Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins

Abscisic acid (ABA), identified in plants in the 1960s, is a small molecule that functions to inhibit growth and to regulate plant stress responses. Genetic analyses have identified many factors involved in ABA signaling (1), including the group A type 2C protein phosphatases (PP2Cs), which negatively regulate ABA signaling at an early step in the pathway (2), and the SNF1-related kinase 2 (SnRK2 kinases), which are positive regulators (3–5). Several (+)-ABA (1) binding proteins have been reported (6–8) (Fig. 1A); however, their roles are not fully understood, and they do not appear to work via the genetically defined signaling pathway (9) or bind to the nonnatural but bioactive stereoisomer (–)-ABA (2) (10–12). The genetic dissection of ABA perception has not identified proteins resembling receptors, which suggests that the ABA receptor(s) may be functionally redundant or may be required for viability (13). We therefore pursued a chemical genetic strategy (14), because chemicals can bypass redundancy by inducing phenotypes not revealed by single-locus mutations (15). For example, an antagonist with low selectivity can perturb the function of an entire protein family, whereas a selective agonist can illuminate the function of one member of normally redundant receptors, as we describe here with pyrabactin (3) (Fig. 1A), a synthetic seed germination inhibitor (14). The analysis of analogs revealed that pyrabactin’s activity requires its pyridyl nitrogen, because the analog apyrabactin (4) is biologically inactive (fig. S1) (16). Further investigation of pyrabactin’s action revealed reduced sensitivity in ABA-insensitive mutants, but not ABA biosynthesis or gibberellic acid–perception mutants (fig. S2), which suggests it is an agonist of ABA signaling that inhibits germination in response to environmental stress (17). Aside from ABA analogs, no synthetic agonists of this stress pathway are known. Microarray analyses of the ABA and pyrabactin responses of seeds and seedlings revealed that, in seeds, both compounds induce highly correlated transcriptional responses (r = 0.98; Fig. 1B; table S1). Three unrelated germination inhibitors (18) failed to induce ABA-like effects (fig. S2), which demonstrates that an indirect germination effect is not sufficient to account for pyrabactin’s agonist activity in seeds. In contrast to seed responses, seedling ABA and pyrabactin responses show poorer correlation (r = 0.72) (Fig. 1C), and few ABA-responsive genes significantly respond to pyrabactin (table S1). Thus, pyrabactin affects some, but not all, of the pathways regulated by ABA and is therefore a selective agonist.

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Selective agonists have a long history as reagents for receptor identification, so we used pyrabactin for genetic dissection and isolated 12 PYRABACTIN RESISTANCE 1 (Pyr1) mutant alleles. Pyr1, isolated by map-based cloning, encodes a member of the cyclase subfamily of the START domain superfamily. START proteins share a conserved hydrophobic ligand–binding pocket (19–21). There are 13 genes in the Arabidopsis genome having marked similarity to Pyr1 (fig. S1), which we have named Pyl1 to Pyl13 (for PYR1-Like). This 14-member gene family (Fig. 4B) has been independently identified as ABI1-interacting proteins (called “regulatory component of ABA receptor,” RCAR1 through RCAR14) by Ma et al. (22), and their role in ABA signaling is characterized there. The pyr1 mutant alleles we isolated are predicted to produce a variety of defects in PYR1 (fig. S3). Gene expression databases (23–27) show that Pyr and Pyl mRNAs are expressed at high levels in seeds and guard cells and respond to ABA (Fig. 2A), which is consistent with a role in ABA signaling. However, all of the pyr1 mutants isolated, including putative null alleles, reduce pyrabactin, but not ABA, sensitivity. To examine if functional redundancy might obscure a role for Pyr1 in ABA signaling, we isolated Pyl1, Pyl2, and Pyl4 insertion alleles and constructed multilocus mutants. Triple (pyr1;pyl1;pyl4) and quadruple (pyr1;pyl1;pyl2;pyl4) mutant lines display strong ABA insensitivity (Fig. 2B), which can be reversed by introducing PYR1- or PYL4-expressing transgenes (fig. S3). The quadruple mutant is less sensitive to (+)-ABA as measured by seed germination, root growth, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Fig. 2C), and SnRK2 kinase assays (Fig. 2D and fig. S4). Collectively, our genetic data show that Pyr1 and Pyls are necessary for multiple ABA responses in vivo and illustrate the differential behavior that natural and synthetic agonists can display in genetic screens.

