Function Analysis Of PYR/PYL Interacting Protein POSF21 In Arabidopsis

ABA (Abscisic acid) is a plant hormone containing sesquiterpene structure, which plays important roles in different developmental stages as well as stress responses, such as drought and salt stresses, therefore it is also called "stress hormone". With the discovery of PYR/PYL/RCARs as ABA receptors, great progresses have been made in the ABA signal pathway. As an important plant hormone, the ABA signal regulatory network is complicated. Therefore, it is of vital importance to identify and characterize new components in ABA signal transduction pathway. In this study, proteins which interact with ABA receptors are obtained by screening the cDNA library of Arabidopsis transcription factors via yeast two hybrid approach. POSF21, a bZIP transcription factor which interacts with PYR/PYL, is selected as a candidate protein for the further study. The interactions between POSF21 and PYR/PYR are verified by yeast two hybrid and bimolecular fluorescence complementation. The transgenic lines and mutants are constructed to analyze the roles of POSF21 in ABA signal pathway and plant stress response. The main results are listed as following:(1) The PYR/PYL/RCAR gene family is divided into three subclades according to their gene sequences. Among them, one or two genes from each subclade are selected to construst the DBleu vector and used to screen the cDNA library of Arabidopsis transcription factors. Proteins putatively intereact with ABA receptors were identified, including POSF21, which belongs to the Basic leucine zipper (bZIP) transcription factor.(2) The yeast two hybrid result shows that POSF21 interacts with PYL1, PYL2, PYL5 and PYL13, and the interaction with PYL13 is the strongest. In addition, the interactions are ABA independent. The interactions between POSF21 and PYL1, PYL2, PYL5, PYL13 are also verified by bimolecular fluorescence complementation.(3) The pGFP2-POSF21 vector is constructed and transfected into the Arabidopsis protoplasts. The Subcellular localization result shows that the transcription factor POSF21 is located in the nuclear.(4) Gene expression analysis shows that POSF21 expression level increase 2.86 and 1.49 folds by 10μM ABA for 1 or 6 hour treatments. Cold stress treatment increase the expression of POSF21 for 2.31 and 6.03 folds at 1 and 6 hour, respectively. These results indicate that ABA and cold stress treatment positively modulate the expression of POSF21.(5) The seeds of wild type, transgenic lines and mutants are treated with different concentration of ABA. Compared with wild type, the transgenic lines are hypersensitive to ABA treatment while the mutants are insensitive to ABA treatment.(6) Protoplasts transfection assays are conducted using the pUBQ10::GUS as internal control gene and the pRD29A::LUC as reporter gene. Compared with control (the pGFP2 vector), POSF21-pGFP2 promotes the expression of pRD29A::LUC reporter gene, indicating that POSF21 plays a positive role in ABA signal pathway.