miRNA and mRNA Expression Profiles Reveal Insight into Chitosan

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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

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Journal of Agricultural and Food Chemistry

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miRNA and mRNA expression profiles reveal insight

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into the chitosan-mediated regulation of plant growth

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

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Haoyue Yang†,‡, Pengcheng Li*,†,‡

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†

Key Laborotory of Experimental Marine Biology, Institute of Oceanology,

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Chinese Academy of Sciences, Qingdao 266071, China

8

‡

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Laboratory for Marine Science and Technology, Qingdao 266237, China

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§

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.

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Pengcheng Li

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

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E-mail address: [email protected].

19

Kecheng Li

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

21

E-mail address: [email protected].

22 1

ACS Paragon Plus Environment


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ABSTRACT

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

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

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plant and unravel its possible metabolic responses mechanisms, we

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simultaneously investigated mRNAs and microRNAs (miRNAs) expression

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

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chitosan-responsive

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

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photosynthesis, primary carbon and nitrogen metabolism, defense responses

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and

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

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

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chitosan

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miRNA171a, miRNA319 and miRNA1127. The integrative analysis of miRNA

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and mRNA expression profiles in this case provides fundamental information

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for further investigation of regulation mechanisms of chitosan on plant growth

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and will facilitate its application in agriculture.

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

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seedlings

transcription

heptamer,

differentially

factors.

such

expressed

Moreover,

as

genes,

miRNAs

miRNA156,

also

including

participate

miRNA159a,

42

2


268

in

miRNA164,

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INTRODUCTION

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On account of various problems such as growing population, worsening

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environmental pollution and decreased soil fertility, conventional crop

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

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

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promote crop growth and increase crop yield. One current agricultural

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

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

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

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such as protein hydrolysates, seaweed extracts and microbial fermentation,

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greatly enhanced the plant growth and plant productivity over past

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decades.2,3

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Recently various carbohydrates have also attracted increasing attention

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

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abundant carbohydrate biopolymers in the world, which is mainly consisting

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of

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

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

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been numerously studied as a plant growth regulator and stress tolerance

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

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

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

β-1,4-linked

D-glucosamine

and

partially

3


of

β-1,4-linked


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

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production, it had been also reported that chitosan oligosaccharides could

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

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

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

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respectively, which suggested that the differentially expressed genes induced

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by chitosan were involved in different biological processes including defense,

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

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

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14,15

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

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promoting the morphological growth parameters of plant under stress or

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

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regulatory networks still remain unknown.

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Recently, high-throughput transcripts profiling greatly facilitated to

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explore the system change of complex biological processes with the

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advantages of high resolution, deep coverage and dynamic landscapes,

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which

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

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

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

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short

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post-transcriptional levels by targeting mRNAs for cleavage or translational

is

considerably

small

RNAs

more

(~24

nt)

efficient

that

than

regulate

4


microarray

gene

analysis.18

expression

at

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

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

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

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diverse plant biological processes, including leaf and root development,

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signal transduction, biotic or abiotic stress responses.21,

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

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the potential action mode of chitosan in plant system.

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Therefore, the

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

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production, wide geographical range and high proportion of human

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

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

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plant growth. We simultaneously profiled mRNA and miRNA expression in

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chitosan-treated

wheat

seedlings

using

high-throughput

sequencing

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technology，and further performed an integrative analysis of mRNAs and

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miRNA expression in order to reveal the complicated network of metabolic

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and signaling pathways involved in chitosan-mediated regulation on plant

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

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regulation mechanism of chitosan on wheat seedlings at mRNA and miRNA

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

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

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regulator and resistance inducer, which will provide and significant guidance

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for its application in agriculture. 5



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MATERIALS AND METHODS

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

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

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

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excellent characteristics with high yield, strong resistance to lodging and

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

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

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

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

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

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into two experimental groups randomly with three replicates each, including

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

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

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functional leaves of twenty randomly-selected plants from two treatment

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groups were collected and immediately frozen with liquid nitrogen and stored

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

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

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

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used to determine fresh weight and dry weight.

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Determination carbohydrates, and total antioxidant activity. The total

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

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


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

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

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described by Zrenner et al. (1995).26

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RNA isolation, library preparation and sequencing. Total RNA was

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

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

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

138

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

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

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

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

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the Agilent Bioanalyzer 2100 system. Then each sequencing library was

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

144

USA).

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Expression analysis of mRNAs. For the transcriptome libraries, after

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

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

148

collection

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

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and

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

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

IWGSC

wheat

genomic

7


DNA

sequences


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

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

155

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

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

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

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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

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sequencing data, we randomly choose 15 differentially-expressed mRNAs

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

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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

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

190

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

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

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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


Mireap

prediction

qvalue

miRNA and mRNA Expression Profiles Reveal Insight into Chitosan

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