The Function Analysis Of The Interaction Between ROP9and ABI2in ABA Signaling Pathway

ROP9is a member of the type Ⅱ subfamily of ROPs which belong to small GTP-binding proteins. As a negative regulator of ABA （abscisic acid） response, ROP9functions in ABA signaling by inhibiting the seed germination and root growth. ABI2is a member of the protein phosphatases, also plays a negative regulatory role in ABA response. Our previous work displayed that ABI2may interact witht ROP9by yeast two hybrid screening. This research focused on the functional analysis of protein interaction between ROP9with ABI2in the ABA signaling pathway. Our data provide cues in disclosing the fine molecular mechanism of ABA signaling pathways, and in stress-resistant researches of plants.The yeast two hybrid system was used for further identification of protein interaction between ROP9and ABI2. We built the activating state mutant CA-ROP9and continuous inactivating state mutant DN-ROP9by PCR point mutation of ROP9. The normal ROP9and activated CA-ROP9interact with ABI2, and the deactivated DN-ROP9cannot interact with ABI2, suggesting that ROP9interacting with ABI2in an activated state.To further validate the interaction in vivo between ROP9and ABI2in plant cells, a bimolecular fluorescent complementation （BiFC） assay was performed in Arabidopsis mesophyll protoplasts. The results indicated that not only ROP9can interact with ABI2in Arabidopsis cells, but the interaction occurred in the protoplasm membrane as well.The ROP9pro-GUS transgenic plants were obtained by agrobacterium mediated floral dip method. ROP9was found mainly expressing in the root tip, vascular system, stigma, receptacle by GUS staining, but no expressing in the guard cells. Three ROP9RNAi transgenic lines were constructed, and the transcriptional expression levels of ROP9were detected by semi-quantitative RT-PCR analysis. The results displayed that the transcriptional levels were lowered to different degrees, indicating that the ROP9expression were suppressed in the ROP9RNAi lines. Unfortunately, a ROP9T-DNA insertion mutant （salk₁11788） was found to be invalid since the ROP9expression was unchanged, compared with the wild type plants.The ROP9overexpression transgenic plants were constructed by transform ROP9-pEGAD to wild type plants. The expressions of ROP9in the ROP9overexpression plants were increased by semi-quantitative analysis.The ROP9RNAi and the overexpression transgenic plants were cultured on MS plates containing ABA to observe their germination rate and growth status. We found that under treatment of ABA, the germination rate of the ROP9RNAi transgenic plants was lower than that of wild type, while that of the ROP9overexpression plants was higher than wild type.We expressed ROP9, CA-ROP9, DN-ROP9and ABI2proteins in prokaryotic cells, and purified. By incubating these proteins with ABI2to measure the phosphatase activity of ABI2, we found that ROP9and CA-ROP9significantly activated the phosphatase activity of ABI2.In conclusion, our data suggest that ROP9negatively regulating ABA signaling pathways through interacting with ABI2and activating ABI2phosphatase.