miRNA and mRNA Expression Profiles Reveal Insight into Chitosan

6 days ago - ... miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case ...
10 downloads 10 Views 2MB Size
Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Bioactive Constituents, Metabolites, and Functions

miRNA and mRNA expression profiles reveal insight into the chitosan-mediated regulation of plant growth Xiaoqian Zhang, Kecheng Li, Ronge Xing, Song Liu, Xiaolin Chen, Haoyue Yang, and Pengcheng Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06081 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 53

Journal of Agricultural and Food Chemistry

1

miRNA and mRNA expression profiles reveal insight

2

into the chitosan-mediated regulation of plant growth

3 4

Xiaoqian Zhang†,§, Kecheng Li*,†,‡, Ronge Xing†,‡, Song Liu†,‡, Xiaolin Chen†,‡,

5

Haoyue Yang†,‡, Pengcheng Li*,†,‡

6



Key Laborotory of Experimental Marine Biology, Institute of Oceanology,

7

Chinese Academy of Sciences, Qingdao 266071, China

8



9

Laboratory for Marine Science and Technology, Qingdao 266237, China

10

§

Laboratory for Marine Drugs and Bioproducts of Qingdao National

University of Chinese Academy of Sciences, Beijing 100049, China

11 12 13 14 15

*Corresponding author.

16

Pengcheng Li

17

Tel.: +86 532 82898707; fax: +86 532 82968951.

18

E-mail address: [email protected].

19

Kecheng Li

20

Tel.: +86 532 82898641; fax: +86 532 82968780.

21

E-mail address: [email protected].

22 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 53

23

ABSTRACT

24

Chitosan has been numerously studied as a plant growth regulator and stress

25

tolerance inducer. To investigate the roles of chitosan as bio-regulator on

26

plant and unravel its possible metabolic responses mechanisms, we

27

simultaneously investigated mRNAs and microRNAs (miRNAs) expression

28

profiles of wheat seedlings in response to chitosan heptamer. We found 400

29

chitosan-responsive

30

up-regulated and 132 down-regulated mRNAs, many of which were related to

31

photosynthesis, primary carbon and nitrogen metabolism, defense responses

32

and

33

chitosan-mediated regulation on plant growth. We identified 87 known and 21

34

novel miRNAs, among which 56 miRNAs were induced or repressed by

35

chitosan

36

miRNA171a, miRNA319 and miRNA1127. The integrative analysis of miRNA

37

and mRNA expression profiles in this case provides fundamental information

38

for further investigation of regulation mechanisms of chitosan on plant growth

39

and will facilitate its application in agriculture.

40

KEYWORDS : Chitosan; mRNA; miRNA; Transcription factor; Wheat

41

seedlings

transcription

heptamer,

differentially

factors.

such

expressed

Moreover,

as

genes,

miRNAs

miRNA156,

also

including

participate

miRNA159a,

42

2

ACS Paragon Plus Environment

268

in

miRNA164,

Page 3 of 53

Journal of Agricultural and Food Chemistry

43

INTRODUCTION

44

On account of various problems such as growing population, worsening

45

environmental pollution and decreased soil fertility, conventional crop

46

production is increasingly being challenged.1 In order to realize the

47

sustainable increase in crop yield, it is crucial to develop techniques to

48

promote crop growth and increase crop yield. One current agricultural

49

practice to increase crop yield is the application of exogenous biostimulants,

50

which are able to increase crop productivity and alleviate the negative effect

51

of biotic or abiotic stress. Mounting studies have revealed that biostimulants,

52

such as protein hydrolysates, seaweed extracts and microbial fermentation,

53

greatly enhanced the plant growth and plant productivity over past

54

decades.2,3

55

Recently various carbohydrates have also attracted increasing attention

56

for their possible roles as resistance inducers. Chitosan is the second most

57

abundant carbohydrate biopolymers in the world, which is mainly consisting

58

of

59

N-acetyl-D-glucosamine.4 Actually, chitin and chitosan derivatives have been

60

assigned as one kind of biostimulants.5,6 In the last decades, chitosan had

61

been numerously studied as a plant growth regulator and stress tolerance

62

inducer. The physiological responses to chitosan in plants have been largely

63

investigated in lab, greenhouse or field conditions, such as stimulating seed

64

germination,7 improving chlorophyll contents,8 inducing salt and drought

β-1,4-linked

D-glucosamine

and

partially

3

ACS Paragon Plus Environment

of

β-1,4-linked

Journal of Agricultural and Food Chemistry

Page 4 of 53

65

tolerance,8, 9 and activating plant antimicrobial activity.10 In large-scale field