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Because PYR1 is predicted to be a ligand-binding START protein necessary for pyrabactin activity, we hypothesized that pyrabactin might promote a protein-protein interaction between PYR1 and a downstream effector. To test this, ~2 million prey cDNA clones were screened against PYR1 yeast two-hybrid bait on medium containing pyrabactin. This revealed that the PP2C HAB1 (28, 29) interacts with PYR1 in response to pyrabactin and (+)-ABA, but not the inactive analog apyrabactin or several other plant hormones (Fig. 3A and fig. S4). HAB1 resides in the group A subfamily of plant PP2Cs, which contains nine partially redundant members that negatively regulate ABA signaling (30, 31). To examine the relevance of the yeast two-hybrid data, we next performed a series of control experiments. First, we investigated the effects of defective PYR1 and PP2C amino acid substitution mutants on ABA-responsive interactions in the yeast two-hybrid assay (32). PYR1^S152L and PYR1^P88S, isolated because they cause pyrabactin insensitivity in planta, reduce ABA-induced PYR1-PP2C interactions (Fig. 3A); similarly, the dominant ABA-insensitive ABI2^G168D mutant (Fig. 3B) disrupts the PYR1-ABI2 interaction. Second, we investigated in planta interactions by coexpressing a panel of yellow fluorescent protein (YFP)–tagged PP2Cs and hemagglutinin (HA)-tagged PYR1 constructs in N. benthamiana. Coimmunoprecipitation experiments performed on ABA or mock-treated plants recapitulated the PYR1-PP2C interactions observed in yeast and also showed that PYR1 does not interact with ABI1^G180D, encoded by abi1-1 (Fig. 3C). Third, we used PYR1 and ABI1 as bait in a yeast two-hybrid screen against a set of 258 ABA-responsive open reading frames and observed that interactions between PYR1 and group A PP2C proteins occur only in the presence of ABA (Fig. 3D and table S2). Fourth, we successfully reconstituted the ABA-induced PYR1-PP2C interactions in vitro using recombinant proteins (Fig. 3E). Thus, we conclude that ABA promotes a biologically meaningful interaction between PYR1 and group A PP2Cs.

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Given the redundancy observed in our genetic analyses, it is likely that other PYR/PYLs interact with PP2Cs in response to ABA. We therefore used the yeast two-hybrid assay to explore interactions of HAB1 and PP2CA (AHG3) (ABA-hypersensitive germination gene 3) with a panel of 12 PYR/PYLs, which shows that (+)-ABA promotes interactions between PYR1, PYL1 to PYL4, and HAB1 (Fig. 4A). We next used this PYR/PYL panel to examine ligand response selectivities, which shows that these five (+)-ABA–responsive PYR/PYLs do not all bind HAB1 in response to nonnatural agonists. For example, PYL2, PYL3, and PYL4 respond to both (+)-ABA and (–)-ABA (Fig. 4A), which makes these proteins candidates for the dual-stereoisomer receptors predicted by earlier studies (12). Consistent with this hypothesis, our quadruple mutant has greatly reduced (–)-ABA sensitivity (fig. S3). Ligand-selective interactions are also observed for pyrabactin, which promotes interactions between HAB1 and PYR1, PYL1, or PYL3 (Fig. 4A). Of these, only Pyr1 is highly transcribed in seeds (Fig. 2B), which likely explains why mutations in Pyr1 cause the seeds to be insensitive to pyrabactin. Last, we observe that PYL12 interacts with PP2CA (AHG3) in response to ABA (fig. S4). Thus, at least 6 of the 14 PYR/PYLs confer ABA responsiveness to yeast. We hypothesize that the entire PYR/PYL gene family participates in ABA-promoted interactions, as these six genes are distributed across the PYR/PYL phylogenetic tree (Fig. 4B). The specificity with which pyr/pyl genes control which ligands trigger ABA signaling suggests that the ABA pathway may be dissected using selective PYR/PYL agonists.

