The hunt for the receptor for abscisic acid, initially marked by false starts and lingering doubts, has met with success. Converging studies now reveal the details of how this plant hormone transmits its message.
To survive and flourish in rapidly changing environmental conditions, plants use a complex family of hormones to regulate growth and reproduction. One plant hormone, abscisic acid (ABA), coordinates responses to stressors such as drought, extreme temperature and high salinity, as well as regulating non-stress responses including seed maturation and bud dormancy. Because of its essential function in plant physiology, targeting the ABA signalling pathway holds enormous promise for future application in agriculture. A burst of papers, four in this issue1,2,3,4 and two published elsewhere5,6, provide structural and mechanistic insights into ABA signalling that take us closer towards realizing this promise.
Plant hormone research has been the subject of recent attention owing to the identification of receptors for two other hormones, gibberellin and auxin, that like the current model for ABA have soluble (non-membrane-bound) receptors. But identification of the ABA receptor has been unusually challenging. Since 2006, several proteins have been suggested as possible ABA receptors, but their exact roles in ABA signalling remained controversial7. In May 2009, a new family of proteins was reported as candidate ABA sensors8,9. Members of this family, known as PYR/PYL/RCAR proteins, were found to bind ABA and inhibit the activity of specific protein phosphatase enzymes, the type 2C plant PP2Cs, which were previously implicated in the ABA response. Six independent groups1,2,3,4,5,6 now simultaneously define the structural and functional mechanisms by which ABA is sensed by this newly identified protein receptor.
Fujii et al.3 (page 660 of this issue) report elegant reconstitution assays that pinpoint the minimal pathway sufficient to recapitulate ABA signalling in plant protoplasts (cells with their cell wall removed) and in vitro, tying together the receptor, phosphatase and downstream kinase signalling components. Phosphatases and kinases exert opposite regulatory effects by respectively removing and adding phosphate groups to substrate proteins. As described in Figure 1, in the absence of ABA, the phosphatase PP2C acts as a constitutive negative regulator of a family of kinases (SnRK2) whose autophosphorylation is required for kinase activity towards downstream targets. When ABA binds, it enables the PYR/PYL/RCAR receptor to subsequently bind to and repress PP2C. Sequestration of PP2C permits auto-activation of the kinase, which phosphorylates downstream transcription factors and facilitates transcription of ABA-responsive genes. This pathway is attractive in its simplicity and offers a seamless complement to the known body of ABA literature.
The crucial step in this pathway is perception of ABA by the PYR/PYL/RCAR proteins and the inhibition of PP2C by the ligand (ABA)-bound receptor. Five crystallographic studies1,2,4,5,6, including those on pages 602, 609 and 665, have converged to paint a complete picture of these events. Together, they reveal the atomic structures of several PYR/PYL/RCAR proteins in different functional states. Studies by Melcher et al.1, in particular, have captured the structures of PYL2 in all critically relevant forms (ligand-free, ligand-bound and ligand/phosphatase-bound), and allow detailed analyses of the conformational changes that PYL2 undergoes on binding first to the hormone, and subsequently to the phosphatase.
The first highlight of these studies is the ligand-binding mechanism, which is regulated by the opening and closing of a gating loop on the ABA-binding pocket. In the absence of the hormone, the PYR/PYL/RCAR proteins present an open and accessible cavity. Two flexible surface loops, together with several nearby structural elements, guard the entrance of the cavity. When this water-filled pocket is occupied by ABA, one of the loops closes like a gate, approaching the other loop and sequestering ABA within the pocket (Fig. 2). Because most of the amino-acid residues in contact with ABA, as well as the sequences of the two entrance loops, are evolutionarily conserved among all PYR/PYL/RCAR proteins, the ABA-binding and open-to-close gating mechanisms are likely to be common for all members of the receptor family.
Three of the five groups1,2,6 extend their crystallographic studies to describe the architecture of the complex formed between an ABA-bound receptor and PP2C. These structures reveal that the ABA-bound receptors dock onto PP2Cs through a large complementary interface that involves the active site of the phosphatase and the two entrance loops of the receptor. A conserved tryptophan residue of the phosphatase inserts its side chain next to the gating loop, locking it closed. In turn, the gating loop interacts closely with the substrate-binding and active site of the phosphatase, blocking its ability to bind and dephosphorylate its substrate. Together, these structural features comprehensively explain how the PYR/PYL/RCAR proteins inhibit PP2C activity in an ABA-dependent manner, and how PP2Cs act as a potent co-receptor to enhance the affinity of the hormone for its receptor.
These reports present us with a consistent view of ABA signalling that also raises questions for future work. In three of the structural studies4,5,6, receptor dimerization is observed in the absence of PP2C; and although it is not discussed in the text, dimerization is also present in structural models of the remaining two studies1,2. But the exact purpose of receptor dimerization remains unclear. In the structure of homodimeric PYR1, only one molecule of ABA can bind per dimer, whereas in the related structure of PYL homodimers, both subunits are occupied. In all of the structural models, the dimer interface involves the flexible gating loop of the receptor, suggesting that dimerization may be functionally relevant. However, receptor dimerization is clearly not required for the final action of the hormone, because only monomeric ABA-bound receptor is found in complex with PP2C.
The movement of the gating loop in PYR/PYL/RCARs to create a hormone-dependent PP2C-binding site is reminiscent of the 'closing lid' mechanism used by the receptor GID1 to sense gibberellin. In this case, hormone binding induces a movement of parts of GID1 that cover the hormone-binding pocket, creating a site for GID1 to bind to substrate proteins and initiate another form of chemical modification, ubiquitylation10,11. Both ABA and gibberellin allosterically remodel their respective receptors, in contrast to the 'molecular glue' mechanism used by auxin12. Although the precise mechanistic details may differ, there is a common feature in plant hormone action at soluble receptors: the hormone signal enhances protein–protein interactions to modulate critical modifications — either phosphorylation or ubiquitylation — that will alter the activity of the target protein. In addition, in all cases, the hormone binds to a site that is directly at or near the protein–protein interface, engaging the associating protein as a co-receptor.
Melcher, K. et al. Nature 462, 602–608 (2009).
Miyazono, K. et al. Nature 462, 609–614 (2009).
Fujii, H. et al. Nature 462, 660–664 (2009).
Santiago, J. et al. Nature 462, 665–668 (2009).
Nishimura, N. et al. Science 10.1126/science.1181829 (2009).
Yin, P. et al. Nature Struct. Mol. Biol. 10.1038/nsmb.1730 (2009).
McCourt, P. & Creelman, R. Curr. Opin. Plant Biol. 11, 474–478 (2008).
Ma, Y. et al. Science 324, 1064–1068 (2009).
Park, S. Y. et al. Science 324, 1068–1071 (2009).
Murase, K. et al. Nature 456, 459–463 (2008).
Shimada, A. et al. Nature 456, 520–523 (2008).
Tan, X. et al. Nature 446, 640–645 (2007).
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