A G Protein-Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid

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Science  23 Mar 2007:
Vol. 315, Issue 5819, pp. 1712-1716
DOI: 10.1126/science.1135882


The plant hormone abscisic acid (ABA) regulates many physiological and developmental processes in plants. The mechanism of ABA perception at the cell surface is not understood. Here, we report that a G protein–coupled receptor genetically and physically interacts with the G protein α subunit GPA1 to mediate all known ABA responses in Arabidopsis. Overexpressing this receptor results in an ABA-hypersensitive phenotype. This receptor binds ABA with high affinity at physiological concentration with expected kinetics and stereospecificity. The binding of ABA to the receptor leads to the dissociation of the receptor-GPA1 complex in yeast. Our results demonstrate that this G protein–coupled receptor is a plasma membrane ABA receptor.

Abscisic acid (ABA) is an important hormone that mediates many aspects of plant growth and development, particularly in response to the environmental stresses (13). Several components involved in the ABA signaling pathway have been identified (4). Two recent reports have shown that the nuclear RNA binding protein flowering time control protein (FCA) (5) and the chloroplast protein Mg chelatase H subunit (6) are ABA receptors (6). In contrast, several earlier experiments had suggested that extracellular perception is critical for ABA to achieve its functions (79). Thus, other ABA receptors, especially plasma membrane–localized receptors, may be the major players for perceiving extracellular ABA and mediating the classic ABA signaling responses.

Ligand-mediated signaling through G protein–coupled receptors (GPCRs) is a conserved mechanism for the extracellular signal perception at the plasma membrane in eukaryotic organisms (10). The GPCR-mediated signaling pathway plays a central role in vital processes such as vision, taste, and olfaction in animals (11). However, the higher plant Arabidopsis thaliana has only one canonical Gα (GPA1) subunit, one Gβ subunit, and two Gγ subunits (1216). The significance of these subunits in plant systems is poorly understood; only one Arabidopsis putative GPCR protein (GCR1) has been characterized in plants (1720), and no ligand has been defined for any plant GPCR.

To identify previously unrecognized GPCR proteins in Arabidopsis, we started by searching the Arabidopsis genome and found a gene (GCR2, GenBank accession code At1g52920) encoding a putative GPCR. Transmembrane structure prediction suggests that GCR2 is a membrane protein with seven transmembrane helices (fig. S1, A and B). The subsequent cellular localization analysis confirmed its plasma membrane localization in the transgenic plant root (fig. S1C). GCR2–yellow fluorescent protein (YFP) is detected in the membrane fraction isolated from the GCR2-YFP transgenic plant. Similar to GCR1 (19), GCR2 is mostly associated with the membrane fraction (fig. S1D). Furthermore, even after washing with detergent or a higher pH buffer, GCR2 is retained with the membrane fraction, suggesting that GCR2 is an integral membrane protein (fig. S1D).

One feature of the GPCR is its ability to interact with G protein to form a complex. To confirm the physical interaction between GCR2 and Gα, we used four different approaches to detect their interaction. We first used surface plasmon resonance spectroscopy to investigate the interaction between GCR2 and GPA1. For this purpose, we expressed and purified recombinant GCR2 and GPA1 proteins in bacteria (fig. S2). This in vitro assay clearly indicated that GPA1 is capable of binding to GCR2, whereas no binding activity was detected between GPA1 and bovine serum albumin (BSA) (fig. S3, A and B). The dissociation binding constant (Kd) for GCR2 and GPA1 is 2.1 × 10–9 M (fig. S3C).

The commonly used yeast two-hybrid system does not work for the GCR2-GPA1 interaction assay because of their membrane localization, so we used a split-ubiquitin system (21) instead to investigate the GCR2-GPA1 interaction in yeast. This assay confirmed the reported interaction of the full-length GCR1 with GPA1 (19), and the full-length GCR2 also interacted with GPA1 (fig. S4A). The interaction was abolished when the C terminus of the receptor was blocked by fusing the ubiquitin fragment (CubPLV) to the C terminus of GCR2 (fig. S4A). We also found that the C terminus of GCR2 (C290–401, containing the free C terminus and the predicted third cytoplasmic loop) interacted with GPA1, whereas the N terminus (N1–289) of GCR2 did not (fig. S4, B and C), indicating that the C terminus of GCR2 is necessary and sufficient for its interaction with GPA1.

