Administrative Supplement to R01GM060396 Project Summary of the Parental Award Abscisic acid (ABA) is a central stress hormone in Arabidopsis that down-regulates cell proliferation and causes cell cycle arrest. Early signal transduction networks that down-regulate cell proliferation are of key importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. The long- term goal of this research is to achieve a quantitative understanding of the network of events that mediate early abscisic acid signaling making use of the potent Arabidopsis and guard cell systems. We will characterize newly identified key cellular signaling mechanisms hypothesized to control the early ABA signaling module consisting of ABA receptors, PP2C protein phosphatases and SnRK2 and Raf-like protein kinases. Aims: Aim I. PP2C phosphatases dephosphorylate and shut off SnRK2 protein kinases and thus ABA signaling. Whether SnRK2 kinase re-activation is accomplished by auto-phosphorylation or by other protein kinases remained unknown. Our recent findings show that dephosphorylated SnRK2 kinases cannot re-activate themselves. These findings reveal that re-activation of the SnRK2 kinases, which orchestrate downstream ABA signaling, requires phosphorylation of a specific site in SnRK2 kinases. Via a genetic redundancy screen, we have identified the long-sought SnRK2 re-activation mechanism as Raf-like-kinases (Raf-Ks). We will determine the mechanisms by which Raf-Ks integrate within ABA receptor - PP2C - SnRK2 and osmotic stress signaling using interdisciplinary approaches, including new dynamic SNRK2-FRET activity nano-sensors. Aim II. Through our recent development of an innovative genome-wide artificial microRNA-screening platform, that is designed to silence redundant homologous genes, we have identified previously uncharacterized E3 ligase F-Box proteins that are required for ABA signal transduction. Candidate F-Box targets have been isolated through a dominant F-Box decoy approach. The functions of the identified F-Box proteins and their targets, including GASA signaling peptides, will be determined in the dynamic ABA signaling network through a combination of guard cell signaling, genetic, proteomic, biochemical and ubiquitination analyses. Aim III. Through a forward genetics screen, we have identified a chromatin remodeling factor SYD, as required for ABA responses. We will investigate the hypothesis that rapid ABA-triggered chromatin remodeling plays a critical role in directing the massive ABA-induced SnRK2-driven transcriptional response, based on new findings, including genomic scale rapid differential ABA-induced chromatin accessibility shifts. Mechanisms by which Snf2 chromatin remodeling factors mediate the ABA-induced chromatin accessibility response and how SYD functions in specificity of ABA-induced SnRK2-driven transcriptional reprogramming will be determined. Results from these integrated approaches will provide new and mechan...