66

production, it had been also reported that chitosan oligosaccharides could

67

significantly increase the yield of wheat.11 Moreover, cDNA microarray

68

analyses of gene expression in rice and Brassica napus treated with chitosan

69

oligosaccharides elicitor were conducted by Tomiyama et al. and Yin et al.,

70

respectively, which suggested that the differentially expressed genes induced

71

by chitosan were involved in different biological processes including defense,

72

primary metabolism, transcription, and signal transduction.12, 13 Actually, the

73

activities of chitosan are closely related with its degree of polymerization (DP).

74

14,15

Based on our prior studies, chitosan heptamer (GlcN)7 was efficient in

75

promoting the morphological growth parameters of plant under stress or

76

non-stress conditions.16,17 However, the exactly molecular mechanism and

77

regulatory networks still remain unknown.

78

Recently, high-throughput transcripts profiling greatly facilitated to

79

explore the system change of complex biological processes with the

80

advantages of high resolution, deep coverage and dynamic landscapes,

81

which

82

Unquestionably, RNA sequencing (RNA-Seq) would be a powerful tool to

83

reveal the global genetic and molecular responses of plants to chitosan. On

84

the other hand, microRNAs (miRNAs) are a class of endogenous non-coding,

85

short

86

post-transcriptional levels by targeting mRNAs for cleavage or translational

is

considerably

small

RNAs

more

(~24

nt)

efficient

that

than

regulate

4

ACS Paragon Plus Environment

microarray

gene

analysis.18

expression

at

Page 5 of 53

Journal of Agricultural and Food Chemistry

87

repression.19 Functionally, miRNAs could regulate the expression of many

88

important genes, and a majority of these genes are transcriptional factors

89

(TFs).20 Numerous studies have suggested that miRNAs are associated with

90

diverse plant biological processes, including leaf and root development,

91

signal transduction, biotic or abiotic stress responses.21,

92

expression analysis of miRNA would also be a valuable tool to demonstrate

93

the potential action mode of chitosan in plant system.

22

Therefore, the

94

Wheat is one of the most important crops in the world because of its high

95

production, wide geographical range and high proportion of human

96

consumption.1 In the present experiment, chitosan heptamer was further

97

used as a representative to investigate the mode of action of chitosan on

98

plant growth. We simultaneously profiled mRNA and miRNA expression in

99

chitosan-treated

wheat

seedlings

using

high-throughput

sequencing

100

technology,and further performed an integrative analysis of mRNAs and

101

miRNA expression in order to reveal the complicated network of metabolic

102

and signaling pathways involved in chitosan-mediated regulation on plant

103

growth. To our best knowledge, this is the first study to investigate the

104

regulation mechanism of chitosan on wheat seedlings at mRNA and miRNA

105

expression level via deep sequencing analysis. Our results are expected to

106

provide a new insight into the molecular mechanisms of chitosan as a growth

107

regulator and resistance inducer, which will provide and significant guidance

108

for its application in agriculture. 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

109

MATERIALS AND METHODS

110

Plant growth conditions and treatments. (GlcN)7 (≥93%) was prepared

111

following the method described by Li et al. (2013).23 Seeds of wheat (Triticum

112

aestivum L. Jimai 22) was used in the present study, which has some

113

excellent characteristics with high yield, strong resistance to lodging and

114

disease. It has become the most popular wheat cultivar in China since

115

2010.24 Seeds were germinated at 25℃ for 24 h in the dark, and then

116

transplanted into Petri dishes (11.5 cm in diameter) with Hoagland solution in

117

a light growth chamber with 25℃/20℃ and 14-h/10-h light/dark photoperiod.

