microRNA268 overexpression affects rice seedling growth under

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microRNA268 overexpression affects rice seedling growth under cadmium stress Yanfei Ding, Yi Wang, Zhihua Jiang, Feijuan Wang, Qiong Jiang, Junwei Sun, Zhixiang Chen, and Cheng Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01164 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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Journal of Agricultural and Food Chemistry

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microRNA268 overexpression affects rice seedling growth

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under cadmium stress

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Yanfei Ding†#, Yi Wang†#, Zhihua Jiang†, Feijuan Wang†, Qiong Jiang†, Junwei Sun†,

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Zhixiang Chen†‡, and Cheng Zhu†*

5

†

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Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou

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310018, People’s Republic of China

8

‡

9

47907-2054 USA

Key Laboratory of Marine, Food Quality and Hazard Controlling Technology of

Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN

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Correspondence: Cheng Zhu

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Tel: 86-571-86836090

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e-mail: [email protected]

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# Yanfei Ding and Yi Wang share the first authorship

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ACS Paragon Plus Environment


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Abstract: MicroRNAs (miRNAs) are 21–24 nucleotide RNAs that function as

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ubiquitous post-transcriptional regulators of gene expression in plants and animals.

19

Increasing evidence points to the important role of miRNAs in plant responses to

20

abiotic and biotic stresses. Cadmium (Cd) is a non-essential heavy metal highly toxic

21

to plants. Although many genes encoding metal transporters have been characterized,

22

the mechanisms for the regulation of the expression of the heavy metal transporter

23

genes are largely unknown. In this study, we found that the expression of rice miR268

24

was significantly induced under Cd stress. By contrast, expression of NRAMP3

25

(natural resistance-associated macrophage protein 3), a target gene of miR268, was

26

dramatically decreased by Cd treatment. Overexpression of miR268 inhibited rice

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seedling growth under Cd stress. The transgenic miR268-overexpressing plant leaves

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contained increased levels of hydrogen peroxide and malondialdehyde and their

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seedlings accumulated increased levels of Cd when compared to those in wild-type

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plants. These results indicate that miR268 acts as a negative regulator of rice tolerance

31

to Cd stress. Thus, miRNA-guided regulation of gene expression plays an important

32

role in plant responses to heavy metal stress.

33 34

Key words: cadmium, miR268, NRAMP, rice, transgenic plants

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Abbreviations: Al, aluminum; ARE, anaerobic responsive element; Cd, cadmium;

36

CSD, Cu/Zn superoxide dismutase; DCL1, Dicer-like1; Hg, mercury; H2O2, hydrogen

37

peroxide; hpt, hygromycin phosphotransferase; HSE, heat shock responsive element;

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MBS, MYB binding site; MDA, malondialdehyde; miRNA, microRNA; Mn,

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manganese; NRAMP, natural resistance-associated macrophage protein; Pb, lead;

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qPCR, quantitative real-time PCR; WT, wild-type; Zn, zinc.

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Introduction

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Toxic heavy metals such as cadmium (Cd), lead (Pb) and mercury (Hg) are

43

major contaminants due to their significant release into the environment from

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anthropogenic activities.1 Due to their unbiodegradable nature, heavy metals can

45

accumulate in plants and ultimately affect human health.2-3 Plants have evolved

46

various mechanisms that regulate the uptake and accumulation of heavy metals.4-6

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One such mechanism involves the synthesis of transport proteins such as channels,

48

carriers or pumps that transport heavy metals across cellular membrane systems.

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Genes encoding metal transporters including heavy metal ATPases and natural

50

resistance-associated macrophage proteins (NRAMPs) have been isolated and

51

characterized.7-9 However, the regulatory mechanisms for the expression of plant

52

heavy metal transporter genes are less clear.

