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