Bimolecular fluorescence complementation was used to detect the interaction between GCR2 and GPA1 inplant cells. GCR2 and GPA1 interacted in Arabidopsis protoplasts (Fig. 1A). Removal of the C terminus of GCR2 (C290–401) abolished the interaction (Fig. 1, A and B). This indicated that the interaction between GCR2 and GPA1 is specific and that the C terminus of GCR2 is necessary for its interaction with GPA1. We also confirmed their in vivo interaction by a coimmunoprecipitation assay. GCR2 and GPA1 can be coimmunoprecipitated; and we could detect GPA1 in the immunocomplex precipitated with an antibody to FLAG from GCR2-FLAG transgenic plants (Fig. 1C). Thus, results from four distinct assays all supported the interaction between GCR2 and GPA1.

Fig. 1.

Physical interaction between GCR2 and GPA1. (A) Bimolecular fluorescence complementation assays indicating the interaction of GCR2: hemagglutinin (HA) with a C-terminal HA tag, but not the corresponding C-terminal deletion construct GCR2IL:HA (N1–289) with GPA1 in vivo. Auto-, autofluorescence. The scale bar represents 20 μm. (B) The GCR2:HA-YFCC, GCR2IL:HA-YFPC, and GPA1:cMYC-YFPN proteins were expressed in the protoplast. Proteins from Arabidopsis protoplasts expressing, GCR2IL:HA-YFPC, or GPA1:cMYC-YFPN were extracted and the sample was analyzed on a 12% SDS–polyacrylamide gel electrophoresis gel. GCR2 or GPA1 was detected with the use of anti-HA for GCR2-YFCC or GCR2IL-YFPC, or anti–c-Myc for GPA1-YFPN. (C) Coimmunoprecipitation (Co-IP) assays verifying the interaction between GCR2 and GPA1 in planta. Total proteins from wild-type (Col-0) and GCR2:FLAG transgenic plants were used for in vivo Co-IP with antibodies to FLAG. Immunoprecipitated proteins were probed with antibodies (α) to GPA1 and to FLAG. An anti-FLAG cross-reacting band is shown as the loading control.

To analyze the function of GCR2 in Arabidopsis, we characterized three independent Arabidopsis lines harboring the transferred DNA insertions in the GCR2 locus, gcr2-1, gcr2-2, or gcr2-3 (fig. S5). Transcript analysis by reverse transcription polymerase chain reaction (RT-PCR) detected no full-length GCR2 transcripts in any of the three mutant alleles, but all had the truncated transcripts (fig. S5).

Arabidopsis seeds require desiccation or dormancy to prevent premature germination before harvesting. The freshly harvested wild-type seeds seldom germinate; however, freshly harvested seeds from all three alleles of gcr2 plants germinated well under the same condition (Fig. 2A), indicating that the seeds from gcr2 mutants lost seed dormancy. The seed dormancy is mainly controlled by phytohormone ABA. This result suggested that gcr2 mutants are defective in ABA signaling. We therefore reasoned that the mutations in GCR2 lead to a decreased sensitivity to ABA. We examined other ABA responses in both wild-type and gcr2 plants. We first checked the effect of GCR2 mutations on seed germination in the presence of ABA and found that all three gcr2 mutants were insensitive, whereas the seeds from GCR2 overexpressor lines were hypersensitive to the ABA-inhibition compared with the wild-type seeds (Fig. 2B). We also found that the seedling growth inhibition by ABA was substantially reduced in the gcr2 mutants, but increased in GCR2 overexpressor lines compared with that of the wild type (Fig. 2C). In the absence of ABA, the gcr2 seedlings developed normally and were indistinguishable from the wild-type seedlings (Fig. 2C). ABA mediates plant development by controlling the expression of ABA-mediated genes, and we thus compared the expression of ABA marker genes in wild-type and gcr2 plants. Three well-known ABA marker genes, RD29A, KIN1, and ABI5, were tested. This analysis confirmed that the expression of ABA marker genes was substantially repressed in the gcr2 mutants (fig. S6).

Fig. 2.

The gcr2 mutants exhibit all known ABA defects. (A) The gcr2 seeds lose the seed dormancy observed in the wild type (WT). Freshly harvested seeds germinated on water-soaked filter paper for 7 days. The scale bar represents 2 mm. (B) The gcr2 seeds are insensitive to ABA-inhibited seed germination (mean ± SD; n = 4). The germination percentage was determined 3 days after growing in 1/2 Murashige and Skoog (MS) media. (C) The seedling development in gcr2 is insensitive to ABA. Seeds were grown on 1/2 MS media with (right) or without (left) 0.3 μM ABA for 10 days. GCR2OE, GCR2 overexpressor line. The scale bar represents 1 cm. (D) The gcr2 plants show defects in ABA-induced stomatal closure (mean ± SD; n = 3). (E) The gcr2 and gpa1 plants are insensitive to ABA for stomatal opening (mean ± SD; n = 3). WS, Wassilewskija. (F) The inward K+ channel in gcr2 guard cells is insensitive to ABA. The data in the graph (bottom) are presented as mean ± SE (n = 4, 3, 5, and 6 for wild type, wild type+ABA, gcr2-1, and gcr2-1+ABA, respectively). (G) Overexpressor of GCR2 confers increased ABA sensitivity for stomatal closure (mean ± SD; n = 3).