53

MicroRNAs

(miRNAs)

are

small

non-coding

RNAs

that

act

as

54

post-transcriptional regulators of target genes in eukaryotic organisms. miRNAs are

55

generated from larger precursors with self-complementary stem-loop structures

56

through the processing by a ribonuclease III nuclease termed Dicer in animals or

57

Dicer-like1 (DCL1) in plants.10-11 There are at least 35828 mature miRNAs in the

58

major miRNA database (miRBase Registry, Release, 21.0).12 Several computational

59

programs are available for predicting miRNA genes in Arabidopsis, rice and other

60

plant species.13-14 In rice, for example, 2100 novel miRNA candidates were identified

3



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computationally from known rice mRNAs, a majority of which were not

62

evolutionarily conserved.15 In plants, a large number of studies have reported that

63

miRNAs play important regulatory roles in growth, development and plant responses

64

to various abiotic stresses including heavy metals.16-19 In Cd hyperaccumulator Sedum

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alfredii, deep sequencing identified 356 miRNAs, of which 79 miRNAs were

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differentially expressed under Cd stress.20 In rice seedling roots, almost 70

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As-responsive miRNAs have also been identified.21 In radish (Raphanus sativus L.),

68

Solexa sequencing identified 54 known and 16 novel miRNAs to be differentially

69

expressed under Cr stress.22 Some of the target genes for metal-responsive miRNAs

70

encode transcription factors and metal transporters.16, 23-24 For example, a target gene

71

of miR192 encodes an ATP-binding cassette transporter involved in transport of

72

heavy metals in plants.23 These results indicate that miRNAs are important regulators

73

in plant responses to heavy metals.

74

In our previous study, we identified 12 Cd-responsive miRNAs based on

75

microarray assays.16 Here, we reported analysis of miR268, which was strongly

76

up-regulated under Cd stress. Furthermore, miR268 was predicted to target NRAMP3

77

(natural resistance-associated macrophage protein 3), which encoded a metal

78

transporter involved in uptake and detoxification of heavy metals in plants.25-26 To

79

further analyze miR268 in Cd stress response in rice, we employed a transgenic

80

approach through overexpression of the miRNA in transgenic rice plants.

81

Overexpression of miR268 significantly led to increased inhibition of seedling growth

82

under Cd stress, when compared to that of wild-type (WT) plants. These results

4


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demonstrated that miR268 acted as a negative regulator of seedling growth under Cd

84

stress.

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Materials and Methods

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Growth Conditions

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Zhonghua 11 (Oryza sativa L. subsp. japonica), a conventional variety rice, is widely

89

used in studying the physiological and molecular mechanisms of heavy metal

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response. Rice seeds were sterilized with 10% NaClO (v/v) for 20 min, rinsed three

91

times with sterilized H2O2 and soaked at 37°C. The germinated seeds were then

92

transferred to pots containing water in a growth chamber with a photoperiod of 13-h

93

light (29°C)/11-h dark (22°C) and with 80% relative humidity. For gene expression

94

analysis, roots of one-week-old seedlings were immersed in 60 µM cadmium chloride

95

(CdCl2) for 0 to 24 h. After treatment, the roots were harvested for total RNA

96

isolation.

97

RNA Isolation and Quantitative Real-time PCR (qRT-PCR)

98

Total RNA was separately isolated from rice roots with TRIzol reagent (Invitrogen).

99

First-strand cDNA was synthesized in 10 µL using 1 µg RNA by PrimeScript reverse

100

transcriptase (TaKaRa). The reaction products were diluted by 5-fold and used as the

101

template for qRT-PCR analysis. qRT-PCRs were performed on a Rotor-Gene Q

102

machine (Qiagen) (parameters: 95°C 1min, followed by 45 cycles of 95°C 10s, 58°C

103

15s and 72°C 15s). All PCR reactions were done in triplicate. Gene expression was

104

quantified using the comparative CT method. Rice β-Actin was used as an internal

105

control. Gene-specific primers used in qRT-PCR were as follows: β-Actin forward,

106

5′-GCCGTCCTCTCTCTGTATGC-3′

and

5


reverse,


107

5′-GGGGACAGTGTGGCTGAC-3′;

108

GATTAGCACCGACTCTA-3′

109

NRAMP3 (LOC_Os03g11010) forward, 5′- CTTCATCTTTTTATTCCTG-3′ and

110

reverse, 5′- TTGTTTGTGTCAATCTTCC-3′.

