A Bifurcating Pathway Directs Abscisic Acid Effects on Stomatal Closure and Opening in Arabidopsis

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Science  14 Apr 2006:
Vol. 312, Issue 5771, pp. 264-266
DOI: 10.1126/science.1123769


Terrestrial plants lose water primarily through stomata, pores on the leaves. The hormone abscisic acid (ABA) decreases water loss by regulating opening and closing of stomata. Here, we show that phospholipase Dα1 (PLDα1) mediates the ABA effects on stomata through interaction with a protein phosphatase 2C (PP2C) and a heterotrimeric GTP-binding protein (G protein) in Arabidopsis. PLDα1-produced phosphatidic acid (PA) binds to the ABI1 PP2C to signal ABA-promoted stomatal closure, whereas PLDα1 and PA interact with the Gα subunit of heterotrimeric G protein to mediate ABA inhibition of stomatal opening. The results reveal a bifurcating signaling pathway that regulates plant water loss.

Abscisic acid (ABA) mediates plant response to environmental stresses (14). During drought, ABA levels in plants increase, and ABA promotes the closing of opened stomata and inhibits the opening of closed stomata. The resulting closure of stomata is crucial to reducing water loss and maintaining the plant's hydration status for survival. The ABA effects on stomatal closure and opening are genetically separable (5). Several signaling proteins have been implicated in mediating the ABA effects in signaling networks perhaps linked through G proteins, protein phosphatases, kinases, and phospholipases (514).

Phospholipase D (PLD) hydrolyzes membrane lipids to generate phosphatidic acid (PA), a lipid signaling mediator (1517). Arabidopsis has 12 PLD genes that form a family of enzymes with heterogeneous regulatory, structural, and biological properties (15). We showed that in response to ABA treatments, PLDα1 produces PA that binds to the ABI1 PP2C (14). ABI1 is a negative regulator of ABA response (4, 8, 18), but its specific function in ABA-regulated stomatal closure and opening is not defined. It was proposed that PLD-derived PA in the plasma membrane interacted with ABI1 and removed its inhibition of ABA response (14). In addition, PLDα1 binds to GPA1, the Gα subunit of a heterotrimeric GTP-binding protein (G protein) (19). Knockout of GPA1 impedes ABA inhibition of stomatal opening but not promotion of stomatal closure (5). These findings prompted us to determine the role of the PLDα1-PA interaction with ABI1 and GPA1 in stomatal movements.

To characterize the interaction between PA and ABI1, we purified ABI1 and the mutant protein ABI1R73A (in which Arg73 is replaced with Ala) expressed in Escherichia coli (fig. S1A). Analysis with isothermal titration calorimetry (ITC) indicates that PA interacts with ABI1 at 1:1 ratio with the dissociation constant at ∼0.3 μM (Fig. 1A), a PA concentration attainable in Arabidopsis cells (14). The mutant protein ABI1R73A has lost its ability to bind PA (Fig. 1B), indicating that Arg73 in ABI1 is essential for PA-ABI1 interaction. Mutation ABI1R73A does not alter the phosphatase activity in the absence of PA, but renders enzyme insensitive to PA inhibition (fig. S1B).

Fig. 1.

PA and ABI1 interaction and its role in ABA effect on stomatal closure and opening. (A and B) Measurements of PA binding to Arabidopsis ABI1 and ABI1R73A, respectively, by isothermal titration calorimetry. ABI1 and ABI1R73A proteins were purified from proteins expressed in E. coli. (C) Positions of T-DNA insertion in abi1-ko and sequence changes of ABI1R73A and the abi1 mutant. Boxes denote exons of ABI1; underlined letters show mutated amino acid residues. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; G, Gly; H, His; I, Ile; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; and V, Val. (D) Reverse transcriptase–polymerase chain reaction analysis of ABI1 or ABI1R73A expression in wild type (WT), abi1-ko, and abi1-ko carrying the ABI1WT or ABI1R73A transgene. (E) Effect of ABI1 and PLDα1 mutations on ABA promotion of stomatal closure. Stomata were induced open by 2.5-hours light treatment, then 50 μM ABA was added, and epidermal peels were incubated for another 2 hours under light. Aperture size is expressed as difference from that of open stomata from wild-type plants before ABA treatment. (F) Effect of mutations on ABA inhibition of stomatal opening. Stomata were closed after 2.5-hours treatment in dark and then incubated for 2 hours under light with or without 50 μM ABA. Values are means ± SD (n = 20). ABI1WT and ABI1R73A denote abi1-ko plants carrying the wild-type ABI1 and mutant ABI1R73A transgene, respectively.