118

After the second leaf was fully developed, the wheat seedlings were divided

119

into two experimental groups randomly with three replicates each, including

120

the control group (CK, sprayed with distilled water) and (GlcN)7 treatment

121

group (sprayed with 15 mg/L (GlcN)7). After forty-eight hours, the second

122

functional leaves of twenty randomly-selected plants from two treatment

123

groups were collected and immediately frozen with liquid nitrogen and stored

124

in -80℃ for further physiological and molecular analyses. For the analysis of

125

the biomass, ten plants from each 3 replications in the control and

126

(GlcN)7-treated groups were selected randomly and their shoot fractions were

127

used to determine fresh weight and dry weight.

128

Determination carbohydrates, and total antioxidant activity. The total

129

antioxidant activity was measured according to Ertani et al. (2017).25 The

130

sucrose was extracted from 0.1g fresh leaves of wheat seedlings with 2M 6

ACS Paragon Plus Environment

Page 6 of 53

Page 7 of 53

Journal of Agricultural and Food Chemistry

131

NaOH, boiled for 5 min and determined based on resorcinol hydrochloric acid

132

method. The content of starch was determined according to the method

133

described by Zrenner et al. (1995).26

134

RNA isolation, library preparation and sequencing. Total RNA was

135

isolated using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). The RNA

136

quality was monitored on 1% agarose gel and the concentration was

137

determined using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life

138

Technologies, CA, USA). mRNA and small RNA libraries were generated

139

using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) and

140

NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.)

141

following manufacturer’s instructions, and the library quality was assessed on

142

the Agilent Bioanalyzer 2100 system. Then each sequencing library was

143

performed using an Illumina Genome Analyzer (Illumina, San Diego, CA,

144

USA).

145

Expression analysis of mRNAs. For the transcriptome libraries, after

146

removing adaptors and low-quality reads from raw reads, clean reads were

147

mapped to the reference genome of wheat released by NCBI wheat EST

148

collection

149

(http://ftp.ncbi.nih.gov/repository/UniGene/Triticum_aestivum/Ta.seq.uniq.gz)

150

and

151

(http://plants.ensembl.org/Triticum_aestivum) to searched for CATG sites. All

152

the possible CATG + 17 nt sequences were used as a reference tag library for

IWGSC

wheat

genomic

7

ACS Paragon Plus Environment

DNA

sequences

Journal of Agricultural and Food Chemistry

Page 8 of 53

153

following clean tags mapping. All clean tags that were uniquely mapped to the

154

reference sequences (with ≤1 mismatch) were used for differential expression

155

analysis. The gene expression level of mRNA was calculated by the RPKM

156

(reads per kb per million reads) method. Differential gene expression analysis

157

between two mRNA libraries was performed using the R packages of DESeq

158

and those genes with an adjusted P-value of 1 were considered to be differentially expressed.

184

Validation of mRNA and miRNA expression by qRT-PCR. To validate the

185

sequencing data, we randomly choose 15 differentially-expressed mRNAs

186

and 8 miRNAs for quantitative real-time RT-PCR (qRT-PCR) analysis. The

187

primers for the mRNA and miRNA RT-PCR were designed by primer premier

188

5.0 and shown in supplementary Table S1. For mRNA quantification, total

189

RNA was extracted with Takara MiniBEST Plant RNA Extraction Kit (Takara,

190

Dalian, China) according to the manufacturer’s instructions. Then total RNA

191

was used as template to synthesis complementary DNA (cDNA) by

192

PrimeScriptTM RT reagent Kit (Takara, Dalian, China), which contains 5 ×

193

gDNA Eraser Buffer, gDNA Eraser, PrimeScript RT Enzyme Mix, RT Primer

194

Mix and 5 × PrimeScript Buffer 2. Then, RT-PCR was performed with SYBR®

195

Premix Ex TaqTM (Tli RNaseH Plus) (Takara, Dalian, China). In brief, the

196

qRT-PCR were conducted in a total volume of 20 µL as follows: 10 µL SYBR

the

using

miRNA

qvalue.

prediction

Small

software

miRNA

RNAs

target

with

9

ACS Paragon Plus Environment

Mireap

prediction

qvalue