111

Prediction of miR268 Target Genes and Analysis of their cis-acting Elements

112

The target mRNAs of plant miRNAs are recognized through perfect or near-perfect

113

base pairing with their corresponding mRNAs and, therefore, can be identified by

114

computational sequence similarity algorithms. All potential rice sequences

115

complementary to the mature miR268 sequence with limited mismatches were

116

identified using a web-based computing system, psRNATarget27 with a preferred

117

lower false-positive prediction rate of 3. The number of mismatches was limited to no

118

more than 3 nt, indels no more than 1 nt and G-U pairs no more than five. For

119

cis-acting element analysis, 1500-bp DNA sequence upstream of the transcription

120

initiation site of the pre-miR268 gene were obtained from the TIGR Rice Genome

121

Annotation site (http://www.tigr.org/tdb/e2k1/osa1/data_download.shtml). Putative

122

cis-acting elements in the miR268 promoter were then identified using PlantCARE

123

(http://bioinformatics.psb.ugent.be/webtools/plantcare/).28

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Generation of Transgenic Rice Plants

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The miR268 precursor sequence was amplified from genomic DNA using

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gene-specific primer pairs (5′-CGGGGTACCTAACAGGAGAGCTGGACC-3′ and

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5′- AACTGCAGCATCCGAGTGACAATCAG-3′). The PCR products were cloned

128

into the KpnI and PstI sites of p1301-35S-NOS between the 35S promoter and NOS

and

miR268 reverse,

forward,

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5′-

5′-GAAGCCATCTGACATAG-3′;

6


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terminator. The construct was introduced into Agrobacterium tumefaciens strain

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EHA105 for transformation into japonica rice Zhonghua 11. Transgenic plants were

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re-planted in a greenhouse to the T2 generation. All transgenic plants were screened

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for hygromycin resistance and confirmed by PCR analysis with leaf genomic DNA as

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templates. The PCR was performed with primers hpt-F and hpt-R in a 20-µL reaction

134

system

135

5′-GGAGCATATACGCCCGGAGT-3′). Expression levels of miR268 and NRAMP in

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transgenic rice plants were examined using qRT-PCR. Histological assays of GUS

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activities, which were also used to confirm the transgenic plants, were performed at

138

37°C using a reaction mixture containing 50 mM phosphate buffer (pH: 7.0)

139

containing 1 mM 5-bromo-4-chloro-3-indolyl glucuronide, 5% methanol, 10 µg/ml

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cycloheximide and 1 mM dithiothreitol. The GUS reaction was terminated by the

141

addition of ethanol.

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Seedling Growth Assay Under Cd Stress

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Seeds of WT and transgenic miR268-overexpressing plants were sterilized with 10%

144

sodium hypochlorite and soaked for 3 d at 37°C in darkness. Scoring of WT and

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transgenic plant seed germination was based on the radicle protruded through the seed

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coat. The germinated seeds were then sown on agar medium (left: WT seeds, right:

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transgenic plant seeds) and placed under light/dark of 13/11 h and 29/22°C. For

148

determination of the effect of Cd stress on seedling growth, the agar medium was

149

supplemented with 0, 60 and 100 µM CdCl2, respectively. EDTA or other chelator

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was not added in the agar medium. After 4 d in the Cd medium, seedling size, shoot

(hpt-F:

5′-GTTTATCGGCACTTTGCATCG-3′;

7


hpt-R:


151

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length and fresh weight were determined.

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For physiological and biochemical analysis, WT and transgenic rice seedlings

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were grown with Yoshida’s culture solution for two weeks and then treated in a

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modified Yoshida’s solution with 60 µM CdCl2. The free Cd2+ in this solution was

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approximately

156

http://www.plantmineralnutrition.net/geochem.php).29 After exposure to 60 µM CdCl2

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for 14 d, Chlorophyll, MDA, H2O2 and Cd content were determined in triplicate as

158

described below.

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Chlorophyll Content Measurement

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The chlorophyll content was determined as described by Knudson et al.30 Fresh rice

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leaf (0.1 g) was extracted in 2 mL 95% ethanol for 36 h in the dark and the extract

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was analyzed spectrophotometrically.

