Functional Analyses Of MiR528in Responses To Arsenite Stress In Rice

Arsenic contamination in rice is a worldwide issue of environmental and food safety. Improvingthe rice plants ability to arsenic tolerance is demonstrated to be one of the effective ways to solve thisproblem. However, only a few of studies related to this issue are reported so far. In rice, a few ofprotein-coding genes but not miRNAs that may be associated with arsenite tolerance have beenidentified. In our previous studies, we used high-throughput sequencing to find that miR528wassignificantly induced in root under arsenite stress in minghui86which is sensitive with arsenite. But it isstill unclear that how miR528responses to arsenite stress.In order to investigate the physiological mechanism and the effect of miR528in arsenite tolerance,we constructed the over-expression vector of miR528, and transformed it into Nipponbare which istolerant with arsenite. We finally obtained miR528over-expressed transgenic plants. In result, there isno significant difference between miR528transgenic and wild-type (WT) rice plants in normal plantingcondition. However, after1day treatment with25μM concentration of arsenite, transgenic plants beganto show curled leaves, yellowing and other typical symptoms of arsenite poisoning, which means theybecome much more sensitive to arsenite than WT. Moreover, it is found that in transgenic plants, theamount of arsenic in root tissue is only48%, while the arsenic transfer rate is2.27times higher than thatin WT plants. The MDA content has greatly increased1.49times as much as that in WT plants.Importantly, the activities of two anti-oxidative enzymes (SOD and POD) are significantly decreased,whereas the Proline contents in roots and leaves of transgenic plants have increased by11.7%and126.2%, respectively.To further elucidate the molecular mechanisms of miR528-regulated arsenite sensitiveness, wequantitatively analyzed the expression level of the predicted target genes and several other genes thatare related to absorption and tolerance of arsenite. As a result, we found that the expression level of fourtargets (LOC_Os07g38290.1、LOC_Os01g03640.1、LOC_Os01g62600.1and LOC_Os07g09690.1) isnegatively correlated with that of the miR528gene, which indicates that these4genes should havemainly been involved in the processes in response to arsenite stress mediated by miR528. Interestingly, after72h arsenite treatment, the expression level of PIP2;4、PIP2;6and PIP2;7is significantly inhibited.All of these show that the over-expression of miR528will have directly or indirectly affected theexpression of its targets and the PIP subfamily member genes that are reported to be related to arsenictolerance, and then leading to the sensitivity of arsenite in rice.It is the first time that we have clarified the biological function of miR528in arsenite tolerance andinvestigated its underlying preliminary physiological and molecular mechanisms in rice, which lays asolid foundation for further studies to reveal the molecular mechanisms of responses for rice to arsenitestress, as well as the interaction of genes network. And it provides excellent target genes resources andintermediate materials for rice resistance breeding or varieties improvement.