IC4R001-miRNA-2011-21362738

Project Title

Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa)

The Background of This Project

Figure 1. GWA Analysis of Al Tolerance within and across Rice Subpopulations.

• MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate specific target mRNAs at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptations to environmental stresses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants. To investigate the responsive functions of miRNAs under Cd stress, miRNA expression in Cd-stressed rice (Oryza sativa) was profiled using a microarray assay. A total of 19 Cd-responsive miRNAs were identified, of which six were further validated experimentally. Target genes were also predicted for these Cd-responsive miRNAs, which encoded transcription factors, and proteins associated with metabolic processes or stress responses. In addition, the mRNA levels of several targets were negatively correlated with the corresponding miRNAs under Cd stress. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a novel molecular mechanism of heavy metal tolerance in plants.
• Cadmium (Cd) is a widespread heavy metal pollutant in the environment, originating mainly from industrial processes and phosphate fertilizers. Cd can be easily taken up by plants, resulting in various toxicity symptoms,such as chlorosis, wilting, growth reduction, and cell death. The cellular toxicity caused by Cd might result from interactions with the carboxyl or thiol groups of proteins and the generation of reactive oxygen species (ROS), inducing oxidative stress. Plants possess a range of mechanisms involved in Cd detoxification, including metal transport, chelation, and sequestration. For example, the natural resistance-associated macrophage protein (Nramp) transporter was reported to function in Cd uptake in Arabidopsis thaliana, whereas the intracellular chelation of Cd by glutathione (GSH) and phytochelatins (PCs) represented a ubiquitous detoxification strategy adopted by many plant species. Detoxification can also be achieved by proline synthesis. Although some of the molecules involved in Cd tolerance have been identified, the regulatory mechanisms involved are still largely unknown.

Plant Culture & Treatment

• Wild-type rice Zhonghua 11 (O. sativa L. subsp. japonica) seeds were sterilized with 3% sodium hypochlorite for 20 min and washed thoroughly with distilled water. After germination at 30 ?C in the dark, the seeds were grown under a 13 h light (29 ?C)/11 h dark (22 ?C) photoperiod. For Cd stress experiments, 7-day-old seedlings were exposed to 60 lM CdCl 2 for 24 h. Roots were harvested as a pool for each sample at 0, 3, 6, 12, and 24 h after Cd treatment. Seedlings not treated with Cd served as controls. Total RNA was extracted from 7-day-old rice roots with Trizol reagent (Invitrogen).

Research Findings

• In this study, miRNA microarrays were used to investigate the expression patterns of miRNAs in rice seedlings under Cd stress (Figure. 1). In general, many miRNAs showed significant alterations in expression in response to Cd treatment. Nineteen miRNAs belonging to 10 families were identified to be responsive to Cd. It was found that miRNA members of the same family had similar expression profiles, probably owing to highly homologous sequences, which were difficult to distinguish even by using hybridization-based methods. Among the 19 Cd-responsive miRNAs, only miR528 was significantly up-regulated; the other 18 miRNAs (miR162a, miR168a, miR168b, miR166m, miR166i, miR166e, miR166k, miR166g, miR171b, miR171a, miR171g, miR396d, miR390, miR156l, miR156k, miR156a, miR1432, and miR444b.1) were down-regulated by Cd stress. These down-regulated miRNAs belong to the miR162, miR168, miR166, miR171, miR396, miR390, miR156, miR1432, and miR444 families.

Figure 2. Haplotype analysis of the Nrat1 gene region.

• To investigate whether the predicted target genes were actually regulated by Cd-responsive miRNAs, the expression levels of three targets [AGO (the target of miR168), HD-Zip (the target of miR166), and RLK (the target of miR390)] were measured in rice seedlings exposed to 60 lM Cd from 0 h to 24 h by using qPCR. As shown in Figure. 3A and B, the abundance of AGO and HD-Zip transcripts increased, whereas the expression of miR168 and miR166 was inhibited from 3 h to 24 h after exposure to Cd. Additionally, changes in miR390 abundance had a negative effect on the abundance of RLK at the four treatment time points during Cd stress (Figure. 3C). The profiles of miRNAs (miR168, miR166, and miR390) and target transcripts (AGO, HD-Zip, and RLK) were complementary, but not exactly opposite, to each other (i.e. the time point showing the higher level of miRNA accumulation did not coincide with the lower accumulation of target mRNA). Together, the negatively correlated expression patterns between miRNAs and their targets further validated the regulatory role of miRNAs on their targets, and indicated the role of miRNAs in Cd tolerance of rice seedlings.

Labs working on this Project

• State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, China
• Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China JiLiang University, Hangzhou 310018, China

Corresponding Author

• Cheng Zhu (pzhch@cjlu.edu.cn)