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Characterization of wheat miRNAs and their target genes responsive to cadmium stress
Accepted Manuscript Characterization of wheat miRNAs and their target genes responsive to cadmium stress ZongBo Qiu, BenZhai Hai, JunLi Guo, YongFang Li, Liang Zhang PII:
S0981-9428(16)30019-5
DOI:
10.1016/j.plaphy.2016.01.020
Reference:
PLAPHY 4392
To appear in:
Plant Physiology and Biochemistry
Received Date: 1 December 2015 Revised Date:
25 January 2016
Accepted Date: 28 January 2016
Please cite this article as: Z. Qiu, B. Hai, J. Guo, Y. Li, L. Zhang, Characterization of wheat miRNAs and their target genes responsive to cadmium stress, Plant Physiology et Biochemistry (2016), doi: 10.1016/ j.plaphy.2016.01.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Characterization of wheat miRNAs and their target genes responsive
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to cadmium stress ZongBo Qiua*, BenZhai Haia,b, JunLi Guoa, YongFang Lia, Liang Zhanga
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b
College of Life Sciences, Henan Normal University, Xinxiang 453007, P R China
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College of information engineering, Wuhan University of Technology, Wuhan 430070, P R China
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* Corresponding author:
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ZongBo Qiu
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College of Life Science, Henan Normal University
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147 Jianshe Road
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P R China
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Phone: +86-373-3326340
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Xinxiang 453007
E-mail: [email protected]
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Abstract A increasing number of microRNAs have been shown to play important
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regulatory roles in plant responses to various metal stresses. However, little
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information about miRNAs especially miRNAs responsive to cadmium (Cd) stress is
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available in wheat. To investigate the role of miRNAs in responses to Cd stress, wheat
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seedlings were subjected to 250 µM Cd solution for 6, 12, 24 and 48 h, and analyses
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of morphological and physiological changes as well as the expression of five miRNAs
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and their corresponding targets were carried out. Our results demonstrated that
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miRNAs and their targets were differentially expressed in leaves and roots of wheat
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seedlings exposed to Cd stress. Furthermore, miR398 may involve in oxidative stress
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tolerance by regulating its target CSD to participate in Cd stress. Among ten
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miRNA-target pairs studied, nine pairs showed complex regulation relationship in
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leaves and roots of wheat seedlings exposed to Cd stress. These findings suggested
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that miRNAs are involved in the mediation of Cd stress signaling responses in wheat.
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The characterization of the miRNAs and the associated targets in responses to Cd
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exposure provides a framework for understanding the molecular mechanism of heavy
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metal tolerance in plants.
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Keywords: Wheat, microRNA, Target gene, Cadmium stress, Gene regulation
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1. Introduction Heavy metal contamination is an increasing environmental problem worldwide.
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Cadmium (Cd) is one of the most toxic heavy metals in the environment, posing
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serious threat to food security and crop production (Qiu et al., 2013)．Plants exposed
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to excess heavy metals can result in reduced biomass, retarded root growth, inhibition
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of cell proliferation, alterations in the photosynthesis rate (Fang et al., 2013;
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Jozefczak et al., 2014; Pérez-Chaca et al., 2014). To survive, plants respond and adapt
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to heavy metal stress by changes at morphological, physiological, genetic, and
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molecular levels (Tang et al., 2014). Great progress has been made in elucidating the
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complex response mechanisms involved in metal stress tolerance and in identifying a
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lot of metal stress responsive genes. To date, a number of heavy metal-responsive
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genes have been identified in various plant species, such as Oryza sativa (Shimo et al.,
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2011), Solanum nigrum (Xu et al., 2012), and Triticum aestivum (Qiu et al., 2013).
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However, the regulation of gene expression is a complex process, especially at the
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transcriptional level. MicroRNAs (miRNAs), recently recognized as important
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regulators of gene expression at the post-transcriptional level, have been found to be
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involved in plants response to a wide range of stresses (Sunkar et al., 2012; Budak et
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al., 2015b).
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miRNAs are a class of small non-coding RNA molecules with 20-24 nucleotides
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in length that regulate gene expression at the transcriptional and post-transcriptional
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levels through mRNAs cleavage or repression of translation, depending on the
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complementarity between miRNA and their target genes (Akpinar et al., 2015). In 3
ACCEPTED MANUSCRIPT plants, miRNAs have been shown to play essential roles in plant growth and
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development and environmental stress responses (Zhou et al., 2012; Srivastava et al.,
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2013; Qiu et al., 2015). Increasing evidence has revealed that miRNA-mediated gene
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regulation plays a significant role in heavy metal regulatory networks. Using a direct
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cloning strategy, 13 conserved miRNAs representing 9 families have been isolated
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from Brassica napus and showed different responses to Cd stress (Huang et al., 2010).
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Ding et al. (2013) identified 12 Cd-responsive miRNAs in rice using
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microarray-based assay and most of these miRNAs were differentially regulated by
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Cd stress. A recent study using high-throughput sequencing found 15 known and 8
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novel Cd-stress regulated miRNA families in radish roots exposed to CdCl2 stress for
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12 h. Furthermore, several Cd-responsive miRNAs have been found to target
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transcripts associated with metal transport and signaling (Xu et al., 2013). All of these
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findings demonstrate that a number of miRNAs play crucial roles in the regulation of
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plant responses to heavy metal stress.
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As one of the world’s most important crop species, some progress has been made
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in characterizing and analyzing miRNAs in the wheat genome (Budak et al., 2014,
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2015a). Furthermore, several miRNAs have been found to play important roles in
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wheat response to a wide range of stresses such as salt (Wang et al., 2014), nutrient
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deficiency (Zhao et al., 2013), drought (Zhao et al., 2015). However, until now, little
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is known about miRNA expression patterns of wheat seedlings response to Cd stress,
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and there is even less understanding on miRNA targets for this regulation. In this
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study, physiological changes and the expression patterns of five miRNAs, which have
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targets in wheat were investigated under Cd stress. The knowledge obtained from this
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study could be helpful in elucidating the potential roles of miRNAs in plant response
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to heavy metal stress and further understanding miRNA regulation in response to
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abiotic stress.
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2. Materials and methods
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2.1. Plant materials and treatment
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The uniform seeds of wheat (Triticum aestivum L. cv. Zhengmai No. 004,
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obtained from Henan Academy of Agricultural Sciences) were surface sterilized in
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0.1% HgCl2 and germinated at 25 °C for 2 d in the dark. Germinated seeds were
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grown in a Petri dishes (diameter 18 cm, each containing 80 seeds) floating on a
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half-strength Hoagland’s solution, in a growth chamber under a 12 h photoperiod at
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800 µmol m-2 s-1, 70% relative humidity and 25°C/18°C (day/night). The plants were
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grown hydroponically for 7 d (with one fully expanded leaves) and then transferred to
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the same nutrient solution containing 250 µM CdCl2 ·2.5H2O for 6, 12, 24 and 48 h,
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respectively. Seedlings grown in Cd-free nutrient solution were treated as the control.
