Summary-Analysis of a synthetic ABA agonist uncovers a new family of ABA binding proteins that control signal transduction by directly regulating the activity of type 2C protein phosphatases.-PP2Cs are vital phosphatases that play important roles in abscisic acid (ABA) signaling. Using chemical genetics, we previously identified a synthetic growth inhibitor called pyrabactin. Here we show that pyrabactin is a selective ABA agonist that acts through PYR1, the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We therefore suggest that PYR/PYLs are ABA-receptors that function at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results

Key TermsBiFC: Bimolecular fluorescence complementation; a method for monitoring in vivo protein interactions by formation of a functional fluorescent protein DELLA: family of proteins that function as negative regulators of GA signaling, and are destabilized by GA infrared thermography: method for viewing infrared light emitted by objects due to their thermal condition; excessive transpiration results in "cool" leaves osmocompatible solutes: small molecules accumulated by cells to permit osmotic adjustment to a dehydrating environment without interfering with cellular function pyrabactin: a selective ABA agonist that is not an ABA analog Abstract Abscisic acid regulates numerous developmental processes and adaptive stress responses in plants. Many ABA signaling components have been identified, but their interconnections and a consensus on the structure of the ABA signaling network have eluded researchers. Recently, several advances have led to both the identification of ABA receptors and an understanding of how key regulatory phosphatase and kinase activities are controlled by ABA. A new model for ABA action has been proposed in which the soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases. This model unifies many previously defined signaling components and highlights the importance of future work focused on defining the direct targets of SnRK2s and PP2Cs, dissecting the mechanisms of hormone interactions (i.e. cross-talk) and defining connections between additional known signaling components and this pathway, and determining how many other pathways control ABA signaling.Abscisic Acid: A brief history ABA was discovered in the 1960s. Reviews of its discovery and early chemistry and biology were published in 1969 and 1974 (2, 105). Briefly, ABA was isolated by several groups using activity-guided purification approaches to isolate endogenous growth regulators. Addicott's group at the USDA was searching for compounds isolated from cotton that promote leaf abscission, using a cotyledon abscission assay to guide purification (122). The compound isolated, originally named abscisin II, was also determined to inhibit Avena coleoptile growth (122). ABA's abscission promoting effect was subsequently determined to be partly an indirect consequence of inducing ethylene biosynthesis (26). The Wareing and Cornforth groups in the UK searched for compounds that promote bud dormancy, reasoned that such compounds would be general growth inhibitors, and ultimately isolated dormin as a wheat embryo germination inhibitor present in sycamore leaf extracts. Chemical analyses showed dormin and abscisin II to be the same compound (25), which was ultimately renamed abscisic acid. A third growth inhibitory activity originally isolated from Aegopodium tubers in the 1950s and named -inhibitor (8) was also determined to be ABA (104); thus, the widespread occurrence and importance of ABA as a plant growth regulator was esta...

The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants and plays critical roles in stress resistance, and growth and development1-7. Several proteins have been reported to function as ABA receptors8-13 and many more are known to be involved in ABA signaling3,4,14. However, the identities of ABA receptors remain controversial and the mechanism of signaling from perception to downstream gene expression is unclear15,16. Here we show that by combining the recently identified ABA receptor PYR1, with the protein phosphatase 2C ABI1, the serine/threonine protein kinase SnRK2.6/OST1, and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. The protoplast and test tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase, and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2Cs-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signaling mechanisms and define a minimal set of core components of a complete major ABA signaling pathway.