Expression, Purification And Crystallographic Analysis Of The Abscisic Acid Receptor

Plant hormones are an endogenous chemical substance with vital physiological functions and they are indispensable throughout the whole life. Abscisic acid(ABA), as one of the first confirmed five major plant hormones, is a significant natural plant hormone. ABA plays a major role in a range of different physiological and developmental processes, such as the promotion of abscission of leaves, flowers and fruit, introduction of the buds and tuber dormancy, inhibition of seed germination, promotion of stomatal closure, improvement of adversity resistance and so on. ABA was discovered half a century ago, however, its receptor and working mechanism has been unclear. In recent years, there were various types of ABA receptors reported, such as FCA, CHLH, GCR2 and so on, but all of them has been shrouded in controversy due to related problems including data explanation and biological methods. April 2009, two separate research groups happened to publish similar research achievement in the journal of Science, that is some of the START protein family called the PYR/PYLs/RCAR in Arabidopsis has been described as ABA receptor, which interacts with the downstream protein(phosphatase PP2C) and inhibit its phosphatase activity once the ABA binding and further influence the physiological activities of plants. After the obvious significance and the loads of controversy about the identification of ABA receptor for a long time are both considered, we chose the project, hoping to get the related structures of ABA receptors. They will not only help confirm whether PYR / PYLs / RCAR are the ABA receptors or not, but also explain the mechanism of ABA sensing from the atomic level.In the course of our project, the gene of ABA receptor PYL / PYR / RCAR and the downstream protein PP2C are cloned, then they are recombined into the prokaryotic expression vector (PET series) and highly expressed in Escherichia coil. Through single gene expression and co-expression of different gene combinations, we obtained related single protein and protein complex with high purity for next crystallization. We mainly used hanging drop vapor diffusion method to screen and optimize the crystallization conditions. Finally, we got the high quality crystals for data collection and after phase problem was solved through heavy atom soaking and molecular replacement methods, we determined three high-resolution structure of ABA receptor PYLs in different states, including 1) no combination of ABA, 2) combined with ABA, 3) protein complex combined with ABA and downstream PP2C. By comparison,we find that ABA can bind to the'ligand binding pocket'of PYLs. And the binding of ABA initiate a simultaneous conformational change of the PYLs, which creates appropriate binding sites for PP2C and further induces the downstream physiological response of plants. These results provide valuable information for the future application in agricultural production.