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At each time point, roots and leaves of wheat seedlings from Cd-treated and control
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groups were separately harvested, immediately frozen in liquid nitrogen and stored at
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-80°C until use. For all physiological experiments, fresh leaf and root tissues
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harvested at five time points viz., 0, 6, 12, 24 and 48 h, were used. Then, plant height
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and root length of wheat seedlings were also measured using a scale.
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2.2. Estimation of relative water content
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treatment and it was calculated according to formula (Barrs and Weatherley, 1962): RWC (%) = [(fresh weight - dry weight)/( turgid weight - dry weight)]× 100
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Leaves of the control and Cd stressed seedlings were collected and immediately
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weighed (fresh weight, FW). They were rehydrated in water for 24 h until fully turgid,
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surface dried, and re-weighed (turgid weight, TW). The tissues were then oven dried
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at 105°C for 24 h and re-weighed (dry weight, DW).
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2.3. Determination of malondialdehyde (MDA) and photosynthetic pigments
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concentration
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The concentration of MDA which is a product of lipid peroxidation was estimated
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by the thiobarbituric acid (TBA) as described by Predieri et al. (1995). Samples of
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leaves (0.2 g fresh weight, FW) were ground in 50 mM phosphate buffer (pH 7.8),
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and then centrifuged at 8,000×g for 15 min. One mL supernatant was combined with
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2.5 mL thiobarbituric acid (TBA) incubated in boiling water for 30 min and then
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quickly cooled in an ice-bath. The mixture was centrifuged at 10,000×g for 5 min and
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the absorbance of supernatant was monitored at 532 and 600 nm. The value for
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non-specific absorbance at 600 nm was subtracted. MDA concentration was
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calculated by its molar extinction coefficient (155 mM-1cm-1) and the results
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expressed as µmol MDA g-1 FW.
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Measurement of chlorophyll a and b content of the control and Cd exposed plants
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was done as described by Lichtenthaler (1987). The photosynthetic pigments were
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extracted from seedling leaves with 10 mL of 80% acetone for 24 h in dark at room 6
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using a spectrophotometer (Lambda35, Perkin Elmer, USA). The experiment was
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repeated three times and the concentration of chlorophyll a and b expressed as mg g-1
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FW.
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2.4. Prediction of miRNA targets
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To identify putative targets of miR156, miR159, miR164, miR398 and miR408,
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we used mature miR156, miR159, miR164, miR398 and miR408 as custom miRNAs
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and the wheat-expressed sequence tag (EST) database (TIGR Wheat Gene Index 9) as
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custom mRNAs to search for complementary hits by using psRNATarget
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(http://bioinfo3.noble.org/psRNATarget/index.php?function=function3) with default
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parameters settings (Zhang, 2005). Sequences with a penalizing score ≤ 3 were
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chosen as putative targets. Functional groups of predicted target genes were
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determined based on the annotations of the wheat EST database and BLAST search
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analysis.
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2.5. Total RNA isolation and cDNA synthesis
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Total RNA was isolated from each sample using RNAsimple total RNA kit
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(Tiangen, China) according to the manufacturer’s instructions. Total RNA was treated
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with DNase I to remove contaminating genomic DNA. The integrity of the total RNA
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was checked by 2% agarose gel electrophoresis with ethidium bromide (EB) staining,
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and the concentration was determined with a NanoDropTM 1000 spectrophotometer
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(Thermo Fisher Scientific, USA). First-strand cDNA synthesis was performed using 7
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Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s
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instructions, using 2 µg of total RNA and oligo (dT) primers.
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2.6. Expression analysis of miRNAs and their target mRNAs by RT-qPCR Quantitative real-time PCR (RT-qPCR) was performed using a Rotor-Gene 3000
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real-time PCR detection system (Qiagen) according to the manufacturer’s protocol.
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Gene-specific primers were designed using the Primer 5.0 (File S2). RT-qPCR
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reaction mix included 2 µL diluted cDNA, 0.3 µM of corresponding forward and
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reverse primers, and 10 µL of the Thunderbird SYBR Green PCR Master Mix in a
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final reaction volume of 20 µL with the following cycling conditions: 95°C for 2 min,
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40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. After amplification, a
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thermal denaturing cycle at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s was
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carried out to determine the dissociation curves and verify the specificity of the
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amplifications. All reactions were performed in biological triplicates, and the results
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were expressed relative to the expression levels of an internal reference gene,
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elongation factor 1 alpha-subunit (TEF1, GenBank accession No. M90077.1) in each
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sample using the 2-∆∆Ct method (Livak and Schmittgen, 2001).
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2.7. Statistical analysis
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The experiment was performed in a completely random design with three
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replications. The data of morphological and physiological characteristics were
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subjected to a two-way analysis of variance (ANOVA) followed by the Duncan’s
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multiple range tests at the 5% probability level using SPSS 16.0.
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3. Results
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3.1. Effect of CdCl2 stress on plant height, root length and relative water content in
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wheat seedlings
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Table 1 Changes of plant height, root length and relative water content in wheat
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seedlings subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Treatment
0h
CK
9.37±1.03bc
6.58±0.21cd
95.02±1.02a
6h
CK
9.47±1.51bc
6.82±0.32cd
94.21±2.04a
Cd stress
9.05±0.53c
6.47±0.62d
78.32±1.25b
CK
9.53±0.54bc
7.03±0.52bc
94.35±1.54a
Cd stress
9.23±0.84c
6.56±0.24cd
66.24±0.98d
CK
10.08±0.62ab
7.32±0.38b
95.67±1.54a
Cd stress
9.47±0.32bc
6.66±0.34cd
71.42±0.84c
10.52±0.84a
8.58±0.52a
96.02±0.68a
9.85±0.25b
6.72±0.62cd
67.28±0.95d
48h
CK Cd stress
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Relative water content (%)
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Plant height (cm)
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Data are means ± standard error of 20 replicates, and each mean in a column followed
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by the same letter indicates that there are no significant differences at 0.05 level
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according to Duncan’s multiple range test.
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Table 1 illustrates changes in plant height and root length in wheat seedlings
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under Cd stress in a time-dependent manner. Cd stress markedly hindered root and
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shoot elongation in the growth of wheat seedlings (Table 1). Compared with the
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control, Cd stress induced a marked decrease (p < 0.05) in the plant height and root
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length with prolonged Cd stress treatment. To further evaluate the responses to the Cd
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0, 6, 12, 24 and 48 h was estimated. The results indicated that the relative water
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content was strongly reduced in leaves of wheat seedlings with prolonged Cd
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exposure. Under the control conditions, it was maintained at 94-96%, whereas it
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gradually decreased to about 67% after 48 h under Cd stress.
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3.2. Effect of CdCl2 stress on MDA, Chl a and Chl b concentration in wheat
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seedlings
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Chl b concentration (mg g-1 FW)
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7 6 5 4 3 2
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Fig.1. Changes of MDA, Chl a and Chl b concentration in wheat seedlings subjected
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to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Data are means ± standard error of 6
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replicates.
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Changes of MDA, Chl a and Chl b concentration in wheat seedlings under
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continuous CdCl2 stress were presented in Fig.1. As shows in Fig.1, with the
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elongation of Cd stress, MDA concentration gradually increased in wheat seedlings
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compared with the control. However, CdCl2 stress resulted in a significant decrease in 10
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the concentration of Chl a and Chl b in wheat seedlings compared with the control.