The ABA-induced closing and ABA-inhibited opening of stomata are classical ABA responses. We observed that both ABA-induced stomata closing and ABA-inhibited stomata opening were insensitive in gcr2 compared with gcr2 sensitivity in wild-type plants (Fig. 2, D and E), and the stomata width is larger in gcr2 plants than in wild-type plants (Fig. 2E). ABA regulates inward K+ (K+in) channels to control the stomatal aperture (22, 23). To determine whether GCR2 mediates the ABA regulation of K+in channels in stomatal guard cells, we applied patch-clamping techniques to analyze the whole-cell K+in currents in guard cells from wild-type and gcr2-1 plants. ABA inhibited K+in currents in wild-type guard cells but had no effect on the K+in currents in gcr2-1 guard cells (Fig. 2F), thus indicating the involvement of GCR2-mediated K+in in ABA-induced stomatal closure. We also found that stomata from GCR2-overexpressing plants were hypersensitive to ABA-induced closure compared with the sensitivity of the wild-type plants (Fig. 2G). The stomata closure defects resulted in a greater water loss in the leaves of the three gcr2 alleles compared with that in wild-type leaves, whereas water loss was reduced in GCR2-overexpressing plant leaves (fig. S7). All of these results indicate that gcr2 plants are insensitive to ABA, whereas GCR2-overexpressing plants are hypersensitive to ABA, demonstrating the involvement of GCR2 in all major ABA responses in Arabidopsis.

The gcr2 plants exhibit all known major ABA defects. The GPA1 null mutant (gpa1) is also defective in ABA-induced stomatal opening and inward K+-channel regulation in guard cells (22). Expression pattern analysis shows that GCR2 and GPA1 share a very similar expression pattern (fig. S8, A and B). We also examined the functional significance of GCR2-GPA1 interaction by checking the ABA response in the gcr2gpa1 or overexpressor of GCR2 or GPA1 in different genetic background using stomatal closure as an assay. We found that GPA1 was involved in both ABA-controlled stomatal opening and closing (Fig. 2E and fig. S8, C to E). The defect exhibited by gcr2gpa1 in ABA-induced stomatal closure was similar to the defect exhibited by gcr2 or gpa1 (fig. S8C). Transgenic lines overexpressing GCR2 or GPA1 were hypersensitive to the ABA response; however, the effects caused by the overexpression of GCR2 or GPA1 were dependent on GPA1 or GCR2, respectively (fig. S8, B, C, and E), indicating that GCR2 and GPA1 function together to transduce the ABA signal.

Given that gcr2 exhibits defects in all known ABA responses and that GCR2 is a plasma membrane receptor, we examined whether GCR2 is an ABA receptor. For this purpose, we first checked whether GCR2 could bind ABA. We observed that ABA bound to GCR2 but did not bind to denatured GCR2 protein (Fig. 3A). As a positive and negative control, ABA also bound to FCA but not BSA or GPA1 (Fig. 3A). The binding of ABA to GCR2 protein is dependent on pH, and the optimum pH is between 7.0 and 7.5 (fig. S9). Stereospecificity of GCR2 binding to the biologically active (+)-ABA was tested in competition assays. Trans-ABA and (–)-ABA analogs did not compete for the GCR2-binding site (Fig. 3B).

Fig. 3.

Binding of ABA to GCR2. (A) ABA binds to the purified GCR2 protein (mean ± SD; n = 3). (B) GCR2 binding of ABA is stereospecific (mean ± SD). (C) ABA binding to GCR2 behaves according to saturation kinetics. The specific and nonspecific bindings are represented by top and bottom curves, respectively (mean ± SD; n = 5). (D) Scatchard plot analysis of ABA binding shows a linear relationship with the square of the correlation coefficient r2 = 0.99, and maximum binding is 0.8 mol mol–1 protein with Kd = 20.1 nM (mean ± SD; n = 5). (E) ABA disrupts the GCR2-GPA1 interaction in yeast. (Top) Co-IP assay. After 10 μM ABA was added to the medium for 20 min, proteins were isolated. The relative intensity of the immunosignal to the control is indicated by the number below each band. WB, Western blot. (Bottom) Yeast growth assay. The proliferation was observed 7 days after the addition of 10 μM (±)-ABA and 1 mM methionine. NubG, ubiquitin N terminus I 13 G mutation. The scale bar represents 2 mm. (F) Schematic model for GCR2-mediated ABA signaling pathway. GDP, guanosine diphosphate; GTP, guanosine 5′-triphosphate; PLD, phospholipase D.

