Research ArticleResearch Article

ABI1 Protein Phosphatase 2C Is a Negative Regulator of Abscisic Acid Signaling

Françoise Gosti, Nathalie Beaudoin, Carine Serizet, Alex A. R. Webb, Nicole Vartanian, Jérôme Giraudat

Published October 1999. DOI: https://doi.org/10.1105/tpc.11.10.1897

Abstract

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.

INTRODUCTION

Abscisic acid (ABA) plays a major role in various aspects of plant growth and development, including seed maturation and germination, as well as adaptation to abiotic environmental stresses (Zeevaart and Creelman, 1988; McCarty, 1995; Rock and Quatrano, 1995; Leung and Giraudat, 1998). Although the nature of the ABA receptor(s) remains unknown, substantial progress has been made recently in the characterization of more downstream elements of the ABA signaling pathways (Busk and Pagès, 1997; Bonetta and McCourt, 1998; Grill and Himmelbach, 1998; Leung and Giraudat, 1998). In particular, genetic screens based on the inhibition of seed germination by applied ABA have led to the isolation of several Arabidopsis mutants with altered ABA responsiveness. Mutations in the ABA-insensitive (ABI) loci ABI1 to ABI5 reduce the sensitivity of seed germination to exogenous ABA (Koornneef et al., 1984; Ooms et al., 1993; Finkelstein, 1994a; Nambara et al., 1995). Conversely, mutations in the ERA1 (ENHANCED RESPONSE TO ABA) locus increase the sensitivity of seed germination to applied ABA (Cutler et al., 1996).

The abi3, abi4, and abi5 mutants exhibit additional defects in various aspects of seed maturation but do not seem to be altered in vegetative responses to ABA (Koornneef et al., 1984; Finkelstein and Somerville, 1990; Ooms et al., 1993; Finkelstein, 1994a; Parcy et al., 1994; Nambara et al., 1995). The corresponding gene products thus may belong to a seed-specific branch of the ABA signaling network. The ABI3 and ABI4 genes have been cloned and encode putative transcriptional regulators (Giraudat et al., 1992; Finkelstein et al., 1998).

In contrast, the abi1, abi2, and era1 mutations are more pleiotropic in that they affect ABA sensitivity in both seeds and vegetative tissues. The ERA1 gene encodes the β subunit of a protein farnesyl transferase (Cutler et al., 1996). Loss-of-function era1 mutants display prolonged seed dormancy and improved ability to withstand drought, which suggests that farnesylation is essential for negative regulation of ABA action (Cutler et al., 1996; Pei et al., 1998). The ABI1 and ABI2 genes encode homologous proteins that belong to the 2C class of protein serine/threonine phosphatases (PP2Cs; Leung et al., 1994, 1997; Meyer et al., 1994; Bertauche et al., 1996; Leube et al., 1998; Rodriguez et al., 1998a). The only mutant alleles available for these genes have been the abi1-1 and abi2-1 mutants isolated by Koornneef et al. (1984). These two mutants carry precisely the same amino acid substitution at equivalent positions in the ABI1 and ABI2 PP2C domains, respectively (Leung et al., 1997; Rodriguez et al., 1998a). The abi1-1 and abi2-1 mutations are both dominant and lead to largely overlapping sets of phenotypic alterations including ABA-resistant seed germination and seedling growth, reduced seed dormancy, abnormal stomatal regulation, and defects in various responses to drought stress (Koornneef et al., 1984; Finkelstein and Somerville, 1990; Vartanian et al., 1994; Gosti et al., 1995; Leung et al., 1997).

The functions of the ABI1 and ABI2 phosphatases, however, could not be fully understood solely on the basis of the dominant mutant alleles identified thus far. First, it remained unclear whether these proteins actually are involved in ABA signaling. Indeed, it is possible that the wild-type ABI1 and ABI2 proteins do not normally contribute to ABA action and that only gain-of-function mutant forms can interfere with ABA responsiveness. Even if ABI1 and ABI2 are components of ABA signaling cascades, dominant mutant alleles do not permit us to conclude whether these proteins are positive or negative regulators of ABA responses. Furthermore, the possible contribution of PP2C proteins to ABA signaling cannot be easily investigated by pharmacological approaches because no potent inhibitor of this class of phosphatases has been identified (Hunter, 1995). It thus is required to characterize the phenotype of recessive loss-of-function mutants of these genes to elucidate the roles of ABI1 and ABI2 in ABA signal transduction.

In this study, we isolated seven distinct loss-of-function alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In marked contrast to the ABA-resistant abi1-1 mutant, all of these intragenic revertants were more responsive to ABA than were wild-type plants, and this ABA supersensitivity phenotype was recessive. Moreover, each of these revertant alleles encodes an ABI1 protein that lacked any detectable phosphatase activity in an in vitro enzymatic assay. These results thus provide genetic evidence that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA, and hence that the wild-type ABI1 phosphatase is a negative regulator of ABA responses.