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3.3. Target prediction of miRNAs in wheat seedlings Table 2 Predicted targets of microRNA in wheat seedlings under Cd stress
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Function
miR156
Development, flowering time
TC447879
Signaling
TC368630
Squamosa
promoter-binding
Accession number
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miRNA Predicted targets
protein (SBP) MYB3 transcription factor
pathway
and
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development NAC domain transcription factor
Signaling
pathway,
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development,
response
root
TC410195
to
oxidative stress
miR398
Cu-Zn
superoxide
(CSD)
Copper homeostasis, response
TC388772
to oxidative stress
Chemocyanin-like protein (CLP)
Stress-related proteins
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dismutase
The identification of miRNA targets is an essential step towards the understanding
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of their regulatory function. Target prediction of miR156, miR159, miR164, miR398
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and miR408 was carried out using the Web-based program psRNATarget. As
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expected, most of the predicted targets for T. aestivum have homologues of known
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miRNA targets in other plant species, such as squamosa promoter-binding protein
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(SBP) for miR156, MYB3 transcription factor for miR159, NAC domain transcription
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factor for miR164, Cu-Zn superoxide dismutases for miR398 or chemocyanin-like
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protein (CLP) for miR408 (Table 2). Putative target prediction for the miRNAs
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suggested that they may regulate various developmental processes and involve in
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oxidative stress tolerance.
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CK
6h
12h
24h
48h
2
0
CK
6h
12h
6h
12h
24h
3 miR408 2 1 0
CK
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12h
24h
48h
12h
24h
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4 miR398 3 2 1 0
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2
Relative expression
4 miR164 3
Leaf Root
Relative expression
Relative expression
4 miR156 3
Relative expression
3.4. Effect of CdCl2 stress on miRNA expression in wheat seedlings
Relative expression
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24h
48h
Fig.2. Expression level of five miRNAs in leaves and roots of wheat seedlings
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subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. The expression level of
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each miRNA in the control was arbitrarily set at 1. Error bars represent the standard
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deviations of three biological replicates.
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In order to study the dynamic expression pattern of miRNAs under Cd stress,
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real-time PCR was performed on the leaves and roots of wheat seedlings at different
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time points (6, 12, 24 and 48 h) after Cd stress for five miRNAs (miR156, miR159,
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miR164, miR398 and miR408). Under Cd stress, the expression patterns of miR156,
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miR159, miR164, miR398 and miR408 in leaves were different from those in roots 12
ACCEPTED MANUSCRIPT (Fig.2). The expression of miR159, miR164, miR398 and miR408 were
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down-regulated in leaves, reaching their lowest expression level at 12 h, and then
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reached a relatively high expression level at 24 h. miR156 was down-regulated and
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remained at an extremely low expression level at all time points ( 6, 12, 24 and 48 h)
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in leaves. In the roots, the expression of miR156, miR159, miR164 and miR398 were
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rapidly up-regulated from 12 to 24 h, and then steadily decreased at 48 h. miR408 was
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up-regulated and remained at an extremely high expression level at all time points ( 6,
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12, 24 and 48 h) in the roots. Thus, expression of miRNAs changed with time and
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also varied in different organs
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3.5. Effect of CdCl2 stress on target mRNAs expression in wheat seedlings
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To further understand the mechanism of the interaction between miRNAs and
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their target genes, the expression levels of miRNA target genes (SBP, MYB, NAC,
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CSD and CLP) at different time points (6, 12, 24 and 48 h) after Cd stress were
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examined by RT-qPCR in leaves and roots of wheat seedlings. In the leaves, the
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transcript levels of MYB, NAC, CSD and CLP, as targets of miR159, miR164,
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miR398 and miR408 were down-regulated, gradually declined at 6 h, and then
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steadily increased at 12 and 24 h with the exception of slightly decreased at 24 h in
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CLP for miR408. The expression levels of MYB and NAC in the roots were sharply
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up-regulated at 6 h, and then gradually declined. CSD was significantly increased at 6
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h, then sharply decreased at 12 h and again sharply increased at 24 and 48 h in roots
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(Fig. 3). The expression of CLP were up-regulated after 24 and 48 h in roots, reaching
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their highest expression at 48 h. SBP, as targets of miR156 remained at an extremely
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6
6h
24h
CLP (miR408)
4 2 0
12h
24h
CK
CK
48h
6h
12h
24h
48h
12h
24h
48h
CSD (miR398)
CK
6h
12h
24h
48h
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5 4 3 2 1 0
48h
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CK
12h
MYB (miR159)
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NAC (miR164)
6h
5 4 3 2 1 0
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5 4 3 2 1 0
CK
Leaf Root
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Relative expression
2.5 2.0 1.5 1.0 0.5 0.0
Relative expression
leaves.
Relative expression
250
Relative expression
low expression level at all time points (6, 12, 24 and 48 h) in roots compared in
Relative expression
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Fig.3. Expression level of five target mRNAs in leaves and roots of wheat seedlings
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subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Corresponding miRNAs
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are indicated in brackets. The expression level of each target mRNA in the control
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was arbitrarily set at 1. Error bars represent the standard deviations of three biological
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replicates.
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3.6. Correlation between miRNAs and their target mRNAs
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2 1 0
CK
6h
12h
24h
48h
3
1 0
CK
6h
12h
24h
48h
6
miR159 MYB3
4 2 0
CK
6h
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CK
Relative expression
D miR159 MYB3
6h
12h
24h
48h
12h
24h
48h
12h
24h
48h
F
Relative expression
E miR408 CLP
CK
6h
12h
24h
48h
8 6
miR408 CLP
4 2 0
CK
6h
TE D
Relative expression
miR156 SBP
2
C
5 4 3 2 1 0
B
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miR156 SBP
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10 8 6 4 2 0
A
Relative expression
Relative expression
3
Relative expression
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Fig.4. Negatively and positively correlated miRNA-target pairs in leaves (a, c, e) and
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roots (b, d, f) of wheat seedlings grown at 250 µM Cd solutions for 0, 6, 12, 24 and 48
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h. The expression level of miRNA and corresponding target mRNA in the control was
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arbitrarily set at 1. Error bars represent the standard deviations of three biological
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replicates.
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In plants, miRNAs regulate gene expression majorly through targeting mRNAs
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for cleavage; thus, there might be an inverse correlation between a given miRNA and
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its targets. As expected, there are three miRNA/target pairs which show negative
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correlation in expression pattern under Cd treatment, including miR398-CSD in root
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Similarly, miR164 showed an inverse relationship with its target NAC in the leaves of
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wheat seedlings under Cd stress except on the 24 h. However, the expression of SBP
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was positively correlated to miR156 in both leaf and root tissues of wheat seedlings
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under Cd stress (Fig.4b). Interestingly, miR164-NAC also showed positive correlation
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in root tissue of wheat seedlings under Cd stress except on the 24 h. These results
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suggest that gene regulation may be more complicated under Cd stress condition.
