| Abscisic acid(ABA),as a typical plant hormone,is involved to various stress responses in plants.Previous studies have shown that OsMPK1 as a positive regulatory factor takes part in ABA-induced antioxidant defense.However,the molecular mechanism by which how OsMPKl is involved in ABA-induced antioxidant defense remains to be determined.In order to determine the molecular mechanism of OsMPK1 in ABA-induced antioxidant defense,we used yeast two hybrid(Y2H)technology to screen the cDNA library of rice leaves by OsMPK1 as a bait protein,and obtained the OsMPK1-interacting protein OsBBX17,a BBox zinc finger protein in rice.Further,we validated the interaction between OsMPK1 and OsBBX17 in vivo and in vitro.Finally,the roles of both OsMPK1 and OsBBX17 in ABAinduced antioxidant defense were investigated.The main results of this study are as follows:We used Y2H assay,LCI and Co-IP assay to verify the authenticity of the interaction between OsMPK1 and OsBBX17 in vivo.All experimental results proved that OsMPK1 does interact with OsBBX17.The Y2H assay and LCI assay further confirmed that OsMPK1 does interact with the N-terminal(1-93aa)of OsBBX17.To determine the subcellular localization of OsBBX17,we transformed pXZP008OsBBX17 in tobacco leaves,onion epidermis cell,and rice protoplasts.Our results showed that OsBBX17 is located in the nucleus.We also found that OsMPK1 is located in the nucleus,cytoplasm,and plasma membrane,and OsMPK1 overlapped with OsBBX17 in the nucleus.We further proved that the interaction between OsMPK1 and OsBBX17 is in the nucleus by BiFC assay.We also analyzed the tissue-specific expression of OsMPK1 and OsBBX17 by qRT-PCR.The results showed that the expression of OsMPK1 was the highest in the roots,followed by that in the leaves,and the expression of OsBBX17 was the highest in the stamens and pistils,which were relatively consistent in the mature leaves.In order to further verify that OsBBX17 is involved in ABA-induced antioxidant defense,we analyzed the expression of OsBBX17 in rice treated with ABA,H2O2,NaCl and polyethylene glycol(PEG)by qRT-PCR.The results showed that the expression of OsBBX17 was up-regulated by all treatments.However,after pretreatments with dimethylthiourea(DMTU)and diphenylene iodonium(DPI),the up-regulation of OsBBX17 expression induced by ABA was inhibited,suggesting that H2O2 is required for ABA-induced expression of OsBBX17 in rice.Using the protoplast transient expression system,the expression and the activities of superoxide dismutase(SOD)and catalase(CAT)were analyzed in the rice protoplasts transiently overexpressing and silencing OsBBX17.The results showed that the expression and the activities of SOD and CAT were up-regulated by OsBBX17 in ABA signaling.In addition,we also analyzed the expression of OsBBX17 in the OsMPK1 mutant and the expression of OsMPK1 in the rice protoplasts transiently overexpressing and silencing OsBBX17.The results showed that the expression of OsBBX17 is regulated by OsMPK1 in ABA signaling.We further analyzed the expression and the activities of SOD and CAT in the OsMPK1 mutant protoplasts transiently overexpressing and silencing OsBBX17,and we found that OsBBX17 could affect the enzyme activity and gene expression of SOD through OsMPK1 in ABA signaling in rice.In this study,we identified the interaction between OsMPK1 and OsBBX17 in ABA signaling.We found that the interaction between OsMPK1 and OsBBX17 is located in the nucleus,and OsMPK1 can act upstream of OsBBX17 to regulate the enzyme activity and gene expression of SOD in ABA signaling.This study is helpful for our understanding on the mechanism of OsMPK1 in ABA signaling in plants. |
