfokine 4)53, WRKY254,

452

WRKY2755 and WRKY3454, displayed differential expression levels between the

453

35S::bol-miR171b transgenic broccoli and the vector controls, implying that these

454

genes may be associated with the sterility of 35S::bol-miR171b transgenic broccoli.

455

However, the expression levels of other genes, such as MS2 (male sterility 2)56,

456

CDM1 (CALLOSE DEFECTIVE MICROSPORE1)57, ARF17 (AUXIN RESPONSE

457

FACTOR17)58 and PPRD2 (polyprenol reductase 2)59, did not significantly change in

458

the 35S::bol-miR171b transgenic broccoli compared with the vector controls (Figure

459

S5). These findings were consistent with the fact that the molecular mechanism

460

underlining the plant sterility is complicated. Any DNA sequence mutation or

461

abnormal transcription, even abnormal protein modification of genes involved in

462

growth and development (especially reproductive development) can result in plant 22


Page 22 of 41

Page 23 of 41


463

sterility. Nevertheless, the molecular mechanism by which bol-miR171b-3p and/or

464

bio-miR171b-5p functions in chloroplast development, sulfate homeostasis and

465

occurrence of sterility in broccoli should be further elucidated.

466

In conclusion, this study identified that bol-miR171b plays an important role in

467

leaf development and fertility in broccoli. The overexpression of bol-miR171b could

468

significantly increase the chlorophyll content of transgenic plants. BolSCL6 and

469

BolSCL27 were negatively regulated by bol-miR171b-3p. APSR3 might be cleaved

470

by bol-miR171b-5p. Genes involved in chloroplast development and sulfate

471

homeostasis should participate in the bol-miR171b-mediated regulatory network.

472

These findings provided new insights into the function and regulation of

473

bol-miR171b for organ development in broccoli, and implied that bol-miR171b is a

474

potential small RNA molecule to breed plants with high chlorophyll by genetic

475

engineering.

476

Conflict of interest The authors declare that they have no conflict of interest.

477

Author contributions

478

CG Wang conceived the research project; H Li performed the gene clone and

479

overexpression analysis in Arabidopsis and broccoli; QL Zhang performed

480

degradome data analysis; LH, Li and JY Yuan conducted transcriptome data analysis;

481

Y Wang conducted to qRT-PCR; ZP Hang and M Wu preformed the phenotype

482

analysis. M Liu, CB Chen and WQ Song performed the tissue culture and genetic

483

transformation of broccoli; H Li, CW Wang wrote the manuscript.

484

Acknowledgements 23



Page 24 of 41

485

We thank Dr. Deling Sun and Dr. Hanmin Jiang of Tianjin Kernel Vegetable

486

Research Institute, Tianjin, China, for kindly providing the homozygous broccoli

487

seeds.

488

Funding

489

This work was funded by grants from the Natural Science Foundation of China (No.

490

31470669

491

No.14JCZDJC34000), and Graduate education quality improvement projects of

492

Tianjin Agricultural University (No.2017YPY004).

493

Supporting Information This material is available free of charge via the Internet at

494

http://pubs.acs.org.

495

Figure S1 Expression levels of the bol-miR171b precursor in 35S::bol-miR171b

496

transgenic Arabidopsis and broccoli. Figure S2 Phenotypes of the overexpressed

497

bol-miR171b in Arabidopsis. Figure S3 GO enrichments of the differentially

498

expressed genes between the 35S::bol-miR171b transgenic broccoli and the vector

499

controls. Figure S4 Expression levels of the genes involved in chloroplast

500

development and sulfate homeostasis. Figure S5 Expression levels of the marker

501

genes involved in plant sterility. Table S1 Primers used in the present study. Table

502

S2 The representative phenotypic data and chlorophyll content in the

503

35S::bol-miR171b transgenic Arabidopsis lines. Table S3 The differentially

504

expressed genes between the 35S::bol-miR171b transgenic broccoli and vector

505

controls. Table S4 GO functional annotation of the down-regulated genes detected in

506

the 35S::bol-miR171b transgenic broccoli. Table S5 GO functional annotation of the

and

No.

31401889)

and

Tianjin

(No.

24


15JCQNJC15100

and

Page 25 of 41


507

up-regulated genes detected in the 35S::bol-miR171b transgenic broccoli. Table S6

508

The relative expression levels of genes involved in chloroplast development and

509

sulfate homeostasis.

510

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(52) Makkena, S.; Lee, E.; Sack, F. D.; Lamb, R. S. The R2R3 MYB transcription

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factors FOUR LIPS and MYB88 regulate female reproductive development. J

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Exp Bot. 2012, 63, 5545-5558.

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(53) Yu, L.; Liu, Y.; Liu, Y.; Li, Q.; Tang, G.; Luo, L. Overexpression of

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phytosulfokine-α induces male sterility and cell growth by regulating cell wall

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development in Arabidopsis. Plant Cell Rep. 2016, 35, 2503-2512.

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(54) Lei, R.; Li, X.; Ma, Z.; Lv, Y.; Hu, Y.; Yu, D. Arabidopsis WRKY2 and WRKY34

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transcription factors interact with VQ20 protein to modulate pollen 32


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development and function. Plant J. 2017, 91, 962-976. (55) Mukhtar, M. S.; Liu, X.; Somssich, I. E. Elucidating the role of WRKY27 in male sterility in Arabidopsis. Plant Signal Behav. 2017, 12, e1363945.

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(56) Aarts, M. G.; Hodge, R.; Kalantidis, K.; Florack, D.; Wilson, Z. A.; Mulligan, B.