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4. Discussion
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Cadmium (Cd) is a non-redox toxic heavy metal present in the environment. In
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plants, Cd could disturb various biochemical and physiological processes, leading to
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cell death and inhibition of growth (Fang et al., 2013; Qiu et al., 2013; Jozefczak et al.,
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2014). In accordance with previous work, our studies indicated that plant height, root
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length and chlorophyll content in wheat seedlings were markedly inhibited by Cd
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stress. Furthermore, Cd stress led to oxidative damage, measured by lipid
282
peroxidation. The increase in MDA content and the decrease in chlorophyll content
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were observed in wheat seedlings exposure to Cd stress, suggesting that the plant
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growth inhibition must be, directly or indirectly, attributable to the Cd induced
285
oxidative damage. Much progress has been made in unraveling the complex
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mechanisms behind Cd stress. The discovery of the role of miRNAs as gene
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regulators has led to a paradigm shift in the understanding of post-transcriptional gene
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regulation in plants. Recent findings illustrate that miRNAs might play a crucial role
289
in regulation of plant genes at the posttranscriptional level in responding to metal
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(2011) found that most of the Cd-responsive miRNAs were down-regulated along
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with miR156, miR162 and miR390. Tang et al. (2014) also identified expression of
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miR156 to be suppressed upon exposure to Cd. In the present investigation, we found
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that miR156 was down-regulated and remained at an extremely low expression level
295
in the leaves of wheat seedlings in response to the elongation of Cd stress, whereas
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the expression of miR398 were rapidly up-regulated from 12 to 24 h and miR408 was
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up-regulated and remained at an extremely high expression level at all time points in
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the roots of wheat seedlings under Cd stress. This result was in agreement with that of
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Ding et al. (2011), who found that miR398 and miR408 were reported to be
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up-regulated in response to Cd exposure in the root of rice. Using RT-qPCR, Zhou et
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al. (2012) also found that expression of miR398 was up-regulated in response to Cd
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exposure in the root of Brassica napus. These results indicated that miRNAs were
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responsive to Cd stress differentially in leaves and roots of wheat seedlings,
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suggesting that miRNAs might play different functions in the regulation of plant
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tolerance to Cd.
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Notably, several key responsive enzymes for heavy metal detoxification were
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identified as target transcripts for miR398. miR398 was identified to detoxify
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superoxide radicals by directing the cleavage of its two targets, CSD1 and CSD2
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(coding cytosolic and chloroplastic Cu/Zn superoxide dismutases, respectively) in
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Arabidopsis thaliana seedlings exposed to several abiotic stresses (Sunkar et al.,
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2006). CSD which is one target gene of miR398 as ROS (reactive oxygen species) 17
ACCEPTED MANUSCRIPT scavenger, is important for stress tolerance and survival in plant. Using RT-qPCR,
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Naya et al. (2014) found that miR398b was decreased and its target gene CSD1 were
314
up-regulated in bean plants under Cu toxicity. The similar results were also detected
315
in mulberry leaf treated with salt stress (Wu et al., 2015). Here, we found that miR398
316
was down-expression in wheat leaves, reaching their lowest expression level at 12 h
317
of Cd exposure, whereas the expression of its target, a CSD gene, was up-expression.
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The decreased miR398 level in wheat seedlings resulted in accumulation of CSD
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mRNAs, which were important for catalyzing the dismutation of superoxide radicals
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into H2O2. Hence, oxidative stress induced miR398 down-regulation was considered a
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major machinery of detoxifying ROS by regulated its target CSD gene in wheat
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seedlings exposed to Cd stress. So we inferred that miR398 may involve in oxidative
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stress tolerance by regulating its target genes to participate in Cd stress.
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In plants, miRNAs regulate gene expression majorly through targeting mRNAs
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for cleavage. Thus, miRNAs usually negatively regulate the accumulation of mRNAs
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and show an inverse correlation expression pattern in the same plant cells. Kantar et al.
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(2010) found that the expression profile of miR156 was negatively correlated with its
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target AV910992 in barley leaves under 8 h of dehydration stress. Our results are
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consistent with previous findings, indicating that three miRNA/target pairs
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(miR398-CSD in root tissue, and miR159-MYB3 and miR408-CLP in both leaf and
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root tissues) showed negative correlation in expression level under Cd stress condition.
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Interestingly, we observed that the transcript level of NAC was positively correlated
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with the accumulation of miR164 in the root of wheat seedlings upon the Cd stress.
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expression of miR164 and its target MesNAC was not inversely regulated in the roots
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of cassava during water deficit. Furthermore, the expression of miR156 and its target
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SBP in both leaf and root tissues of wheat seedlings in our study also showed positive
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correlation under Cd stress. There are several reasons why miRNA/target pairs do not
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show perfect negative correlation. One potential reason is that abiotic stress is a
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complicated progress, and it is also possible that the target is regulated by multiple
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miRNAs, the final expression level of the targets should be combined results for all
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the miRNAs targeting this gene (Khaksefidi et al., 2015). Another reason is that
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miRNAs have been demonstrated to establish temporal and spatial expression patterns
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of target genes (Zhou et al., 2012; Qiu et al., 2015). Therefore, expression patterns in
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a sample comprising multiple tissue types may conceal a reverse expression level for
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the miRNA and its targets. Thus, further investigation of the regulatory functions of
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miRNAs will deepen our understanding on plant response to heavy metal-induced
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toxicity.
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Taken together, the results generated from this study suggested that
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Cd-responsive miRNAs showed time- and organ dependent expression patterns.
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Furthermore, a complex regulation relationship between miRNA and target
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expression profiles was validated in wheat seedlings under Cd stress using RT-qPCR.
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Comprehensive characterisation of the miRNAs and their target genes will contribute
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to our understanding of gene regulatory frameworks in plants, and may provide
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insights into the role of miRNAs and their targets in regulating plant tolerance to Cd
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stress.
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Acknowledgments This research was supported by the National Science Foundation of China
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(U1404304 and No. 31500499), Program for Science
Technology Innovation
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Talents in Universities of Henan Province (16HASTIT019) and Innovative Research
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Team (in Science and Technology) in University of Henan Province (No.
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15IRTSTHN020).
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Highlights
Cd stress severely affected wheat morphological and physiological function
Expression of five known miRNAs and their targets were carried out using
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RT-qPCR
miRNAs and their targets were differentially expressed in leaves and roots of wheat
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miRNA and its target showed complex regulation relationship in response to Cd
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DOI:
10.1016/j.plaphy.2016.01.020
Reference:
PLAPHY 4392
To appear in:
Plant Physiology and Biochemistry
Received Date: 1 December 2015 Revised Date:
25 January 2016
Accepted Date: 28 January 2016
Please cite this article as: Z. Qiu, B. Hai, J. Guo, Y. Li, L. Zhang, Characterization of wheat miRNAs and their target genes responsive to cadmium stress, Plant Physiology et Biochemistry (2016), doi: 10.1016/ j.plaphy.2016.01.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Characterization of wheat miRNAs and their target genes responsive
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to cadmium stress ZongBo Qiua*, BenZhai Haia,b, JunLi Guoa, YongFang Lia, Liang Zhanga
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b
College of Life Sciences, Henan Normal University, Xinxiang 453007, P R China
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College of information engineering, Wuhan University of Technology, Wuhan 430070, P R China
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* Corresponding author:
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ZongBo Qiu
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College of Life Science, Henan Normal University
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147 Jianshe Road
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P R China
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Phone: +86-373-3326340
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E-mail: [email protected]
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Abstract A increasing number of microRNAs have been shown to play important
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regulatory roles in plant responses to various metal stresses. However, little
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information about miRNAs especially miRNAs responsive to cadmium (Cd) stress is
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available in wheat. To investigate the role of miRNAs in responses to Cd stress, wheat
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seedlings were subjected to 250 µM Cd solution for 6, 12, 24 and 48 h, and analyses
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of morphological and physiological changes as well as the expression of five miRNAs
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and their corresponding targets were carried out. Our results demonstrated that
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miRNAs and their targets were differentially expressed in leaves and roots of wheat
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seedlings exposed to Cd stress. Furthermore, miR398 may involve in oxidative stress
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tolerance by regulating its target CSD to participate in Cd stress. Among ten
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miRNA-target pairs studied, nine pairs showed complex regulation relationship in
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leaves and roots of wheat seedlings exposed to Cd stress. These findings suggested
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that miRNAs are involved in the mediation of Cd stress signaling responses in wheat.