To determine the functional significance of PA-ABI1 interaction, we introduced wild-type ABI1WT or the mutant ABI1R73A gene into a T-DNA insertional ABI1-null mutant (abi1-ko) Arabidopsis (Fig. 1, C and D). We reasoned that if the PA binding to ABI1 is critical to ABA response, abi1-ko plants expressing the ABI1R73A that cannot bind PA should disrupt ABA signaling. The expression of ABI1WT and ABI1R73A transgenes were under the control of the native ABI1 promoter to mimic ABI1's normal temporal and spatial expression. ABI1WT and abi1-ko plants showed a normal response to ABA, as did the wild type (Fig. 1, E and F). However, abi1-ko plants carrying the ABI1R73A transgene were insensitive to ABA, but for only part of the ABA signaling responses. ABI1R73A plants were insensitive to the promotion of stomatal closure (Fig. 1E), but retained normal sensitivity to the inhibition of stomatal opening (Fig. 1F). This altered response indicates that PA binding to ABI1 is required for ABA promotion of stomatal closure but not for ABA inhibition of stomatal opening.

We also generated Arabidopsis plants lacking both PLDα1 and ABI1 by crossing pldα1 and abi1-ko mutants. The double mutations, pldα1abi1-ko, were confirmed by real-time polymerase chain reaction (PCR) and by immunoblotting with a PLDα1-specific antibody. The single mutant pldα1 was insensitive to ABA for both promotion of stomatal closure and inhibition of stomatal opening (Fig. 1, E and F). The double mutant pldα1abi1-ko was insensitive to ABA for inhibition of stomatal opening, but was sensitive to ABA for promotion of stomatal closure (Fig. 1, E and F). The ABA responses of pldα1abi1-ko and ABI1R73A plants both indicate the bifurcation of the ABA signaling at this point: The PLDα1 and PA interaction with ABI1 promotes stomatal closure, but does not inhibit stomatal opening. The double mutant resembled abi1-ko in the stomatal-closure pathway because ABI1, which inhibits the ABA response, is downstream of PLDα1. The normal response of pldα1abi1-ko to ABA in closure suggests that an ABA-responsive pathway is operating when both PLDα1 and ABI1 are removed, but this pathway in normal guard cells is controlled by PLDα1 through removal of the ABI1 inhibition of ABA response. This ABA-responsive pathway is not constitutive, which may explain why the abi1-ko mutant has no overt ABA phenotype in stomatal closure. In contrast, inhibition of the stomatal-opening pathway is not governed by ABI1, and thus for this pathway the double mutant behaved like the PLDα1 knockout.

To identify the component that interacts with PLDα1 to inhibit stomatal opening, we studied PLDα1 binding with GPA1. PLDα1 binds to GPA1 through a DRY (Asp-Arg-Tyr) motif that is usually found in animal G protein–coupled receptors (19). We used the yeast two-hybrid system to verify the interaction in cells; mutation at the DRY motif residue Lys564 decreases the ability of PLDα1 to bind to GPA1 (fig. S2A). ITC analysis showed that wild-type PLDα1 binds to GPA1 in equimolar ratio (N = 1.07 ± 0.075). The affinity of wild-type PLDα1 for GPA1 (dissociation constant Kd = 0.3 μM) is ∼150 times that of PLDα1K564A for GPA1 (Kd = 50 μM). The amount of PLDα1K564A that coprecipitated with GPA1 was ∼10% of that of wild-type PLDα1 (19). Thus, the mutant PLDα1K564A diminishes but does not abolish the binding of PLDα1 to GPA1.

To determine the function of PLDα1-GPA1 interaction, we perturbed the interaction by introducing the mutant PLDα1K564A (in which Lys564 is replaced with Ala) and wild-type PLDα1WT genes into PLDα1-null Arabidopsis (fig. S2B). Expression of the transgenes was controlled by the PLDα1 native promoter. Except for the decreased ability to bind GPA1, PLDα1K564A is enzymatically as active as PLDα1 (19). Introducing PLDα1WT should and did restore the normal ABA response (Fig. 2, A and B). By comparison, PLDα1K564A plants were more sensitive than wild-type plants to ABA for inhibition of stomatal opening, responding to 5 μM ABA, a concentration that had no effect on wild-type plants (Fig. 2C). The increase in ABA sensitivity was reflected in the rate of water loss: PLDα1K564A mutant plants lost less water than wild-type plants, whereas pldα1 and gpa1 plants lost more water from their leaves (Fig. 2D). On the other hand, PLDα1K564A plants showed normal sensitivity to ABA for promotion of stomatal closure (Fig. 2A). Thus, blunting the PLDα1-GPA1 interaction renders plants hypersensitive to ABA in inhibiting stomatal opening, but it does not affect the ABA-induced closure of open stomata.

Fig. 2.

Effects of PLDα1-GPA1 interaction and PA on ABA regulation of stomatal closure and opening. (A) ABA promotion of stomatal closure in GPA1 and PLDα1 mutants. Aperture size was measured after 2-hours treatment of open stomata with or without 50 μMABA. (B) ABA inhibition of stomatal opening in GPA1 and PLDα1 mutants. Stomata were closed in the dark and then incubated for 2 hours under light with or without 50 μM ABA. (C) Response of PLDWT and PLDα1K564A plants to increasing ABA concentrations in inhibition of stomatal opening. (D) Water loss from detached Arabidopsis leaves as a function of time. Values are means ± SD (n = 20). FW, fresh weight. (E) PA promotion of stomatal closure. Stomata were induced open by 2.5-hours light treatment and then transferred to a solution with or without 50 μM dioleoyl-PA and incubated for another 2 hours under light. (F) PA inhibition of stomatal opening. Stomata were closed after 2.5-hours treatment in the dark and then incubated for 2 hours under light with or without 50 μM dioleoyl-PA. Values are means ± SD (n = 15).

