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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 established by the late 1960’s. ABA was subsequently documented as an endogenous regulator in some fungi and a variety of animals (for review see: (114, 169)). Over the past 30 years, molecular genetic, biochemical and pharmacological studies have identified over 100 loci and numerous secondary messengers involved in ABA signaling, including Ca 2+ , reactive oxygen species (ROS), cyclic nucleotides, and phospholipids. Due to space constraints, we have focused on recent developments linking ABA perception to known signaling elements, and have excluded discussion of second messengers in ABA signaling, an important topic that has been reviewed extensively elsewhere (19, 29, 48, 145). Major physiological roles of ABA Many key aspects of ABA’s physiological effects were established shortly after its discovery. Wareing’s group (159) noted the ability of ABA to antagonize several GA effects, including promotion of seedling growth and a-amylase synthesis. A role for ABA in water relations (specifically guard cell responses) was suggested by the observations that the wilty tomato flacca mutant was deficient in ABA, its phenotype could be rescued by exogenous ABA treatment (58, 157), and that ABA applications caused stomatal closure in Xanthium(67). Coupled with observations that ABA levels rise substantially after water deprivation, a physiological model for ABA’s critical role in guard cell regulation emerged in the early 1970s. The role of ABA in these processes has been extensively studied and reviewed (118, 145, 152). Root growth maintenance during water deficits is also a key adaptive response that maintains adequate water supply. This process involves ABA and is controlled by the concerted action of different hormonal signaling pathways (148). In cases where water uptake and water loss cannot be balanced by primary adaptive responses, different mechanisms may be exploited to avoid and / or tolerate dehydration, which involve regulation of stressresponsive gene expression through ABA and other signaling pathways (183). In particular, the accumulation of osmocompatible solutes and the regulated synthesis of dehydrins and LEA proteins play important roles in both retaining water and protecting proteins and membranes under stress (59, 165). Recently, ABA has been found to affect pathogen responses; its effects range from promoting resistance by inhibiting pathogen entry via stomata to increasing susceptibility by interfering with defense responses mediated by other signaling pathways (reviewed in (160)). In addition to its role in plant abiotic and biotic stress responses, ABA regulates important aspects of plant growth and development, such as embryo and seed development, promotion of seed desiccation tolerance and dormancy, germination, seedling establishment, vegetative development including heterophylly as well as general growth, and reproduction. For instance, severe ABA-deficient or ABA-insensitive mutants display a stunted phenotype even under well-watered conditions and are severely impaired in seed production (7, 15, 34, 112). Chemical features necessary for ABA action Shortly after its discovery, the structure of ABA was deduced by a combination of spectroscopic methods (121) and ultimately confirmed by chemical synthesis (24). The molecular structure of abscisic acid has a number of features that are important for biological activity in plants (Figure 1). One such feature is the side chain of the ABA molecule, which contains two double bonds conjugated to the carboxylic acid; the configuration of the double bond adjacent to the ring is trans and that proximal to the acid group is cis. On exposure to ultraviolet light, biologically active 2-cis,4-trans ABA is reversibly isomerized to the inactive trans form 2-trans, 4-trans ABA. Thus, under low light conditions 2-trans, 4-trans ABA can be employed as an inactive analog for studies to probe biological processes regulated by ABA. Under high light conditions or for long-term studies, the equilibrium between 2-trans, 4-trans ABA and the 2-cis, 4-trans ABA may shift to afford significant quantities of the active form. The active and inactive forms are readily distinguished by HPLC or GC analyses. The importance of ABA’s stereocenter and the biological activity of unnatural R-(–)-ABA versus natural S-(+)-ABA has been investigated since the discovery of the plant hormone (reviewed in (88, 178)). In many assays, including stomatal closure, (–)-ABA is weakly active. In seed germination