163

MDA Content Measurement

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The MDA content was determined as described by Heath and Packer31 with slight

165

modifications. Rice shoots (0.1 g) were ground in 1 mL of 10% (w/v) trichloroacetic

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acid. After centrifugation at 12,000×g for 10 min at 4°C, the supernatants were

167

collected and mixed with an equal volume of 0.6% TBA solution. The mixtures were

168

incubated at 95°C for 30 min and then cooled quickly on ice. The resulting mixtures

169

were centrifuged at 10,000×g for 10 min and the absorbance values of the

170

supernatants at 450, 532 and 600 nm were measured.

171

H2O2 Content Measurement

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H2O2 levels were determined as described by Jana and Choudhuri32. Leaf segments

0.37

µM

calculated

using

Geochem-EZ

8


(Shaff

et

al.;

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(0.1 g) were ground into powder in liquid nitrogen and homogenized in 3 mL

174

pre-cooled 50 mM sodium phosphate (pH 6.5). The homogenate was centrifuged at

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6000×g for 25 min. The supernatant was collected and mixed with one third of

176

volume of 0.1% titanium sulphate in 20% (v/v) H2SO4. The mixture was immediately

177

centrifuged at 6000×g for 15 min and the absorbance of the supernatant at 410 nm

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was determined. The H2O2 levels were calculated using the extinction coefficient of

179

0.28 µM−1 cm−1.

180

Cd Concentration Measurement

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Two-week-old WT and transgenic rice seedlings were treated with 60 µM CdCl2 for

182

14 d. For determination of Cd content, roots and shoots were harvested and dried at

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105°C for 2 h and then at 70°C until the weight became stable. Dried samples

184

(100–200 mg of roots and 200–300 mg of shoots) were digested with HNO3 at 120°C

185

with a Multiwave (MARSX 240/50, CEM, Matthews, NC, USA). After digestion, the

186

samples were diluted to 25 mL and the Cd content was quantified using an atomic

187

absorption spectrometer (AA-7000, Shimadzu, Tokyo, Japan).

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Statistical Analysis

189

Experimental data of morphological and physiological characteristics of the treated

190

plants were analyzed using SPSS software. Data were presented as the mean±

191

standard deviation (SD) calculated from three replicats. Statistic analysis of the

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significant differences among WT and transgenic lines were performed using

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Student’s t-test.

194

Results

9



195

Cd Stress Up-regulates miR268 Expression

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In our previous study, we exposed one-week-old rice seedlings to Cd-free (control)

197

and 60 µM CdCl2 for 6 h. We then isolated small RNAs from the two samples and

198

analyzed their levels using miRNA microarrays.16 In this study, we focused on a new

199

miRNA, miR268, which was also highly increased under Cd stress based on

200

microarray analysis (Table 1). To confirm the microarray results, we analyzed

201

miR268 expression in one-week-old rice seedlings using qRT-PCR. The results

202

showed that miR268 expression was strongly induced after 6 h exposure to 60 µM

203

CdCl2. These results confirmed the inducible nature of the miRNA from the

204

microarray analysis (Figure 1A).

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Prediction of miR268 Targets and Analysis of their Expression

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miRNAs are involved in diverse biological processes by regulating expression of their

207

target genes. Thus, identification of miRNA target genes is important for

208

understanding of their biological functions. In this study, we used a miRNA target

209

prediction online tool, psRNATarget, to identify putative target genes of miR268.27

210

Using the online tool, we predicted that miR268 targeted the metal transporter gene

211

NRAMP3 (Os03g11010), with a 3.0 score. Sequence alignments of miR268 and

212

NRAMP3 are shown in Figure 1C.

213 214

To investigate whether miR268 indeed targeted the predicted target gene, we

215

determined the expression levels of miR268 and NRAMP3 using qRT-PCR in rice

216

seedlings after treatment with 60 µM Cd for 0–24 h (Figure 1B). The miR268

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expression was elevated after 3 h, and continued to increase over 24 h of the treatment

218

with 60 µM Cd. In contrast, expression of its target gene, NRAMP3, was sharply

219

reduced after exposure to Cd for 24 h (Figure 1B). The opposite changes in the

220

expression patterns of miR268 and NRAMP3 supported the negative regulation of

221

NRAMP3 by miR268.

