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

Stomatal pores are formed by pairs of specialized epidermal guard cells and serve as major gateways for both CO 2 influx into plants from the atmosphere and transpirational water loss of plants. Because they regulate stomatal pore apertures via integration of both endogenous hormonal stimuli and environmental signals, guard cells have been highly developed as a model system to dissect the dynamics and mechanisms of plant-cell signaling. The stress hormone ABA and elevated levels of CO 2 activate complex signaling pathways in guard cells that are mediated by kinases/phosphatases, secondary messengers, and ion channel regulation. Recent research in guard cells has led to a new hypothesis for how plants achieve specificity in intracellular calcium signaling: CO 2 and ABA enhance (prime) the calcium sensitivity of downstream calciumsignaling mechanisms. Recent progress in identification of early stomatal signaling components are reviewed here, including ABA receptors and CO 2 -binding response proteins, as well as systems approaches that advance our understanding of guard cell-signaling mechanisms.

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone-an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation 1 and climate change 2 . The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes 3,4 . Despite the vital role of guard cells in controlling plant water loss 3,4 , ozone sensitivity 1,2 and CO 2 supply 2,5-7 , the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli 3,4,8 . However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO 2 , ©2008 Nature Publishing GroupCorrespondence and requests for materials should be addressed to J.K. (jaakko.kangasjarvi@helsinki.fi). † Present address: Division of Biology, Imperial College London, London SW7 2AZ, UK. * These authors contributed equally to this work Fig. 3d and Supplementary Fig. 6a. N.N. performed experiments in Fig. 3a and Supplementary Fig. 7. Y.-F.W. performed experiments in Fig. 4 and Supplementary Figs 8 and 9. J.K. and J.I.S. wrote the paper. All the authors discussed the results, and commented on and edited the manuscript.The primary microarray data reported has been deposited with the ArrayExpress database under accession number E-MEXP-1388.Reprints and permissions information is available at www.nature.com/reprints. 8,11 by mediating anion efflux and causing membrane depolarization, which controls K + efflux through K + channels. So far, none of the candidates for plant anion channels -the plant homologues to the animal CLC chloride channels -has been localized to the plasma membrane 10 , and the first plant CLC channel that was functionally characterized encodes a central vacuolar proton/nitrate exchanger 12 , rather than an anion channel. Thus, despite their proposed importance in several physiological and stress responses in plants 8,10,11 , the molecular identity of the guard cell plasma membrane proteins that mediate anion channel activity has remained unknown. NIH Public AccessIn a mutant screen for O 3 sensitivity, a series of Arabidopsis ethyl methanesulphonate (EMS) mutants called radical-induced cell death (rcd) was identified 13,14 . One of them, a recessive mutant originally referred ...

The plant hormone abscisic acid (ABA) regulates many key processes in plants, including seed germination and development and abiotic stress tolerance, particularly drought resistance. Understanding early events in ABA signal transduction has been a major goal of plant research. The recent identification of the PYRABACTIN (4-bromo-N-[pyridin-2-yl methyl]naphthalene-1-sulfonamide) RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family of ABA receptors and their biochemical mode of action represents a major breakthrough in the field. The solving of PYR/RCAR structures provides a context for resolving mechanisms mediating ABA control of protein-protein interactions for downstream signaling. Recent studies show that a pathway based on PYR/RCAR ABA receptors, PROTEIN PHOSPHATASE 2Cs (PP2Cs), and SNF1-RELATED PROTEIN KINASE 2s (SnRK2s) forms the primary basis of an early ABA signaling module. This pathway interfaces with ion channels, transcription factors, and other targets, thus providing a mechanistic connection between the phytohormone and ABA-induced responses. This emerging PYR/RCAR-PP2C-SnRK2 model of ABA signal transduction is reviewed here, and provides an opportunity for testing novel hypotheses concerning ABA signaling. We address newly emerging questions, including the potential roles of different PYR/RCAR isoforms, and the significance of ABA-induced versus constitutive PYR/RCAR-PP2C interactions. We also consider how the PYR/RCAR-PP2C-SnRK2 pathway interfaces with ABA-dependent gene expression, ion channel regulation, and control of small molecule signaling. These exciting developments provide researchers with a framework through which early ABA signaling can be understood, and allow novel questions about the hormone response pathway and possible applications in stress resistance engineering of plants to be addressed.

The phytohormone abscisic acid (ABA) acts in seed dormancy, plant development, drought tolerance and adaptive responses to environmental stresses. Structural mechanisms mediating ABA receptor recognition and signaling remain unknown, but are essential for understanding and manipulating abiotic stress resistance. Here we report structures of PYR1, a prototypical PYR/PYL/RCAR protein that functions in early ABA signaling. The crystallographic structure reveals an α/β helix-grip fold and homodimeric assembly, verified in vivo by co-immunoprecipitation. ABA binding within a large internal cavity switches structural motifs distinguishing ABA-free “open-lid” from ABA-bound “closed-lid” conformations. Small angle X-ray scattering suggests that ABA signals by converting PYR1 to a more compact, symmetric closed-lid dimer. Site-directed PYR1 mutants designed to disrupt hormone binding lose ABA-triggered interactions with type 2C protein phosphatase partners in planta.