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Given the role of PYR/PYLs in controlling ligand responses, we explored whether (+)-ABA binds PYR1 by using ¹⁵N-labeled PYR1 and PYR1^P88S in heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments, which probe chemical shifts of protein amide–NH bonds in response to ligands (33). Because START proteins contain a conserved ligand-binding cavity (34), binding should selectively perturb residues lining this cavity. Addition of (+)-ABA altered the HSQC signals for many PYR1 and PYR1^P88S residues (Fig. 4C and figs. S5 and S6), which showed that ABA binds PYR1 and likely induces a conformational change. We also investigated the PYR1-HAB1 interaction in the presence of ABA, because our NMR experiments showed that PYR1^P88S is not defective in ABA binding. Addition of unlabeled HAB1 caused peak broadening for PYR1, but not PYR1^P88S, HSQC signals (fig. S7), which localized the PYR1^P88S defect to binding HAB1 after ABA perception. Because PP2Cs are negative regulators of ABA signaling, we hypothesized that ABA-promoted PYR/PYL–PP2C interactions would inhibit phosphatase activity. To test this, we examined the effects of (+)-ABA on PP2C enzyme kinetics using recombinant HAB1, PYR1, or PYR1^P88S. These experiments show that (+)-ABA acts as a potent and saturable inhibitor of phosphatase activity in the presence of PYR1 [median inhibitory concentration (IC₅₀) = 125 nM], but not PYR1^P88S (IC₅₀ = 50 μM) (Fig. 4D and fig. S8). Similarly, ABA displays saturable inhibition of HAB1 PP2C activity in the presence of recombinant PYL4 (fig. S8). Thus, PYR/PYLs regulate PP2Cs in response to ABA, which defines an unprecedented mechanism for ligand-mediated regulation of PP2C activity.

Collectively, we have shown that PYR1 binds (+)-ABA, PYR/PYLs bind to and inhibit PP2Cs in response to (+)-ABA, and PYR/PYLs control which ligands trigger PP2C interactions. We conclude that the PYR/PYLs are a family of ABA receptors. However, the precise site of ABA binding remains unclear, because the ABA-binding site may be shared with the PP2C. Discriminating between these receptor and co-receptor models will require structural studies of cocrystallized PYR/PYLs, PP2Cs, and ligands. Note that the PYR/PYLs interact directly with PP2Cs, which are core components of the ABA signaling pathway. Because SnRK2 activity is decreased in the PYR/PYL quadruple mutant, we propose a hypothetical model (Fig. 4D) for ABA action in which ABA and PYR/PYLs inhibit PP2Cs, which in turn relieves repression of positive factors, such as the SnRK2s. Consistent with this model, we observed interaction of SnRK2.2 with PP2CA (AHG3), AHG1, and ABI1 when we used the yeast two-hybrid assay (fig. S4). This suggested that the low SnRK2 activity observed in the PYR/PYL quadruple mutant may be a direct consequence of PP2C-SnRK2 interactions. Understanding of the role of PP2Cs in ABA signaling has been complicated by observations from abi1-1 and abi2-1 mutations. Their dominant phenotypes suggest that they encode hypermorphic proteins (35), but they paradoxically reduce, but do not abolish, PP2C activity (36). Our data show that these mutants do not bind PYR1 in response to ABA. We therefore hypothesize that ABA normally lowers wild-type PP2C activity through PYR/PYL proteins, but abi PP2Cs escape this and disrupt signaling because of their residual activity. Consistent with this model, a second site mutation that abolishes abi1-1’s catalytic activity suppresses its dominant ABA-insensitive phenotype (36).

The redundancy in the Pyr/Pyl gene family, typical of many plant genes, has kept these genes from emerging as factors necessary for ABA response. We leveraged pyrabactin’s selectivity for a subset of the PYR/PYL family to bypass the genetic redundancy that masks ABA phenotypes in single mutants. Thus, our results demonstrate the power of synthetic molecules to expose phenotypes for otherwise redundant genes.