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The characterization of the miRNAs and the associated targets in responses to Cd
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exposure provides a framework for understanding the molecular mechanism of heavy
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metal tolerance in plants.
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Keywords: Wheat, microRNA, Target gene, Cadmium stress, Gene regulation
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1. Introduction Heavy metal contamination is an increasing environmental problem worldwide.
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Cadmium (Cd) is one of the most toxic heavy metals in the environment, posing
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serious threat to food security and crop production (Qiu et al., 2013)．Plants exposed
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to excess heavy metals can result in reduced biomass, retarded root growth, inhibition
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of cell proliferation, alterations in the photosynthesis rate (Fang et al., 2013;
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Jozefczak et al., 2014; Pérez-Chaca et al., 2014). To survive, plants respond and adapt
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to heavy metal stress by changes at morphological, physiological, genetic, and
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molecular levels (Tang et al., 2014). Great progress has been made in elucidating the
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complex response mechanisms involved in metal stress tolerance and in identifying a
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lot of metal stress responsive genes. To date, a number of heavy metal-responsive
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genes have been identified in various plant species, such as Oryza sativa (Shimo et al.,
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2011), Solanum nigrum (Xu et al., 2012), and Triticum aestivum (Qiu et al., 2013).
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However, the regulation of gene expression is a complex process, especially at the
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transcriptional level. MicroRNAs (miRNAs), recently recognized as important
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regulators of gene expression at the post-transcriptional level, have been found to be
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involved in plants response to a wide range of stresses (Sunkar et al., 2012; Budak et
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al., 2015b).
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miRNAs are a class of small non-coding RNA molecules with 20-24 nucleotides
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in length that regulate gene expression at the transcriptional and post-transcriptional
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levels through mRNAs cleavage or repression of translation, depending on the
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complementarity between miRNA and their target genes (Akpinar et al., 2015). In 3
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development and environmental stress responses (Zhou et al., 2012; Srivastava et al.,
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2013; Qiu et al., 2015). Increasing evidence has revealed that miRNA-mediated gene
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regulation plays a significant role in heavy metal regulatory networks. Using a direct
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cloning strategy, 13 conserved miRNAs representing 9 families have been isolated
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from Brassica napus and showed different responses to Cd stress (Huang et al., 2010).
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Ding et al. (2013) identified 12 Cd-responsive miRNAs in rice using
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microarray-based assay and most of these miRNAs were differentially regulated by
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Cd stress. A recent study using high-throughput sequencing found 15 known and 8
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novel Cd-stress regulated miRNA families in radish roots exposed to CdCl2 stress for
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12 h. Furthermore, several Cd-responsive miRNAs have been found to target
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transcripts associated with metal transport and signaling (Xu et al., 2013). All of these
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findings demonstrate that a number of miRNAs play crucial roles in the regulation of
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plant responses to heavy metal stress.
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As one of the world’s most important crop species, some progress has been made
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in characterizing and analyzing miRNAs in the wheat genome (Budak et al., 2014,
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2015a). Furthermore, several miRNAs have been found to play important roles in
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wheat response to a wide range of stresses such as salt (Wang et al., 2014), nutrient
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deficiency (Zhao et al., 2013), drought (Zhao et al., 2015). However, until now, little
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is known about miRNA expression patterns of wheat seedlings response to Cd stress,
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and there is even less understanding on miRNA targets for this regulation. In this
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study, physiological changes and the expression patterns of five miRNAs, which have
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targets in wheat were investigated under Cd stress. The knowledge obtained from this
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study could be helpful in elucidating the potential roles of miRNAs in plant response
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to heavy metal stress and further understanding miRNA regulation in response to
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abiotic stress.
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2. Materials and methods
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2.1. Plant materials and treatment
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The uniform seeds of wheat (Triticum aestivum L. cv. Zhengmai No. 004,
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obtained from Henan Academy of Agricultural Sciences) were surface sterilized in
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0.1% HgCl2 and germinated at 25 °C for 2 d in the dark. Germinated seeds were
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grown in a Petri dishes (diameter 18 cm, each containing 80 seeds) floating on a
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half-strength Hoagland’s solution, in a growth chamber under a 12 h photoperiod at
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800 µmol m-2 s-1, 70% relative humidity and 25°C/18°C (day/night). The plants were
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grown hydroponically for 7 d (with one fully expanded leaves) and then transferred to
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the same nutrient solution containing 250 µM CdCl2 ·2.5H2O for 6, 12, 24 and 48 h,
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respectively. Seedlings grown in Cd-free nutrient solution were treated as the control.
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At each time point, roots and leaves of wheat seedlings from Cd-treated and control
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groups were separately harvested, immediately frozen in liquid nitrogen and stored at
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-80°C until use. For all physiological experiments, fresh leaf and root tissues
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harvested at five time points viz., 0, 6, 12, 24 and 48 h, were used. Then, plant height
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and root length of wheat seedlings were also measured using a scale.
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2.2. Estimation of relative water content
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treatment and it was calculated according to formula (Barrs and Weatherley, 1962): RWC (%) = [(fresh weight - dry weight)/( turgid weight - dry weight)]× 100
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Leaves of the control and Cd stressed seedlings were collected and immediately
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weighed (fresh weight, FW). They were rehydrated in water for 24 h until fully turgid,
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surface dried, and re-weighed (turgid weight, TW). The tissues were then oven dried
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at 105°C for 24 h and re-weighed (dry weight, DW).
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2.3. Determination of malondialdehyde (MDA) and photosynthetic pigments
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concentration
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The concentration of MDA which is a product of lipid peroxidation was estimated
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by the thiobarbituric acid (TBA) as described by Predieri et al. (1995). Samples of
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leaves (0.2 g fresh weight, FW) were ground in 50 mM phosphate buffer (pH 7.8),
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and then centrifuged at 8,000×g for 15 min. One mL supernatant was combined with
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2.5 mL thiobarbituric acid (TBA) incubated in boiling water for 30 min and then
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quickly cooled in an ice-bath. The mixture was centrifuged at 10,000×g for 5 min and
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the absorbance of supernatant was monitored at 532 and 600 nm. The value for
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non-specific absorbance at 600 nm was subtracted. MDA concentration was
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calculated by its molar extinction coefficient (155 mM-1cm-1) and the results
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expressed as µmol MDA g-1 FW.