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J.; Stiekema, W. J.; Scott, R.; Pereira, A. The Arabidopsis MALE STERILITY 2

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protein shares similarity with reductases in elongation/condensation complexes.

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Plant J. 1997, 12, 615-623.

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(57) Lu, P.; Chai, M.; Yang, J.; Ning, G.; Wang, G.; Ma, H. The Arabidopsis

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CALLOSE DEFECTIVE MICROSPORE1 gene is required for male fertility

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through regulating callose metabolism during microsporogenesis. Plant Physiol.

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2014, 164, 1893-1904.

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(58) Yang, J.; Tian, L.; Sun, M. X.; Huang, X. Y.; Zhu, J.; Guan, Y. F.; Jia, Q. S.;

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Yang, Z. N. AUXIN RESPONSE FACTOR17 is essential for pollen wall pattern

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formation in Arabidopsis. Plant Physiol. 2013, 162, 720-731.

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(59) Jozwiak, A.; Gutkowska, M.; Gawarecka, K.; Surmacz, L.; Buczkowska, A.;

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Lichocka, M.; Nowakowska, J.; Swiezewska, E. POLYPRENOL REDUCTASE2

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Deficiency is lethal in Arabidopsis due to male sterility. Plant Cell 2015, 27,

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3336-3353.

701 702 703 704 33



705

Figure captions

706

Figure 1 Secondary structure, phylogenetic tree, and expression levels of

707

bol-miR171b. (a) The secondary structure of bol-miR171b in broccoli. The yellow

708

and green sequences indicate the mature 5p and 3p sequences of bol-miR171b,

709

respectively. The sequences that match the bol-miR171b-5p and bol-miR171b-3p

710

detected by small RNA-Seq are displayed, and the count of sequences is showed in

711

the following brackets. (b) The phylogenetic tree of bol-miR171b and other

712

miR171b precursors, which was constructed by neighbor-joining method with

713

bootstrap replicates of 1, 000, from diverse plant species. The bar denotes the genetic

714

distance. (c) and (d) The differential expression levels of bol-miR171b-5p and

715

bol-miR171b-3p in the different organs of broccoli by qRT-PCR.

716

Figure 2 Quantification analysis of the leaves, siliques, chlorophyll content, and

717

shoot branching of the overexpressed bol-miR171b transgenic Arabidopsis and

718

broccoli. (a), (b) and (c) The number of rosette leaves, the length of siliques, and the

719

chlorophyll content in the three independent 35S::bol-miR171b transgenic

720

Arabidopsis lines (line 2, line 5 and line 6) and the vector controls (CK). (Student’s

721

t-test, ** P < 0.01); n ≥ 20; error bars indicated ± SD. (d) and (e) The rosette and

722

cauline branches in the 35S::bol-miR171b transgenic Arabidopsis, respectively. The

723

y-axis shows the percentage of plants with zero (blue), one (dark red), two to three

724

(green), and more than four (purple) rosette (d) and cauline branches (e) in CK and

725

the three independent 35S::bol-miR171b transgenic Arabidopsis lines (line 2, line 5

726

and line 6). (f) The chlorophyll content in the three independent 35S::bol-miR171b 34


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Page 35 of 41


727

transgenic broccoli lines (line 7 (bol), line 12 (bol) and line 26 (bol)) and the vector

728

controls (CK (bol)). (Student’s t-test, ** P < 0.01); n ≥ 15; error bars indicated ± SD.

729

Figure 3 Phenotypes of overexpressed bol-miR171b transgenic broccoli. (a) and (b)

730

The representative 35S::bol-miR171b transgenic broccoli (line 7, line 12 and line 26)

731

and the vector control. (c) The leaves of the 35S::bol-miR171b transgenic broccoli

732

(line 7) and the vector control. (d) and (e) The flower buds of the vector control (d)

733

and the 35S::bol-miR171b transgenic broccoli (e). (f) The stamens and pistils of the

734

35S::bol-miR171b transgenic broccoli (down) and the vector control (up). (g) The

735

siliques of the 35S::bol-miR171b transgenic broccoli (up) and the vector control

736

(down). CK showed the vector control.

737

Figure 4 Identification and quantitative expression assay of targeted genes of

738

bol-miR171b-3p in broccoli. (a) Identification of the targeted genes of

739

bol-miR171b-3p by degradome sequencing. The y-axis showed the count of

740

normalized reads that matched the different positions of the targeted genes. The

741

x-axis showed the sequence positions of the targeted genes. Only the cleavage sites

742

with P-value < 0.05 were counted and showed. The red lines indicated the cleavage

743

sites of bol-miR171b-3p. (b) The relative expression levels of the targeted genes

744

BolSCL6 and BolSCL27 as well as their several homologous genes by qRT-PCR

745

assay.

746

Figure 5 Predicted target of bol-miR171b-5p and its expression patterns in different

747

organs of broccoli and 35S::bol-miR171b transgenic broccoli. (a) The base paring

748

pattern of bol-miR171b-5p and its predicated target, APSR3. (b) The expression 35



749

trends of bol-miR171b-5p and APSR3 in different organs of broccoli. (c) The relative

750

expression levels of bol-miR171b-5p and APSR3 in the 35S::bol-miR171b

751

transgenic broccoli and the vector controls. 35S::bol-miR171b indicated the

752

overexpressed bol-miR171b transgenic broccoli. CK indicated the vector controls.

36


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Figure 1

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Figure 2

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Figure 3

Figure 4

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Figure 5

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TOC Graphic

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Ectopic overexpression of bol-miR171b increases chlorophyll content

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