RESULTS

Isolation of Phenotypic Suppressors of abi1-1

Seeds homozygous for the abi1-1 mutation were treated with ethyl methanesulfonate (EMS), and the M2 population was screened for mutants showing a reversion of the ABA-resistant seed germination phenotype of abi1-1. M2 seeds were plated along with wild-type and abi1-1 controls on agar medium containing 3 μM ABA. After 4 days at 21°C, the wild-type seeds displayed either an intact seed coat or an emerged but growth-arrested seedling (Figure 1A), whereas in contrast, abi1-1 seeds had germinated and produced seedlings with green expanded cotyledons and a well-developed root (Figure 1B). The M2 seeds that resembled wild-type controls were selected and transferred onto ABA-free medium to rescue the viable individuals. The plantlets that developed well on ABA-free medium were retained as putative abi1-1 revertants. These plantlets then were incubated on agar medium containing 10 μM ABA, and their root growth after 3 days was compared with that of wild-type and abi1-1 controls (Figure 1C). The candidate revertants selected in the germination assay then were divided into two classes, depending on whether they had regained wild-type ABA sensitivity for root growth.

Figure 1.
Figure 1.

Phenotypic Criteria Used for the Mutant Screen.

(A) and (B) Sensitivity of seed germination to 3 μM ABA. Seeds of the Ler wild type (A) and the abi1-1 mutant (B) were plated on medium supplemented with 3 μM ABA, chilled for 3 days at 4°C in the dark, and incubated for 4 days at 21°C with a 16-hr-light photoperiod. Bars = 1 mm.

(C) Sensitivity of root growth to 10 μM ABA. Seedlings of the Ler wild type (left) and the abi1-1 mutant (right) were grown for 6 days on ABA-free medium and then incubated vertically for 3 days on an agar plate supplemented with 10 μM ABA. Upon transfer to the ABA-containing plate, the root tips of the seedlings were aligned on the white line. Hence, the portions of the roots that are located below the line were formed during incubation in the presence of ABA. Bar = 5 mm.

From an estimated total of 4000 M1 plants and 139,000 M2 seeds screened, we finally retained 11 independent (originating from distinct pools of M1 plants) lines that showed a significant reversion of abi1-1 phenotypes in the M3 generation. Three lines showed only partial reversion of the abi1-1 phenotype in the germination and/or root growth assays of the screen. These three lines subsequently were shown to contain extragenic suppressor mutations, and their analysis will be described elsewhere. The eight other lines displayed a complete reversion of the ABA resistance of abi1-1 in both the germination and the root growth assays used during the screen. These lines subsequently were found to be intragenic revertants (see below). Hence, as recommended by Meinke and Koornneef (1997), these mutants were designated as abi1-1R1 to abi1-1R8 to indicate that each of these novel alleles of ABI1 contains the abi1-1 mutation and a suppressor mutation (R1 to R8). Because abi1-1R5 and abi1-1R8 contain exactly the same suppressor mutation (see below), only the abi1-1R5 line was included in subsequent studies.

Genetic and Molecular Characterization of the Intragenic Revertants

Each of the abi1-1R1 to abi1-1R7 lines was backcrossed to abi1-1. In all cases, the resulting F1 seeds were resistant to ABA, and the F2 seed from self-fertilized F1 plants showed 3:1 segregation for the ABA-resistant phenotype (Table 1). These results indicate that the suppressor mutations are nuclear, monogenic, and recessive to abi1-1.

To test linkage between the suppressor mutations and the abi1-1 mutation, we outcrossed each of the abi1-1R1 to abi1-1R7 lines to wild-type Arabidopsis Landsberg erecta (Ler). In the F2 generation, individuals having inherited the abi1-1 mutation but not the suppressor mutation would be expected to display the ABA-resistant phenotype of abi1-1. A sample (1 to 1.4 × 105 seeds) of each F2 population was analyzed in the germination assay on 3 μM ABA, as described above. In all cases, the frequency of ABA-resistant individuals was lower than 0.01% (data not shown), indicating that the suppressor mutations are tightly linked to the abi1-1 mutation. The ratio of physical to genetic distance in this region of chromosome 4 is ∼150 kb per centimorgan (Leung et al., 1994; Schmidt et al., 1995), suggesting that the suppressor mutations were located within the ABI1 gene itself.