Arabidopsis plants with abrogation of both PLDα1 and GPA1 were generated by crossing pldα1 and gpa1. The double-mutant pldα1 gpa1 plants resembled the single-mutant pldα1 plants in that both were insensitive to ABA effects on both pathways, inhibition of stomatal opening and promotion of stomatal closure (Fig. 2, A and B). When the PLD product PA was supplied to epidermal peels of the single and double mutants, PA promoted stomatal closure and inhibited opening in wild-type and pldα1 plants (Fig. 2, E and F). However, for gpa1 or pldα1 gpa1 plants, PA promoted only stomatal closure, but had no effect on inhibiting opening (Fig. 2, E and F). By contrast, for ABI1R73A plants that are unable to bind PA, PA did not promote stomatal closure, but inhibited stomatal opening (Fig. 2, E and F). These results indicate that PLDα1 and PA act upstream of GPA1 and ABI1 in the pathway regulating stomatal responses to ABA signals.

We propose that stomatal responses to ABA are regulated by a bifurcating pathway that includes PLDα1, PA, ABI1, and GPA1 (Fig. 3). To promote closure of open stomata, PLDα1 regulates ABI1 with the use of its lipid product PA. PA binds to ABI1, and this interaction is necessary to remove the ABI1 inhibition of the ABA promotion of stomatal closure. PA regulates ABI1 function by inhibiting its phosphatase activity and by sequestering it to the plasma membrane. The membrane tethering by PA decreases ABI1's translocation from the cytosol to the nucleus and promotes ABA signaling (14).

Fig. 3.

A bifurcating model for interaction among PLDα1, PA, ABI1, and GPA1 (Gα) in mediating ABA effects on stomatal closure and opening. PLDα1-produced PA binds to ABI1, and this binding removes ABI1 inhibition of ABA promotion of stomatal closure. On ABA inhibition of stomatal opening, PLDα1-produced PA acts upstream of GTP-bound Gα (Gα-GTP) to inhibit stomatal opening, whereas GDP-bound Gα (Gα-GDP) binds to PLDα1 to suppress PLD activity. This model is not comprehensive and concerns only components studied here.

To inhibit opening of closed stomata, PLDα1 modulates the GPA1 function through multiple interactions (Fig. 3). Biochemical data suggest that PLDα1 activates the intrinsic guanosine triphosphatase activity that converts active Gα-GTP to inactive Gα-GDP (19). In turn, Gα-GDP binds to PLDα1 and decreases its activity (19, 20). Thus, in PLDα1K564A plants, the ABA signal transduction is sensitized because both the GPA1 and PLDα1 functions are less inhibited by the subdued interaction between PLDα1K564A and GPA1. In contrast, the ABA responses of the pldα1 and gpa1 single mutants indicate that both PLDα1and GPA1 are positive regulators in ABA inhibition of stomatal opening. The positive role of GPA1 may result from the exchange of GTP with GDP; the binding of GTP to GPA1 (Gα-GTP) dissociates Gα from PLDα1, thus removing the inhibition of PLDα1 activity (19). PA resulting from PLDα1 activity promotes inhibition of stomatal opening (Fig. 3). Thus, the PLDα1-GPA1 interaction regulates mutually the activity of both proteins. However, the activation of PLDα1 acts upstream of GPA1 because pldα1 plants display a broader alteration in ABA sensitivity than do gpa1 plants, and because PA inhibits stomatal opening in pldα1, but not in gpa1 or pldα1gpa1 plants. One potential target of PA is a sphingosine kinase that acts upstream of GPA1 (7). ABA activates both PLDα1 and sphingosine kinase (7, 14), and PA has been implicated in binding sphingosine kinase in animal cells (21).

Thus, we conclude that the PLDα1 signaling pathway in guard cells bifurcates at ABI1 and GPA1 to mediate the ABA effects on stomatal closure and opening. Such interaction between PLDα1 and Gα would be unusual for animal cells, in which heterotromeric G proteins typically regulate the function of phospholipases (22). However, plants and animals diverge greatly in the PLD and G protein families; Arabidopsis has 10 more PLD genes than do humans, but only one canonical Gα (22, 23), and thus their signaling pathways may be organized differently. ABA can act as an intracellular signal (24), and activation of membrane-associated PLDα1 may be one initial step that directs the ABA response in guard cells. Although Arabidopsis has multiple PLD and PP2C genes, the clear phenotypes from the various mutants in this study indicate that the interaction between PLDα1-PA and ABI1 is specific. The PA binding region in ABI1 is located at its N terminus, which is highly variable among PP2Cs. Our insights into the pathways regulating stomatal function may be used to produce plants with enhanced water-usage efficiency and drought tolerance.

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