The specific binding of ABA to purified GCR2 could be saturated with increasing amounts of ABA (Fig. 3C). Scatchard plot analysis exhibits a linear pattern (Fig. 3D), suggesting a single binding site for ABA in GCR2. The equilibrium dissociation constant (Kd) for the GCR2-ABA complex is 20.1 nM (Fig. 3D), which is consistent with the physiological concentration range of ABA in plants.

Ligand binding to the GPCR induces the dissociation of the GPCR–G protein complex to release Gα and the Gβγ dimer, thus activating the downstream signaling events (16, 24). To test whether the binding of ABA to GCR2 could dissociate the GCR2-GPA1 complex, we used the split-ubiquitin system to reconstitute this initial ABA signaling event in yeast. Application of the physiologically active form of ABA [(±)-ABA or (+)-ABA] indeed disrupted the GCR2-GPA1 interaction, whereas the physiologically inactive forms of ABA analogs [(–)-ABA or trans-ABA] did not affect the interaction between GCR2 and GPA1, as indicated in the coimmunoprecipitation assay (Fig. 3E). This result again clearly confirmed that the binding of ABA to GCR2 is stereospecific.

In the split-ubiquitin yeast protein interaction assay, the downstream reporter genes (HIS3, ADE2, and lacZ) were activated if the two assayed proteins interacted with each other (21). We reasoned that the binding of ABA to GCR2 could repress the expression of reporter genes as a result of the dissociation of GCR2 and GPA1. This prediction was verified (fig. S10). The addition of a physiologically active form of ABA repressed lacZ expression, whereas physiologically inactive forms of ABA [(–)-ABA or trans-ABA] did not (fig. S10). As a control, ABA did not affect the expression of the reporter gene in a reconstituted K+ ATPASE1 (KAT1)–KAT1 pathway in the same split-ubiquitin system (fig. S10). In addition, the expression of another two reporter genes, HIS3 and ADE2, was necessary for yeast proliferation in a selection medium. The addition of ABA substantially repressed the proliferation of yeast expressing GCR2 and GPA1, but did not affect yeast proliferation in expressing KAT1-KAT1 system (Fig. 3E). Together, these results indicated that the effect of ABA on reporter gene expression was specific for the GCR2-GPA1 interaction, further supporting that the binding of ABA to GCR2 results in the dissociation of the GCR2-GPA1 complex.

Terrestrial plants are immobile and incapable of escaping unfavorable environmental conditions such as drought or cold. Instead, they rely heavily on ABA to survive these conditions. Thus, how plants perceive and transduce the ABA signal is a fundamental question. It has been shown that GPA1 was involved in ABA-mediated stomatal response and seed germination (19, 22, 25); a putative GPCR GCR1 interacted with GPA1 to negatively regulate ABA signaling (19). We now describe the characterization of a new GPCR, GCR2, in Arabidopsis. We conclude that GCR2 is a major ABA receptor on the basis of the following evidence: (i) Loss-of-function gcr2 exhibits all known ABA defects; (ii) an overexpressor of GCR2 shows an ABA-hypersensitive phenotype; (iii) GCR2 binds ABA with a high affinity and reasonable dissociation constants, and the binding is stereospecific and abides by receptor kinetics; (iv) GCR2 is localized in the plasma membrane; (v) GCR2 genetically and physically interacts with GPA1; and (vi) the binding of ABA to GCR2 disrupts GCR2-GPA1 interaction. Notably, the gcr2 mutants still display ABA responses. This may be due to the weak nature of the mutations (fig. S5) or functional redundancy with GCR2-related proteins, given that there are two other GCR2 homologous genes in the Arabidopsis genome. Consistent with this latter possibility, the semidominant phenotype and partial complementation of gcr2 by GCR2 cDNA expression (fig. S11) suggests the possible interference between GCR2 and its homologs.

The following model of ABA signaling can be envisaged. A GPCR, GCR2, which is a plasma membrane–localized ABA receptor, interacts with the Gαβγ complex. Binding of ABA to GCR2 results in the release of the G protein and dissociation of the heterotrimeric complex into Gα and the Gβγ dimer to activate downstream ABA effectors and to trigger the ABA responses (Fig. 3F). Thus, our results identified an ABA receptor and its target protein, a heterotrimeric G protein.

Supporting Online Material

Materials and Methods

Figs. S1 to S11


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