222

Overexpression of miR268 reduced NRAMP3 expression in Rice

223

To further determine the regulation of NRAMP3 by miR268, we overexpressed the

224

miRNA in rice plants. The T-DNA region of the binary vector for rice transformation

225

contains a hygromycin phosphotransferase (hpt) gene as a selection marker and a

226

GUS reporter gene (Fig. S1). The T0 transgenic plants were confirmed by both PCR

227

genotyping of the hpt gene and GUS activity assays (Figure 2A). qRT-PCR analysis

228

further detected the overexpression of miR268 in transgenic plants (Figure 2B). Two

229

transgenic lines (#1 and #4), overexpressing miR268 at relatively high levels, were

230

chosen for further analysis. In the miR268-overexpressing plants, qPCR showed that

231

NRAMP3 transcript levels declined drastically when compared with those in WT

232

controls. The large decrease of NRAMP3 mRNA levels in transgenic plants further

233

supported that NRAMP3 mRNAs were targeted for cleavage by miR268.

234

miR268 Has a Negative Effect on Rice Seedling Growth Under Cd stress

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In this study, we examined the seedling growth of miR268-overexpressing rice in the

236

agar medium containing different concentrations of CdCl2. Under the normal

237

conditions without Cd in the medium, there was no noticeable difference in seedling

238

growth between WT and transgenic plants. However, at 60 and 100 µM CdCl2 in the

11



239

growth medium, the growth of transgenic lines was more inhibited than that of WT

240

(Figure 3A). The increased growth inhibition of the transgenic rice plants under Cd

241

stress was also apparent based on a greater reduction of seedling weight and shoot

242

length in the 35S:MIR268 plants than in WT plants when grown with Cd (Figure

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3B,C). Thus, overexpression of MIR268 increased sensitive to Cd.

244 245

We also investigated the physiological basis for the increased Cd sensitivity of the

246

transgenic seedlings that were cultured with Yoshida’s culture solution with or without

247

Cd for two weeks. After exposure to 60 µM CdCl2 for 14 d, chlorophyll, hydrogen

248

peroxide (H2O2), malondialdehyde (MDA), and Cd content were measured. Under the

249

normal condition without Cd in the culture solution, we observed no significant

250

differences in chlorophyll contents between WT and overexpression plants. Under Cd

251

stress however, 35S:MIR268 lines had less chlorophyll than WT controls (Figure 4A).

252

Exposure to Cd also caused a significant increase of MDA level in rice seedlings

253

(Figure 4B). However, this Cd-induced increased in MDA was 60% more in

254

35S:MIR268 lines than in WT plants (Figure 4B). These results indicated that

255

overexpression of miR268 appeared to increase Cd toxicity based on increased MDA

256

levels in leaves.

257 258

To investigate the effect of miR268 overexpression on oxidative stress, we

259

analyzed the H2O2 content in leaves of WT and transgenic plants. As shown in Figure

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4C, there was no significant difference in H2O2 content between WT and transgenic

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rice plants under the normal condition without Cd. However, when treated with 60

262

µM CdCl2 for 14 d, the H2O2 level increased by 1.53-fold in leaves of WT rice, but by

263

3.3- and 2.7-fold in the two transgenic lines (Figure 4C). These results suggested that

264

35S:MIR268 lines either generated more ROS or scavenged less ROS than WT plants

265

under Cd stress.

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35S:MIR268 Plants Accumulated More Cd than WT Plants

267

To assess whether the increase in Cd sensitivity was due to elevated metal

268

accumulation, we analyzed the Cd contents in roots and shoots of 35S:MIR268 and

269

WT plants. Indeed, the Cd levels were higher in shoots and roots of transgenic lines

270

than in WT plants (Figure 5). Thus, miR268 overexpression increased Cd uptake and

271

accumulation in rice, leading to increased sensitivity to Cd.

