| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 107 === The Clade A protein phosphatase 2C Highly ABA-Induced 1 (HAI1) plays an important role in stress signaling yet little information is available on HAI1-regulated phosphoproteins. Quantitative phosphoproteomics identified phosphopeptides of increased abundance in hai1-2 in unstressed plants and in plants exposed to low water potential (drought) stress. One of the phosphosites putatively regulated by HAI1 was S313/S314 of AT Hook-Like10 (AHL10), a DNA binding protein of unclear function. HAI1 could directly dephosphorylate AHL10 in vitro and the level of HAI1 expression affected the abundance of phosphorylated AHL10 in vivo. AHL10 S314 phosphorylation was critical for restriction of plant growth under low water potential stress and for regulation of Jasmonic Acid and Auxin-related gene expression as well as expression of developmental regulators including Shootmeristemless (STM). These genes were also mis-regulated in hai1-2. AHL10 S314 phosphorylation was required for AHL10 complexes to form foci within the nucleoplasm, suggesting that S314 phosphorylation may control AHL10 association with the nuclear matrix or with other transcriptional regulators. These data identify a set of HAI1-affected phosphorylation sites, show that HAI1-regulated phosphorylation of AHL10 S314 controls AHL10 function and localization and also indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses. As this suggested a role for AHL10 phosphorylation in modulating its protein-protein interactions, a yeast two-hybrid screen was conducted to identify AHL10- interacting proteins. This screening identified Ribosomal RNA-processing Protein 6 Like1 (RRP6L1), a protein involved in epigenetic regulation and RNA-directed DNA methylation. In vivo and in vitro assays suggested that AHL10 phosphorylation promotes its interaction with RRP6L1. AHL10 and RRP6L1 regulate an overlapping set of genes during low water potential stress. Chromatin immunoprecipitation found that AHL10 associated with promoter regions of STM, the auxin-amido synthase gene WES1 and JA synthesis gene Allene Oxide Synthase (AOS1). These results suggest that AHL10-RRP6L1 interaction could be important for targeting of these proteins to specific promoter regions.
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