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Measurement of chlorophyll a and b content of the control and Cd exposed plants
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was done as described by Lichtenthaler (1987). The photosynthetic pigments were
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extracted from seedling leaves with 10 mL of 80% acetone for 24 h in dark at room 6
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using a spectrophotometer (Lambda35, Perkin Elmer, USA). The experiment was
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repeated three times and the concentration of chlorophyll a and b expressed as mg g-1
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FW.
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2.4. Prediction of miRNA targets
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To identify putative targets of miR156, miR159, miR164, miR398 and miR408,
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we used mature miR156, miR159, miR164, miR398 and miR408 as custom miRNAs
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and the wheat-expressed sequence tag (EST) database (TIGR Wheat Gene Index 9) as
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custom mRNAs to search for complementary hits by using psRNATarget
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(http://bioinfo3.noble.org/psRNATarget/index.php?function=function3) with default
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parameters settings (Zhang, 2005). Sequences with a penalizing score ≤ 3 were
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chosen as putative targets. Functional groups of predicted target genes were
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determined based on the annotations of the wheat EST database and BLAST search
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analysis.
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2.5. Total RNA isolation and cDNA synthesis
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Total RNA was isolated from each sample using RNAsimple total RNA kit
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(Tiangen, China) according to the manufacturer’s instructions. Total RNA was treated
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with DNase I to remove contaminating genomic DNA. The integrity of the total RNA
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was checked by 2% agarose gel electrophoresis with ethidium bromide (EB) staining,
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and the concentration was determined with a NanoDropTM 1000 spectrophotometer
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(Thermo Fisher Scientific, USA). First-strand cDNA synthesis was performed using 7
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Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s
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instructions, using 2 µg of total RNA and oligo (dT) primers.
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2.6. Expression analysis of miRNAs and their target mRNAs by RT-qPCR Quantitative real-time PCR (RT-qPCR) was performed using a Rotor-Gene 3000
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real-time PCR detection system (Qiagen) according to the manufacturer’s protocol.
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Gene-specific primers were designed using the Primer 5.0 (File S2). RT-qPCR
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reaction mix included 2 µL diluted cDNA, 0.3 µM of corresponding forward and
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reverse primers, and 10 µL of the Thunderbird SYBR Green PCR Master Mix in a
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final reaction volume of 20 µL with the following cycling conditions: 95°C for 2 min,
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40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. After amplification, a
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thermal denaturing cycle at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s was
165
carried out to determine the dissociation curves and verify the specificity of the
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amplifications. All reactions were performed in biological triplicates, and the results
167
were expressed relative to the expression levels of an internal reference gene,
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elongation factor 1 alpha-subunit (TEF1, GenBank accession No. M90077.1) in each
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sample using the 2-∆∆Ct method (Livak and Schmittgen, 2001).
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2.7. Statistical analysis
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The experiment was performed in a completely random design with three
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replications. The data of morphological and physiological characteristics were
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subjected to a two-way analysis of variance (ANOVA) followed by the Duncan’s
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multiple range tests at the 5% probability level using SPSS 16.0.
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3. Results
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3.1. Effect of CdCl2 stress on plant height, root length and relative water content in
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wheat seedlings
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Table 1 Changes of plant height, root length and relative water content in wheat
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seedlings subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Treatment
0h
CK
9.37±1.03bc
6.58±0.21cd
95.02±1.02a
6h
CK
9.47±1.51bc
6.82±0.32cd
94.21±2.04a
Cd stress
9.05±0.53c
6.47±0.62d
78.32±1.25b
CK
9.53±0.54bc
7.03±0.52bc
94.35±1.54a
Cd stress
9.23±0.84c
6.56±0.24cd
66.24±0.98d
CK
10.08±0.62ab
7.32±0.38b
95.67±1.54a
Cd stress
9.47±0.32bc
6.66±0.34cd
71.42±0.84c
10.52±0.84a
8.58±0.52a
96.02±0.68a
9.85±0.25b
6.72±0.62cd
67.28±0.95d
48h
CK Cd stress
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Root length (cm)
Relative water content (%)
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Duration
12h
Plant height (cm)
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175
Data are means ± standard error of 20 replicates, and each mean in a column followed
181
by the same letter indicates that there are no significant differences at 0.05 level
182
according to Duncan’s multiple range test.
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Table 1 illustrates changes in plant height and root length in wheat seedlings
183 184
under Cd stress in a time-dependent manner. Cd stress markedly hindered root and
185
shoot elongation in the growth of wheat seedlings (Table 1). Compared with the
186
control, Cd stress induced a marked decrease (p < 0.05) in the plant height and root
187
length with prolonged Cd stress treatment. To further evaluate the responses to the Cd
9
ACCEPTED MANUSCRIPT stress, the relative water content of wheat seedlings grown at 250 µM Cd solutions for
189
0, 6, 12, 24 and 48 h was estimated. The results indicated that the relative water
190
content was strongly reduced in leaves of wheat seedlings with prolonged Cd
191
exposure. Under the control conditions, it was maintained at 94-96%, whereas it
192
gradually decreased to about 67% after 48 h under Cd stress.
193
3.2. Effect of CdCl2 stress on MDA, Chl a and Chl b concentration in wheat
194
seedlings
65
55 50
195
12
10
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14
0h 6h 12h 24h 48h
8
6
0h 6h 12h 24h 48h
Chl b concentration (mg g-1 FW)
70
8
CK Cd stress
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Chl a concentration (mg g-1 FW)
75
EP
-1
MDA concentration (umol. g FW)
80
45
SC
16
85
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7 6 5 4 3 2
0h 6h 12h 24h 48h
Fig.1. Changes of MDA, Chl a and Chl b concentration in wheat seedlings subjected
197
to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Data are means ± standard error of 6
198
replicates.
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199
Changes of MDA, Chl a and Chl b concentration in wheat seedlings under
200
continuous CdCl2 stress were presented in Fig.1. As shows in Fig.1, with the
201
elongation of Cd stress, MDA concentration gradually increased in wheat seedlings
202
compared with the control. However, CdCl2 stress resulted in a significant decrease in 10
ACCEPTED MANUSCRIPT 203
the concentration of Chl a and Chl b in wheat seedlings compared with the control.