272

Discussion

273

Several recent studies have reported that miRNAs mediate transcriptional and

274

post-transcriptional regulation of gene expression in plant response to heavy metal

275

stress.24, 33-34 Using deep-sequencing analysis of Cd-treated or non-treated Brassica

276

napus roots, Zhou et al.23 identified 84 conserved and non-conserved miRNAs from

277

37 miRNA families, including 19 miRNA members not previously identified. Using

278

microarrays, Ding et al.35 identified 19 Cd-responsive miRNAs from rice seedlings,

279

and predicted that the targets of the Cd-responsive miRNAs included genes encoding

280

transcription

281

metal-responsive miRNAs and their target genes have been identified, their biological

282

functions are still not very clear. Overexpression of miRNAs and their targets or

factors

and

stress-related

proteins.

Although

13


a

number

of


283

knockout mutants of target genes have been widely used for functional analysis of

284

stress-responsive miRNA.17,36 Zhang et al.37 generated transgenic B. napus plants

285

overexpressing miR395d, which accumulated more sulfur-containing compounds and

286

were less sensitive to Cd toxicity than WT. In addition, transgenic rice plants

287

overexpressing miR390 displayed reduced Cd tolerance and higher Cd accumulation

288

than WT plants, supporting an important role of miR390 in rice response to Cd stress.

289

38

290 291

In this study, we functionally analyzed miR268, a rice-specific miRNA, in rice

292

responses to Cd stress. Overexpression of miR268 increased Cd uptake and

293

accumulation in rice, which were associated with increased oxidative stress and Cd

294

sensitivity. The contents of H2O2, a toxic agent in plants, were also substantially

295

higher in the transgenic rice plants than in WT after treatment of CdCl2 (Figure 4 C).

296

The increased levels of H2O2 could result from increased production and/or reduced

297

scavenging in the 35S:MIR268 lines under Cd stress. Potential oxidative damage to

298

membranes under increased oxidative stress was consistent with elevated MDA

299

content, an indicator of lipid peroxidation.39 After exposure to Cd stress, WT rice

300

seedlings displayed increased MDA levels, most likely due to the Cd-induced

301

oxidative stress. In the transgenic plants, the MDA levels in leave tissues were

302

substantially higher than those in WT (Figure 4 B). Therefore, the extent of damage to

303

cell membrane in the transgenic plants was significantly more severe than that in WT

304

plants.

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305 306

Identification of the specific upstream regulatory pathways and downstream

307

targets of miRNAs will shed light on the role of miRNAs in plant response to heavy

308

metals. Upstream cis-acting regulatory elements of miR268 were analyzed using

309

plantCARE. Stress-related elements, such as ARE (anaerobic responsive element),

310

TGA-rich repeats (auxin responsive element) and MBS (MYB binding site, involved

311

in drought-induced expression), were found in the miR268 promoter region. Thus,

312

inducible expression of miR268 was possibly mediated by transcription factors

313

involved in plant stress responses (Table 2). In addition, we investigated that a

314

downstream target gene of miR268, which provided further insights into its role in

315

plant adaptation to the heavy metal. We predicted that miR268 targeted NRAMP3 in

316

rice, which encode a metal transporter belongs to a family of proteins conserved in

317

bacteria, plants and animals. Previously several other genes encoding metal

318

transporters have also been characterized and identified as target genes of miRNAs.16,

319

23

320

ATP-binding cassette transporter and NRAMP, respectively and these metal

321

transporters also played key roles in metal translocation in plants.23 Our study miR268

322

and its targeted NRAMP3 further indicates that metal transporter genes are important

323

target genes of miRNAs in the regulation of plant seedling growth and tolerance

324

under Cd stress.

For example, in B. napus, miR159 and miR167 target the genes encoding a

325 326

NRAMP proteins function as proton-coupled metal ion transporters that can

15



327

transport divalent manganese (Mn), zinc (Zn), aluminum (Al) and Cd.25-26 The rice

328

NRAMP gene family contains seven members, some of which have been functionally

329

characterized at the molecular level. It was reported that NRAMP1 was involved in

330

cellular Cd uptake and transport within the plant.40 NRAMP4 was the first identified

331

transporter of the trivalent Al ion. NRAMP5 is a Mn, Fe and Cd transporter in rice.