studies in cereals (166) and Arabidopsis (115), applied (–)-ABA has been found to have comparable activity to (+)-ABA ( reviewed in (88)). Recent microarray studies in which ABA was supplied to Arabidopsis plants have shown that (–)ABA regulates most (+)-ABA regulated genes (55). In structure/activity studies where stereoisomeric forms of ABA analogs have been compared, the (–)-ABA analogs have been found to be inactive. Genetic studies have shown that (–)-ABA’s action in Arabidopsis seeds requires a functional ABA signaling pathway (115). To explain the activity of (–)-ABA, Nambara et al. (115) hypothesized the existence of dual selectivity ABA-receptors. Since some members of the recently discovered PYR/PYL/RCAR protein family can bind or respond to both stereoisomers (128, 142), this new protein family contains candidates for the dual selectivity receptors hypothesized by Nambara et al. There are several phenomena that can complicate the interpretation of whole plant structure activity relationship studies. For example, (–)-ABA supplied to plant tissues can trigger biosynthesis of natural (+)-ABA, which can accumulate and cause ABA processes to be induced, as documented in induction of the heterophyllous switch in Marselia quadrifolia(88). Furthermore, (–)-ABA is metabolized more slowly than natural (+)-ABA, so the apparent activity of (–)-ABA is magnified. Thus, before drawing conclusions about the physiological mechanism(s) underlying bioactivity of (–)-ABA or other analogs, characterizing their effects on endogenous ABA biosynthesis and / or enlisting the use of mutant strains deficient in ABA biosynthesis should be considered. Because of the confounding effects of metabolism, re-visitation of structure activity relationships using purified receptors in ligand-binding assays should be a productive line of future investigation. Over 30 years ago, Milborrow proposed a structural hypothesis for the activity of (–)-ABA, based on the near symmetry of ABA (105). The (–)-enantiomer can be rotated about its lengthwise plane to effectively “flip” the positions of its 7’ methyl and 8’,9’ dimethyl ring substitutions (Figure 1) and still leave the relative positions of the other polar functional groups relatively intact within a binding pocket. ABA analogs lacking either the 8’, 9’ or 7’ methyl groups have been synthesized to explore this hypothesis (167). These studies showed that the 7’ methyl group is critical to bioactivity. Ultimately, structural investigations of receptors bound to each stereoisomer will be required to resolve this long standing hypothesis for the bioactivity of (–)-ABA. Additional molecules have been identified that may act on ABA receptors. These include analogs of ABA altered at either the 7', 8' or 9'-carbon atoms (reviewed in (178)), an ABA analog that acts as an ABA antagonist and inhibits expression ABA-induced genes in Brassica napus microspore-derived embryos (170) and pyrabactin, a selective ABA agonist that acts through the ABA receptor PYR1 but does not structurally resemble ABA (128) (Figure 1). The structural diversity of these and other ABA signaling modulators raises interesting questions about the nature of the receptor(s)’ ABA-binding pocket(s). ABA Binding Proteins Implicated in Signaling Microinjection studies and treatments with impermeant ABA analogs in the 1990s suggested that ABA may have both intracellular (3, 147) and extracellular sites of perception (4, 42, 61, 146) and several proteins with the properties of either plasma membrane or intracellular ABA receptors have been described (90, 97, 126, 128, 133, 149). We summarize the current data on these proteins below. FCA The first ABA binding protein isolated (ABAP1) was identified in barley aleurone by virtue of its ability to bind an anti-idiotypic ABA antibody (i.e. an antibody against an ABAantibody) (134). Sequence analysis showed this was related to Arabidopsis FCA, an RNA binding protein with a well documented role in regulation of flowering time (133). However, ABAP1 and FCA differ in several fundamental aspects: ABAP1 was initially described as associated with the plasma membrane, whereas FCA is a nuclear protein with two conserved RNA binding domains not present in ABAP1. Attempts to reproduce the FCA ABA-binding data using radioligand binding assays were unsuccessful (138). Based on these findings, the FCA report was retracted. Risk et al. have noted that the filter-based ligand-binding assay employed in the FCA and other receptor studies is prone to artifacts arising from incomplete removal of non-protein bound ABA (137, 138).