Mechanism Of MiR159-mediated Drought Resistance In Arabidopsis

Drought has great influence on plant growth and development, thus on food production and security. Therefore, it is of great importance to explore the molecular mechanism of how plants resist drought in order to develop drought-resistant varieties. Previous research showed that miR159 plays an important role in plant growth and development, and it functions downstream of ABA to regulate plant response to drought. However, the question of how miR159-MYBs regulate downstream signals to respond to drought remains unknown. In this study, we explored the possible molecular mechanism of miR159-mediated drought response of miR159ab and miR159 overexpresser based on the results from qRT-PCR, ChIP(Chromatin immunoprecipitation)-PCR and genetics. The results are as follows:1. Compared with wild type Col-0, the loss-of-funciton miR159ab mutant exhibited significantly increased sensitivity to ABA and it displayed significantly higher resistance to drought; miR159ab also exhibited significantly higher percentage of closed stomata. qRT-PCR result indicated that the expression of ABI5 and its downstream genes, EM1, RD19A and EM6 were all significantly upregulated in miR159ab.2. ABA sensitivity test showed that mirl59ab abi5-2 triple mutant had significantly reduced sensitivity to ABA compared with that of mir159ab, and the sensitivity the triple muant is more close to that of abi5-2 single mutant. This result demonstrated that abi5-2 is epistatic to mir159ab in regarding to the sensitivity to ABA.3.Loss-of-function mutation in MYB33 showed reduced sensitivity to ABA, myb33 could rescue the hypersensitivity of mir159ab to ABA. This result indicated that MYB33 is the key gene responsible for the miR159-mediated sensitivity to ABA.4. Sequence analysis of ABI showed that there are multiple putative MYB protein binding sites in ABI5 promoter. ChIP-qPCR result indicated that MYB33 regulates ABI5 expression by directly binding to its promoter. We also constructed a GUS reporter line p?ABI5::GUS with muated MYB33 binding sequence in ABI5 promoter, and recovered transgenic plants using Agrobacterium-mediated flower dipping method.Results above indicated that miR159 regulate ABA signaling pathway throught its target MYB33, which can directly regulate ABI5, to respond to drought response. Our results here provide a theoretical basis for studying the function of downstream genses of miR159-MYB3 in drought resistance.