332

The suppression of NRAMP5 promoted Cd translocation to rice shoots, making it a

333

potential target of manipulation for Cd phytoremediation.41 NRAMP3 is a vascular

334

bundle-localized Mn-influx transporter with a role in Mn distribution in rice.42-43

335

Although NRAMP3 has been implicated in the regulation of Mn distribution, its role

336

in Cd transport in rice has not been reported. In this study, we predicted NRAMP3 to

337

be a target gene of miR268. After Cd treatment, miR268 expression level was

338

up-regulated, whereas the NRAMP3 transcript level was decreased (Figure 1). The

339

up-regulation of miR268 would lead to the post-transcriptional suppression of

340

NRAMP3 mRNA. A transgenic approach was employed to determine the role of

341

miR268 and NRAMP3 in plant response to Cd stress. miR268-overexpressing

342

transgenic plants accumulated less NRAMP3 mRNA and were consequently more

343

sensitive to Cd stress than WT plants. The decrease of NRAMP3 abundance could

344

contribute to Cd-toxicity, either by inhibiting the vacuolar sequestration of Cd, or by

345

inhibiting Cd efflux.2, 44-45 In conclusion, this study demonstrated miR268-mediated

346

regulation of NRAMP3 and its effect on seedling growth under Cd stress. These

347

results provided new insights into the roles of miRNAs and their target genes in heavy

348

metal response in plants.

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349

Supporting Information Available

350

A Supporting Information is available.

351

Author contribution statement

352

Yanfei Ding and Cheng Zhu designed the experiments and wrote the manuscript. Yi

353

Wang and Zhihua Jiang cultured plant materials, performed the experiments and data

354

analysis. Qiong Jiang, Feijuan Wang, and Junwei Sun revised the manuscript.

355

Zhixiang Chen performed data analysis and improved English of the paper.

356

Acknowledgments

357

This study was supported through funding from Zhejiang Provincial Natural Science

358

Foundation of China (LR17C130001, LZ14C020001, Y15C020003), and the National

359

Natural Science Foundation of China (31401299, 31470368, 31401356).

360

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Table 1 miR268 expression analysis based on microarray assay Chip1

Chip2 log2

CK

Cd

(Sample

Sample

Sample

Cd

Signal

Signal

Sample

log2 CK

Cd

(Sample

Sample

Sample

Cd/

Signal

Signal

Sample

Probe_I Sequence (5' to 3')

/

D

CK)

CK)

CCAGUCAGGGGCUC miR268

165.81

3,492.55

4.40

181.86

2,376.95

GUUGCUGG

468 469

Table 2 Stress-related cis-elements analysis of miR268

miR268

Site name

Loc (-bp)

ARE

1144

HSE

-479

MBS

10

TGA

-701

TGACG-motif

-296

ARE, anaerobic responsive element; HSE, heat shock responsive element; TGACG-motif, MeJA-responsive element; MBS, MYB binding site, involved in drought-inducibility; TGA-element, auxin responsive element.

470 471

20


3.71

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472

Legends to figures

473

Fig. 1 Expression analysis of miR268 in rice roots in response to Cd stress. (A) Expression analysis of

474

miR268 under Cd stress for 6 h by qPCR. CK, untreated control; 6 Cd, 6 h Cd treatment; (B) Temporal

475

expression analysis of miR268 and NRAMP3 transcripts under Cd stress by qPCR. Cd treatment time

476

was 0, 3, 6, 9, 12 and 24 h, respectively. OsActin1 gene was used as the internal standard. Error bars

477

indicate standard deviations of three replicates. (C) Sequence alignments of miR268 and its target gene

478

NRAMP3.

479

Fig. 2 Overexpression of miR268 in transgenic rice plants. (A) GUS activity analysis of transgenic rice.

480

(B) Expression levels of miR268 and NRAMP3 in WT and transgenic lines by qPCR. cDNAs were

481

normalized using OsActin1 gene. Error bars indicate standard deviations of three replicates.

