IC4R010-miRNA-2010-19796675

Project Title

• Identification of novel stress-regulated microRNAs from Oryza sativa L.

The Background of This Project

• A class of small RNA molecules called microRNAs (miRNAs) has been identified in recent years. MiRNAs are endogenous non-coding RNAs, are 20–22 nucleotides long, and regulate gene expression in eukaryotes ranging from animals to plants [1–6].
• Four Dicer-like enzymes (DCL1–DCL4) are encoded in the genome of Arabidopsis thaliana [20]. It has been shown that DCL1 is involved in miRNA accumulation [21]. However, there is no dcl1 mutant available for rice. A recent study has shown that the loss of function of OsDCL1 transformants could be used to identify miRNAs[22].
• MiR399 was shown to down-regulate UBC24 mRNA accumulation and to be involved in plant responses to Pi starvation in planta [24,25]. MiR393 has also been shown to inhibit the expression of TIR1 to down-regulated auxin signaling and seedling growth under abiotic stress conditions [4,26]. Moreover, miR159 was shown to involved in hormone signaling and dehydration responses in Arabidopsis [27,28]. However, these observations were all shown in Arabidopsis, few stress-related miRNAs have been discovered in rice.Among miRNAs discovered in rice, only two have been found to be related to abiotic stress, miR393 and miR169g, both up-regulated by dehydration [29].

Plant Culture & Treatment

• The seeds (O. sativa L. ssp. Japonica cv 9522) were stimulated to break dormancy and germinated. Uniformly germinated seeds were sown in plates, immersed in water for 1 d at room temperature and 1d at 37 °C, and then transferred to grown chamber with a 16-h light and 8-h dark photoperiod at 25 °C. Two-week-old seedlings were treated with 100 μM ABA, 4 °C, dehydration (exposed to dry air), or 300 mM NaCl individually for 24 h. Untreated seedlings were used as controls. Entire seedlings were collected and immediately transferred into liquid nitrogen. The frozen seedlings were used for generation of small RNA libraries, the miRNA array and for Northern hybridization.

Research Findings

• To identify novel miRNAs from rice, we generated five independent small RNA libraries with size range of 18–28 nucleotides from untreated 2-week rice seedlings and 2-week rice seedlings treated with ABA, 4 °C, dehydration, and NaCl, respectively. After sequencing, about 3900 individual small cDNA sequences between 18 and 28 nucleotides in length were isolated (Table 1).

• The ∼3900 small cDNAs were used to predict Oryza sativa miRNAs and eliminate redundancy. A total of 74 new miRNA candidates were predicted in the cloned small RNA libraries as well as seven previously identified rice miRNAs (Table 2).

• Microarray analysis indicated that some miRNAs were significantly over-expressed in treated seedlings with fold changes N1.5, of which 3 miRNAs were over-expressed under ABA treatment,14 miRNAs were over-expressed under drought treatment, 7 miRNAs were over-expressed under NaCl treatment, and no miRNA overexpressed under cold treatment. Among them, eight candidate miRNAs were shown to be up-regulated under stress treatments(Table 3).Meanwhile, fewer under-expressed miRNAs/candidate miRNAs were identified than over-expressed ones. Eight miRNAs were underexpressed under ABA treatment, four miRNAs were under-expressed under cold treatment, two miRNAs were under-expressed under drought treatment, and four miRNAs were under-expressed under NaCl treatment, and in all of them, the change was b1.5-fold. Our 15 down-regulated candidate miRNAs under stress treatments are shown in Table 3.

• To test whether these candidate miRNAs were regulated by abiotic stress, RNA gel blot analysis was performed on 2-week-old seedlings without stress treatment or treated with ABA, cold, drought or NaCl.The researchers found that the expression of several novel miRNAs is regulated by one or more stress treatments (Fig. 1). Two novel miRNAs, miR2001 and miR2004, are strongly up regulated by NaCl treatments (Figs. 1A and B). miR2005 is strongly up-regulated by dehydration (Fig. 1C) and miR2006 is strongly up-regulated by ABA and NaCl treatment (Fig.1D). miR2002 and miR2007 are slightly up-regulated by drought treatment (Figs. 1E and F), and they were expressed under all stress treatments as well as in the control. Furthermore, miR2003 is not only up-regulated but also down-regulated by different stress treatments,showing a complex expression pattern under stress treatments.miR2003 is slightly up-regulated by NaCl, but slightly down-regulated by cold (Fig. 1G). This is the first time that a miRNA has been shown to have opposite expression patterns by different stress treatments.Interestingly, miR2003 has two mature miRNA products of different sizes. The two mature miRNA products were detected by Northern analysis (Fig. 1G) giving lengths of 20 and 21 nt, respectively. This kind of expression pattern is similar to miR156 [32]. The complex expression patterns of the novel miRNAs indicate that they are functional in the stress response pathways of rice.

'Fig. 1. Detection of the miRNAs by Northern blot with oligonucleotide probes complementary to miRNA sequences. The samples were from rice seedlings, as well as seedlings with different treatments, including ABA, cold, drought and NaCl. The 5S rRNA and tRNA bands were visualized by ethidium bromide staining of gels and served as loading control. Each probe is listed.'

Labs working on this Project

• National Centre for Plant Gene Research, Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080, P.R. China
• State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P.R. China
• National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China

Corresponding Author

• Fan Chen:fchen@genetics.ac.cn.