Preliminary Investigation Of The Biological Function Of MiR528 In Regulation Of Salt Stress Responses In Rice (Oryza Sativa L.)

The growth and development of rice plants were severely challenged by salt stress in nature,hence leading to the decrease of rice production. Accordingly, it is very significative to identify and characterize salt stress-responsive genes and further uncover its underlying physiological and molecular mechanisms. A growth number of evidence shows that a kind of small non-coding RNA molecule - miRNA plays important roles in regulation of stress responses in plants. In our previous study, we found that miR528 should be involved in the regulation of arsenite tolerance in rice. However, whether the corresponding miRNA plays a role in responses to salt stress still remains unknown. Therefore,on the basis of the obtained transgenic rice plants that separately carries the overexpressed and inhibited miR528 constructs, and the transgenic plants carrying the construct of the promoter of MIR528 with the fusion of GUS, this study aims at investigating the miR528-mediated salt stress response in rice. The main results are as follows:1. Compared to the wild-type Nipponbare (WT) and the miR528-inhibited transgenic plants,miR528-overexpressed plants were more sensitive under the treatment of the concentration of 120 mM NaCl, and exhibited some typical symptoms of salt stress, including the strong inhibition of root growth, and significant reduction of contents of chlorophyll and soluble proteins. However,the contents of MDA and proline increased remarkably in leaves of miR528-overexpressed plants.In addition, the activities of oxidant enzymes exhibited distinct responses to salt stress in leaves and roots of different tested genotypes.2. Under salt stress, the content of proline increased significantly, but the contents of glutamic acid and ornithine showed the reverse in leaves of different tested genotypes.Furthermore, the expression analysis showed that the key protein-coding genes OsP5CS and OsOAT that were involved in the regulation of different proline synthetic pathways were lowly expressed in miR528-overexpressed plants after 6 d of salt treatment, compared to WT and miR528-inhibited plants. Nonetheless, the expression of the OsProDH gene that was crucial for regulating the degradation of proline was strongly down-regulated in leaves of both tested rice.3. The histochemical staining results showed that the promoter of MIR528 was constitutively expressed in riee roots, stems, and leaves. However, the extent of GUS staining of tested tissues decreased along with the increase of treatment time of salt stress. In addition, both the enzymatic activity and the gene expression level of GUS decreased too, indicative of a decreased tendency of the miR528 expression under salt stress.4. The expression analysis of seven published salt-tolerance-related genes showed that two of them including OsDHODH1 and OsCOIN were highly induced by salt, but their expression in miR528-overexpressed plants were lower than that in WT and miR528-inhibited plants. Moreover,the expression of OsCDPK and OsMYB91 was down-regulated in miR528-overexpressed plants,but was significantly up-regulated in WT and miR528-inhibited plants.Based on these above observations, we infer that under salt stress the expression of some salt-tolerance-related genes should be affected in an indirect way by the overexpression of miR52 8 in rice that might further trigger the changes in the downstream salt-stress signal transduction as well as production of osmotic adjustment substances, and then caused the changes in salt-stress tolerance in rice. These results will be useful for further investigating the molecular mechanisms of salt-stress responses in rice and for stress-resistant rice breeding.