Elsevier

Gene

Volume 459, Issues 1–2, 1 July 2010, Pages 39-47
Gene

Profiling of cold-stress-responsive miRNAs in rice by microarrays

https://doi.org/10.1016/j.gene.2010.03.011Get rights and content

Abstract

MicroRNAs (miRNAs) are small single-stranded RNAs with a length of about 21 nt; these non-coding RNAs regulate developmental and stress responses in plants by cleaving mRNAs. Cold stress is one of the most severe abiotic stresses and adversely affects rice yields by restraining sowing time, causing tissue damage, and stunting growth. Although many miRNAs have been identified in rice, little is known about the role of miRNAs in the response to cold stress. In this study, we identified 18 cold-responsive rice miRNAs using microarrays. Most were down-regulated. Members of the miR-167 and miR-319 families showed similar profiles. Intriguingly, members of miR-171 family showed diverse expression patterns. Three miRNAs derived from transposable element sequence were clustered within an intron and proved to be co-transcribed with the host gene only under cold stress. The existence of hormone-responsive elements in the upstream regions of the cold-responsive miRNAs indicates the importance of hormones in this defense system mediated by miRNAs. Two miRNA target pairs validated by 5′ RACE showed opposite expression profiles under cold stress. Finally, the predicted stress-related targets of these miRNAs provided further evidence supporting our results. These findings confirm the role of miRNAs as ubiquitous regulators in rice.

Introduction

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs of approximately 21 nt in length; these RNAs regulate gene expression at the post-transcriptional level through base-pairing to target mRNAs (Bartel, 2004, Kim, 2005, Jones-Rhoades et al., 2006, Kim & Nam, 2006). miRNAs are generated by polymerase II as pri-miRNAs, which fold to form extensively double-stranded stem–loop structures. In plants, miRNA/miRNA* duplex are formed through multi-steps in the nucleus that requires Dicer-like1 (DCL1). The mature miRNAs are incorporated into the RISC (RNA-induced silencing complex) and guide cleavage of the complementary target mRNAs by the endoribonuclease Argonaute (AGO) proteins (Bartel, 2004, Kim, 2005). Plant miRNAs regulate diverse process, including development, signal transduction, stress response, pathogen invasion, and their own biogenesis (Mallory & Vaucheret, 2006, Sunkar et al., 2007, Liu et al., 2008, Liu et al., 2009, Zhao et al., 2009). Many plant miRNAs have been identified by cloning, bioinformatics, and high-throughput sequencing approaches (Griffiths-Jones et al., 2008, Lu et al., 2008a, Lu et al., 2008b). However, little is known about their functions.

Cold stress, including chilling and frost, affects crop yields worldwide by causing tissue injury and delayed growth (Mahajan and Tuteja, 2005). The signaling pathways used by plants in responding to cold stress and the key genes for modifying the response are of interest. The best characterized regulon of cold-stress responses in plants contains transcription factor CBF/DREB and its cold-inducible target genes, known as COR (cold-regulated gene), KIN (cold-induced gene), RD (responsive gene to dehydration), or LTI (low-temperature-induced gene) (Baker et al., 1994, Dubouzet et al., 2003, Rabbani et al., 2003, Beer & Tavazoie, 2004, Yazaki et al., 2004, Morsy et al., 2005, Agarwal et al., 2006, Benedict et al., 2006, Ito et al., 2006). The precise hierarchical organization of the global network has not been defined. RNA gel blots have been used to analyze expression of some miRNAs in seedlings treated with cold. The expression levels of miR-393 and miR-319c are up-regulated by cold (Sunkar and Zhu, 2004). Liu et al. (2008) and Lu et al. (2008b) used microarray to analysis all known miRNAs expression profiles under cold stress in Arabidopsis and Populus, respectively. Zhou et al. (2008) developed a transcriptome-based approach to annotating cold-inducible miRNAs in plants. Jian et al. (2009) identified several cold-regulated miRNAs by direct cloning and sequencing. Currently, deep sequencing led to the identification of 28 cold response miRNAs in Brachypodium (Zhang et al., 2009). These results show that plant miRNAs can be regulated by cold stress and may function in critical defense systems for cold fitness.

Rice (Oryza sativa) is one of the most economically important crops in the world and its yield is frequently affected by cold stress. Rice is a model system for cold-sensitive plants as the complete genome sequence is known. Currently, 414 rice miRNAs have been identified (http://microrna.sanger.ac.uk) (Griffiths-Jones et al., 2008), of which 37% are non-conserved in Arabidopsis and the other plant species. In this study, we comprehensively examined the expression profiles of rice miRNAs under cold stress and analyzed the regulation patterns of cold-responsive miRNAs based on the rice genome data. The results reported here further our understanding of how the expression levels of these miRNAs are regulated and provide the insight into the role of miRNAs in the rice response to cold stress.

Section snippets

Plant materials

Non-dormant seeds were sterilized in 10% NaClO, rinsed in sterile water, and germinated in moist filter paper. The uniformly germinated seeds were sown in 96-well plates with the bottoms removed. The plates were immersed in a box containing Yoshida's culture solution and grown under a 12-h light (28 °C)/12-h dark (22 °C) photoperiod (photo intensity 240 lm photos m2⋅s1). The culture solution was renewed every 2 days. Seedlings were allowed to grow until the prophyll emergence stage (S3) for 10 

Identification of cold-responsive miRNAs in rice

The cold environment was mimicked by 4 °C incubation. The rice seedlings were kept in the cold for 0, 0.5, 1, 3, 6, 9, 12, and 24 h prior to analysis. Among 316 rice probes presented on the array, 148 miRNAs had detectable expression in analyzed seedlings. Then the miRNAs that gave low signals were removed leaving 64 miRNAs. Significance analysis (t-test, p-value < 0.05) and a criterion of fold change of > 1.5 were used to examine the effects of cold-stress treatment at each time point (Supplemental

Discussion

Cold stress impacts crop yields in many regions of the world and is the primary cause of crop loss. Many cold-induced genes have been identified (Baker et al., 1994, Dubouzet et al., 2003, Rabbani et al., 2003, Beer & Tavazoie, 2004, Yazaki et al., 2004, Morsy et al., 2005, Agarwal et al., 2006, Benedict et al., 2006, Ito et al., 2006). However, little is known about the relation between the miRNAs and cold response (Liu et al., 2008). Our study showed that 18 miRNAs respond rapidly to the cold

Acknowledgments

We thank Ms. Qiulei Lang and Mr. Peng Wu (LC-Bio, Hangzhou, China) for technical assistance. This work was supported by the Key Research Plan of Heilongjiang Province (GA06B103-3), the Innovation Research Group of NEAU (CXT004), the “863” project (2008AA10Z153), and the Scientific Research Foundation of NEAU.

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    These authors contributed equally to this work.

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