204
3.3. Target prediction of miRNAs in wheat seedlings Table 2 Predicted targets of microRNA in wheat seedlings under Cd stress
205
Function
miR156
Development, flowering time
TC447879
Signaling
TC368630
Squamosa
promoter-binding
Accession number
RI PT
miRNA Predicted targets
protein (SBP) MYB3 transcription factor
pathway
and
SC
miR159
development NAC domain transcription factor
Signaling
pathway,
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miR164
development,
response
root
TC410195
to
oxidative stress
miR398
Cu-Zn
superoxide
(CSD)
Copper homeostasis, response
TC388772
to oxidative stress
Chemocyanin-like protein (CLP)
Stress-related proteins
TC395266
TE D
miR408
dismutase
The identification of miRNA targets is an essential step towards the understanding
207
of their regulatory function. Target prediction of miR156, miR159, miR164, miR398
208
and miR408 was carried out using the Web-based program psRNATarget. As
209
expected, most of the predicted targets for T. aestivum have homologues of known
210
miRNA targets in other plant species, such as squamosa promoter-binding protein
211
(SBP) for miR156, MYB3 transcription factor for miR159, NAC domain transcription
212
factor for miR164, Cu-Zn superoxide dismutases for miR398 or chemocyanin-like
213
protein (CLP) for miR408 (Table 2). Putative target prediction for the miRNAs
214
suggested that they may regulate various developmental processes and involve in
215
oxidative stress tolerance.
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11
ACCEPTED MANUSCRIPT
CK
6h
12h
24h
48h
2
0
CK
6h
12h
6h
12h
24h
3 miR408 2 1 0
CK
48h
12h
24h
48h
12h
24h
48h
4 miR398 3 2 1 0
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1
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0
SC
1
10 miR159 8 6 4 2 0 CK 6h
CK
6h
TE D
217
2
Relative expression
4 miR164 3
Leaf Root
Relative expression
Relative expression
4 miR156 3
Relative expression
3.4. Effect of CdCl2 stress on miRNA expression in wheat seedlings
Relative expression
216
24h
48h
Fig.2. Expression level of five miRNAs in leaves and roots of wheat seedlings
219
subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. The expression level of
220
each miRNA in the control was arbitrarily set at 1. Error bars represent the standard
221
deviations of three biological replicates.
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In order to study the dynamic expression pattern of miRNAs under Cd stress,
222 223
real-time PCR was performed on the leaves and roots of wheat seedlings at different
224
time points (6, 12, 24 and 48 h) after Cd stress for five miRNAs (miR156, miR159,
225
miR164, miR398 and miR408). Under Cd stress, the expression patterns of miR156,
226
miR159, miR164, miR398 and miR408 in leaves were different from those in roots 12
ACCEPTED MANUSCRIPT (Fig.2). The expression of miR159, miR164, miR398 and miR408 were
228
down-regulated in leaves, reaching their lowest expression level at 12 h, and then
229
reached a relatively high expression level at 24 h. miR156 was down-regulated and
230
remained at an extremely low expression level at all time points ( 6, 12, 24 and 48 h)
231
in leaves. In the roots, the expression of miR156, miR159, miR164 and miR398 were
232
rapidly up-regulated from 12 to 24 h, and then steadily decreased at 48 h. miR408 was
233
up-regulated and remained at an extremely high expression level at all time points ( 6,
234
12, 24 and 48 h) in the roots. Thus, expression of miRNAs changed with time and
235
also varied in different organs
236
3.5. Effect of CdCl2 stress on target mRNAs expression in wheat seedlings
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To further understand the mechanism of the interaction between miRNAs and
238
their target genes, the expression levels of miRNA target genes (SBP, MYB, NAC,
239
CSD and CLP) at different time points (6, 12, 24 and 48 h) after Cd stress were
240
examined by RT-qPCR in leaves and roots of wheat seedlings. In the leaves, the
241
transcript levels of MYB, NAC, CSD and CLP, as targets of miR159, miR164,
242
miR398 and miR408 were down-regulated, gradually declined at 6 h, and then
243
steadily increased at 12 and 24 h with the exception of slightly decreased at 24 h in
244
CLP for miR408. The expression levels of MYB and NAC in the roots were sharply
245
up-regulated at 6 h, and then gradually declined. CSD was significantly increased at 6
246
h, then sharply decreased at 12 h and again sharply increased at 24 and 48 h in roots
247
(Fig. 3). The expression of CLP were up-regulated after 24 and 48 h in roots, reaching
248
their highest expression at 48 h. SBP, as targets of miR156 remained at an extremely
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13
ACCEPTED MANUSCRIPT
6
6h
24h
CLP (miR408)
4 2 0
12h
24h
CK
CK
48h
6h
12h
24h
48h
12h
24h
48h
CSD (miR398)
CK
6h
12h
24h
48h
EP
251
6h
5 4 3 2 1 0
48h
TE D
8
CK
12h
MYB (miR159)
RI PT
NAC (miR164)
6h
5 4 3 2 1 0
SC
5 4 3 2 1 0
CK
Leaf Root
M AN U
SBP (miR156)
Relative expression
2.5 2.0 1.5 1.0 0.5 0.0
Relative expression
leaves.
Relative expression
250
Relative expression
low expression level at all time points (6, 12, 24 and 48 h) in roots compared in
Relative expression
249
Fig.3. Expression level of five target mRNAs in leaves and roots of wheat seedlings
253
subjected to 250 µM Cd solution for 0, 6, 12, 24 and 48 h. Corresponding miRNAs
254
are indicated in brackets. The expression level of each target mRNA in the control
255
was arbitrarily set at 1. Error bars represent the standard deviations of three biological
256
replicates.
257
3.6. Correlation between miRNAs and their target mRNAs
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252
14
2 1 0
CK
6h
12h
24h
48h
3
1 0
CK
6h
12h
24h
48h
6
miR159 MYB3
4 2 0
CK
6h
M AN U
CK
Relative expression
D miR159 MYB3
6h
12h
24h
48h
12h
24h
48h
12h
24h
48h
F
Relative expression
E miR408 CLP
CK
6h
12h
24h
48h
8 6
miR408 CLP
4 2 0
CK
6h
TE D
Relative expression
miR156 SBP
2
C
5 4 3 2 1 0
B
RI PT
miR156 SBP
SC
10 8 6 4 2 0
A
Relative expression
Relative expression
3
Relative expression
ACCEPTED MANUSCRIPT
258
Fig.4. Negatively and positively correlated miRNA-target pairs in leaves (a, c, e) and
260
roots (b, d, f) of wheat seedlings grown at 250 µM Cd solutions for 0, 6, 12, 24 and 48
261
h. The expression level of miRNA and corresponding target mRNA in the control was
262
arbitrarily set at 1. Error bars represent the standard deviations of three biological
263
replicates.
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In plants, miRNAs regulate gene expression majorly through targeting mRNAs
264 265
for cleavage; thus, there might be an inverse correlation between a given miRNA and
266
its targets. As expected, there are three miRNA/target pairs which show negative
267
correlation in expression pattern under Cd treatment, including miR398-CSD in root
15
ACCEPTED MANUSCRIPT tissue, and miR159-MYB3 and miR408-CLP in both leaf and root tissues (Fig.4 c-f).
269
Similarly, miR164 showed an inverse relationship with its target NAC in the leaves of
270
wheat seedlings under Cd stress except on the 24 h. However, the expression of SBP
271
was positively correlated to miR156 in both leaf and root tissues of wheat seedlings
272
under Cd stress (Fig.4b). Interestingly, miR164-NAC also showed positive correlation
273
in root tissue of wheat seedlings under Cd stress except on the 24 h. These results
274
suggest that gene regulation may be more complicated under Cd stress condition.