482

Fig. 3 Growth of WT and 35S:MIR268 transgenic seedlings on control media or media containing 0, 60,

483

100 µM CdCl2. Pictures were taken after 7 d of growth. (A) Phenotypic comparison of WT and

484

35S:MIR268 transgenic rice plants under Cd stress. (B,C) Shoot length and seedling weight of WT and

485

35S:MIR268 transgenic plants under Cd stress for 4 d. Each column represents the mean ± SD of three

486

independent experiments each with five replicates. The significant level of the difference between WT

487

and transgenic plants is indicated by * for P≦0.05 and ** for P≦0.01.

488

Fig. 4

489

grown under normal conditions (CK) or after 60 µM CdCl2 for 14 d. (A) Chlorophyll content. (B)

490

MDA content (C) H2O2 content. Each column represents an average of three replicates, and bars

491

indicate SDs. * and ** indicate significant differences from between WT and transgenic plants at P≦

492

0.05 and P≦0.01, respectively.

493

Fig. 5

Chlorophyll, MDA, and H2O2 contents in WT and 35S:MIR268 transgenic rice seedlings

Cd contents in shoots and roots of WT and 35S:MIR268 transgenic plants exposed to 60 µM

21



494

CdCl2 for 14 d. (A) Shoot tissue. (B) Root tissue. Data are presented as the mean ± SD. The significant

495

level of the difference between WT and transgenic plants is indicated by * for P≦0.05 and ** for P≦

496

0.01.

497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515

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516

TOC

517

23



Fig. 1 Expression analysis of miR268 in rice roots in response to Cd stress. (A) Expression analysis of miR268 under Cd stress for 6 h by qPCR. CK, untreated control; 6 Cd, 6 h Cd treatment; (B) Sequence alignments of miR268 and its target gene NRAMP3; (C) Temporal expression analysis of miR268 and NRAMP3 transcripts under Cd stress by qPCR. Cd treatment time was 0, 3, 6, 9, 12 and 24 h, respectively. OsActin1 gene was used as the internal standard. Error bars indicate standard deviations of three replicates. 254x190mm (96 x 96 DPI)


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Fig. 2 Overexpression of miR268 in transgenic rice plants. (A) GUS activity analysis of transgenic rice. (B) Expression levels of miR268 and NRAMP3 in WT and transgenic lines by qPCR. cDNAs were normalized using OsActin1 gene. Error bars indicate standard deviations of three replicates. 254x190mm (96 x 96 DPI)



Fig. 3 Growth of WT and 35S:MIR268 transgenic seedlings on control media or media containing 0, 60, 100 µM CdCl2. Pictures were taken after 7 d of growth. (A) Phenotypic comparison of WT and 35S:MIR268 transgenic rice plants under Cd stress. (B,C) Shoot length and seedling weight of WT and 35S:MIR268 transgenic plants under Cd stress for 4 d. Each column represents the mean ± SD of three independent experiments each with five replicates. The significant level of the difference between WT and transgenic plants is indicated by * for P≦0.05 and ** for P≦0.01. 254x190mm (96 x 96 DPI)


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Fig. 4 Chlorophyll, MDA, and H2O2 contents in WT and 35S:MIR268 transgenic rice seedlings grown under normal conditions (CK) or after 60 µM CdCl2 for 14 d. (A) Chlorophyll content. (B) MDA content (C) H2O2 content. Each column represents an average of three replicates, and bars indicate SDs. * and ** indicate significant differences from between WT and transgenic plants at P≦0.05 and P≦0.01, respectively. 254x190mm (96 x 96 DPI)



Fig. 5 Cd contents in shoots and roots of WT and 35S:MIR268 transgenic plants exposed to 60 µM CdCl2 for 14 d. (A) Shoot tissue. (B) Root tissue. Data are presented as the mean ± SD. The significant level of the difference between WT and transgenic plants is indicated by * for P≦0.05 and ** for P≦0.01. 254x190mm (96 x 96 DPI)


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microRNA268 overexpression affects rice seedling growth under

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