Interaction Between Abscisic Acid Receptor (PYL3) And Serine/Threonine Protein Phosphatase Type 2C In Maize

Abscisic acid (abscisic acid, ABA) is one of the five groups of plant hormones, and involved in the regulation of plant growth and development, such as seed germination, maturity and dormancy, root growth, leaf blight. As the "stress hormone", it plays a key role in response of plant to drought, cold, salt and other environmental stresses. The physiological functions of ABA have been elucidated gradually since the 1960s, but little has been known about the signal transduction pathway mediated by ABA. The identity of the cell receptor and its downstream components of ABA remains under debate. Two independent research results in Arabidopsis were published on the same issue of "Science" in 2009. Pyrabactin resistance 1-like (PYL) was found to be ABA receptor by structural biology analysis, and verified by yeast two hybridization and immune coprecipitation. After a configuration change caused by its combination with ABA, PYL enters and stops the substrate protein from entering the catalytic active center of PP2C, resulting in the inhibition of the phosphatase activity of PP2C. This result implies that PP2C is the second messenger of ABA signaling pathway.However, researches showed that there are 14 members of the PYL family, and 80 members of 13 subfamilties of the PP2C family in Arabidopsis. Interaction does not occur between each pairs of these members. Some of them are probably not involved in ABA signal transduction. Our previous bioinformatics analysis showed that there are 13 members of 3 subfamilies of the PYL family, and 130 members of 16 subfamilies of the PP2C family in the maize genome. In this study, bioinformatics analysis and prediction was made for ZmPYL3 and ZmPP2C16 proteins. Their interaction was verified by yeast two hybridization and bimolecular fluorescence complementation, and the differential expression of their encoding genes under the induction of ABA was detected by real-time fluorescence quantitative PCR, in order to provide reference for the study on ABA signaling pathway in maize.The major results are as follows:(1) The bioinformatics prediction for the amino acid composition, transmembrane domains, subcellular localization, secondary and tertiary structures showed that ZmPYL3 and ZmPP2C16 are hydrophilic acidic proteins, distributing in the cytoplasm. The main structure elements are the alpha helices and random coils. The phylogenetic tree analysis showed that ZmPYL3 is relative to the member of the third subfamily of the PYL family, and ZmPP2C16 is relative to the six type A members of the PP2C family in response to ABA induction in Arabidopsis thaliana.(2) The bait vector pGADT7-ZmPYL3 and the prey vector pGBKT7-ZmPP2C16 for yeast two hybridization was constructed, and used for co-transformation of yeast strain AH 109. The transformants, together with the co-transforment by the empty vector pGADT7-T and the vector pGBKT7-53 as the positive control, and the co-transformant by the empty vector pGADT7-T and the vector pGBKT7 as the negative control, were screened on the auxotroph medium SD/Trp-Leu-His-Ade, induced by ABA, and stained by 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside. The results showed that the co-transformant by pGADT7-ZmPYL3 and pGBKT7-ZmPP2C16 grew as the positive control, and stained blue under the induction of ABA, indicating that interaction occurs between ZmPYL3 and ZmPP2C16.(3) The fluorescence complementary vectors pSY736-ZmPYL3 (YFPN) and pSY735-ZmPP2C16 (YFPc) were constructed by the insertion of the ZmPYL3 and ZmPP2C16 genes, and used for co-transformation of maize protoplasts. Fluorescence confocal microscopy showed that interaction occurs between ZmPYL3 and ZmPPP2C16 in the cytoplasm.(4) The expression patterns of the ZmPYL3 and ZmPP2C16 genes under the induction of ABA in maize inbred line "B73" was analyzed by using real-time fluorescent quantitative PCR. The result showed that the expression of ZmPYL3 gene is down-regulated and ZmPP2C16 gene up-regulated under the induction of ABA.In conclusion, ZmPYL3 and ZmPP2C16 interact under the induction of ABA. The expression of their encoding genes is down-regulated or up-regulated in response to ABA induction. These results imply that ZmP YL3 is one of the receptor and ZmPP2C 16 is the second component of ABA signaling pathway in maize. They are involved in the ABA signaling in response to adversity stresses.