275
4. Discussion
M AN U
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Cadmium (Cd) is a non-redox toxic heavy metal present in the environment. In
277
plants, Cd could disturb various biochemical and physiological processes, leading to
278
cell death and inhibition of growth (Fang et al., 2013; Qiu et al., 2013; Jozefczak et al.,
279
2014). In accordance with previous work, our studies indicated that plant height, root
280
length and chlorophyll content in wheat seedlings were markedly inhibited by Cd
281
stress. Furthermore, Cd stress led to oxidative damage, measured by lipid
282
peroxidation. The increase in MDA content and the decrease in chlorophyll content
283
were observed in wheat seedlings exposure to Cd stress, suggesting that the plant
284
growth inhibition must be, directly or indirectly, attributable to the Cd induced
285
oxidative damage. Much progress has been made in unraveling the complex
286
mechanisms behind Cd stress. The discovery of the role of miRNAs as gene
287
regulators has led to a paradigm shift in the understanding of post-transcriptional gene
288
regulation in plants. Recent findings illustrate that miRNAs might play a crucial role
289
in regulation of plant genes at the posttranscriptional level in responding to metal
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16
ACCEPTED MANUSCRIPT stress (Zhou et al., 2012; Srivastava et al., 2013; Budak et al., 2015b). Ding et al.
291
(2011) found that most of the Cd-responsive miRNAs were down-regulated along
292
with miR156, miR162 and miR390. Tang et al. (2014) also identified expression of
293
miR156 to be suppressed upon exposure to Cd. In the present investigation, we found
294
that miR156 was down-regulated and remained at an extremely low expression level
295
in the leaves of wheat seedlings in response to the elongation of Cd stress, whereas
296
the expression of miR398 were rapidly up-regulated from 12 to 24 h and miR408 was
297
up-regulated and remained at an extremely high expression level at all time points in
298
the roots of wheat seedlings under Cd stress. This result was in agreement with that of
299
Ding et al. (2011), who found that miR398 and miR408 were reported to be
300
up-regulated in response to Cd exposure in the root of rice. Using RT-qPCR, Zhou et
301
al. (2012) also found that expression of miR398 was up-regulated in response to Cd
302
exposure in the root of Brassica napus. These results indicated that miRNAs were
303
responsive to Cd stress differentially in leaves and roots of wheat seedlings,
304
suggesting that miRNAs might play different functions in the regulation of plant
305
tolerance to Cd.
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Notably, several key responsive enzymes for heavy metal detoxification were
306 307
identified as target transcripts for miR398. miR398 was identified to detoxify
308
superoxide radicals by directing the cleavage of its two targets, CSD1 and CSD2
309
(coding cytosolic and chloroplastic Cu/Zn superoxide dismutases, respectively) in
310
Arabidopsis thaliana seedlings exposed to several abiotic stresses (Sunkar et al.,
311
2006). CSD which is one target gene of miR398 as ROS (reactive oxygen species) 17
ACCEPTED MANUSCRIPT scavenger, is important for stress tolerance and survival in plant. Using RT-qPCR,
313
Naya et al. (2014) found that miR398b was decreased and its target gene CSD1 were
314
up-regulated in bean plants under Cu toxicity. The similar results were also detected
315
in mulberry leaf treated with salt stress (Wu et al., 2015). Here, we found that miR398
316
was down-expression in wheat leaves, reaching their lowest expression level at 12 h
317
of Cd exposure, whereas the expression of its target, a CSD gene, was up-expression.
318
The decreased miR398 level in wheat seedlings resulted in accumulation of CSD
319
mRNAs, which were important for catalyzing the dismutation of superoxide radicals
320
into H2O2. Hence, oxidative stress induced miR398 down-regulation was considered a
321
major machinery of detoxifying ROS by regulated its target CSD gene in wheat
322
seedlings exposed to Cd stress. So we inferred that miR398 may involve in oxidative
323
stress tolerance by regulating its target genes to participate in Cd stress.
TE D
M AN U
SC
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312
In plants, miRNAs regulate gene expression majorly through targeting mRNAs
325
for cleavage. Thus, miRNAs usually negatively regulate the accumulation of mRNAs
326
and show an inverse correlation expression pattern in the same plant cells. Kantar et al.
327
(2010) found that the expression profile of miR156 was negatively correlated with its
328
target AV910992 in barley leaves under 8 h of dehydration stress. Our results are
329
consistent with previous findings, indicating that three miRNA/target pairs
330
(miR398-CSD in root tissue, and miR159-MYB3 and miR408-CLP in both leaf and
331
root tissues) showed negative correlation in expression level under Cd stress condition.
332
Interestingly, we observed that the transcript level of NAC was positively correlated
333
with the accumulation of miR164 in the root of wheat seedlings upon the Cd stress.
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18
ACCEPTED MANUSCRIPT This result was in agree with that of Phookaew et al. (2014), who found that the
335
expression of miR164 and its target MesNAC was not inversely regulated in the roots
336
of cassava during water deficit. Furthermore, the expression of miR156 and its target
337
SBP in both leaf and root tissues of wheat seedlings in our study also showed positive
338
correlation under Cd stress. There are several reasons why miRNA/target pairs do not
339
show perfect negative correlation. One potential reason is that abiotic stress is a
340
complicated progress, and it is also possible that the target is regulated by multiple
341
miRNAs, the final expression level of the targets should be combined results for all
342
the miRNAs targeting this gene (Khaksefidi et al., 2015). Another reason is that
343
miRNAs have been demonstrated to establish temporal and spatial expression patterns
344
of target genes (Zhou et al., 2012; Qiu et al., 2015). Therefore, expression patterns in
345
a sample comprising multiple tissue types may conceal a reverse expression level for
346
the miRNA and its targets. Thus, further investigation of the regulatory functions of
347
miRNAs will deepen our understanding on plant response to heavy metal-induced
348
toxicity.
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334
Taken together, the results generated from this study suggested that
350
Cd-responsive miRNAs showed time- and organ dependent expression patterns.
351
Furthermore, a complex regulation relationship between miRNA and target
352
expression profiles was validated in wheat seedlings under Cd stress using RT-qPCR.
353
Comprehensive characterisation of the miRNAs and their target genes will contribute
354
to our understanding of gene regulatory frameworks in plants, and may provide
355
insights into the role of miRNAs and their targets in regulating plant tolerance to Cd
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19
ACCEPTED MANUSCRIPT 356
stress.
357
Acknowledgments This research was supported by the National Science Foundation of China
358
(U1404304 and No. 31500499), Program for Science
Technology Innovation
360
Talents in Universities of Henan Province (16HASTIT019) and Innovative Research
361
Team (in Science and Technology) in University of Henan Province (No.
362
15IRTSTHN020).
363
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Zhou, Z.S., Song, J.B., Yang, Z.M., 2012. Genome-wide identification of Brassica
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napus microRNAs and their targets in response to cadmium. J. Exp. Bot. 63, 4597-4613.
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Highlights
Cd stress severely affected wheat morphological and physiological function
Expression of five known miRNAs and their targets were carried out using
RI PT
RT-qPCR
miRNAs and their targets were differentially expressed in leaves and roots of wheat
SC
miRNA and its target showed complex regulation relationship in response to Cd
EP
TE D
M AN U
stress
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