The entire nucleotide sequence of the ABI1 gene was determined from the abi1-1R1 to abi1-1R8 revertant lines. Each of these lines was found to contain the original abi1-1 mutation together with an additional missense mutation within the ABI1 open reading frame (Figure 2). Hence, combined genetic and molecular evidence indicates that these intragenic mutations are responsible for suppression of the abi1-1 phenotype. The same suppressor mutation was found in the independent abi1-1R5 and abi1-R8 lines, whereas all the other suppressor mutations were isolated only once (in a single pool of M1 plants).

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Table 1.

Segregation of the Revertant Phenotype in an Homozygous abi1-1 Background

All the intragenic suppressor mutations identified above translate into amino acid substitutions within the PP2C domain of the ABI1 protein (Figure 2). Remarkably, although these mutations are scattered along the primary structure of the ABI1 PP2C domain, they all occur within blocks of amino acids that are conserved in other PP2Cs (Pilgrim et al., 1995; Das et al., 1996) and are located in proximity to the catalytic site (Das et al., 1996).

The abi1-1R1 to abi1-1R7 Mutant Proteins Have No Detectable PP2C Activity in Vitro

The effect of the intragenic suppressor mutations on the PP2C activity of ABI1 was investigated using an in vitro phosphatase assay. Recombinant proteins that consisted of glutathione S-transferase (GST) fused to the wild type or various mutant forms of the ABI1 protein were produced in Escherichia coli and affinity purified. The amounts of fusion protein present in the various affinity-purified samples were standardized to each other by analysis on SDS–polyacrylamide gels (Figure 3A) and quantified by scanning the Coomassie blue–stained gel, using BSA as a standard. The recombinant proteins then were tested for their ability to dephosphorylate 32P-labeled casein, an artificial substrate commonly used in assaying PP2C activity (MacKintosh, 1993).

Figure 2.
Figure 2.

Localization of the Intragenic Suppressor Mutations within the Sequence of the PP2C Domain of ABI1.

Shown is an alignment of the amino acid sequences of the PP2C domain of ABI1 (ABI1) (Leung et al., 1994; Meyer et al., 1994) and of the human protein PP2Cα (HPP2Ca) (Mann et al., 1992). Residues conserved between ABI1 and HPP2Cα are displayed as white letters on a black background. Dots indicate gaps introduced to maximize alignment of conserved residues. The abi1-1 mutation and the various suppressor mutations (R1 to R8) are indicated above the ABI1 sequence. In the nucleotide sequence of the ABI1 cDNA (GenBank accession number X77116), the suppressor mutations are G-to-A transitions at nucleotides 984 (R4), 1207 (R7), 1351 (R6), and 1678 (R3), and C-to-T transitions at nucleotides 1341 (R1), 1372 (R5 and R8), and 1414 (R2).

When introduced alone (i.e., without the abi1-1 mutation in the ABI1 sequence), each of the four suppressor mutations tested engendered a severe reduction in the PP2C enzymatic activity of ABI1. The R1 and R6 mutations caused 60-fold decreases in the specific activity of the ABI1 protein, and the R2 and R5 mutations resulted in the loss of any detectable phosphatase activity in our experimental conditions (i.e., at least a 1000-fold reduction in specific activity; Table 2 and Figure 3B).

The suppressor mutations inhibited the ABI1 phosphatase activity also when they were combined with the abi1-1 mutation. As previously described (Bertauche et al., 1996; Leube et al., 1998), the abi1-1 mutation alone reduced the specific activity of the ABI1 phosphatase (Table 2). Remarkably, all the double mutant abi1-1R1 to abi1-1R7 proteins lacked any detectable phosphatase activity in our experimental conditions, indicating that their specific activities were at least 1000-fold lower than that of the wild-type ABI1 protein and 40-fold lower than that of the abi1-1 mutant protein (Table 2).

These results indicated that the intragenic suppressor mutations are likely loss-of-function mutations. Hence, it was of interest to analyze the effect of these mutations on ABA responsiveness. Because of the very tight genetic linkage between the suppressor mutations and the abi1-1 mutation, it was difficult to isolate plants containing a suppressor mutation without the abi1-1 mutation. As an alternative, we analyzed in more detail the ABA sensitivity of the abi1-1R1 to abi1-1R7 revertants. The in vitro enzymatic assay described above indicated that each of the abi1-1R1 to abi1-1R7 alleles encodes a catalytically inactive ABI1 protein, or at least an ABI1 protein with extremely reduced phosphatase activity. If the ABI1 phosphatase is not involved in ABA signaling, the ABA sensitivity of the loss-of-function abi1-1R1 to abi1-1R7 alleles should be identical to that of the wild type. Alternatively, the finding that these alleles differ from the wild type in their ABA sensitivity would demonstrate that the ABI1 phosphatase does regulate ABA signaling. Furthermore, a decrease in ABA sensitivity would indicate that ABI1 is a positive regulator of ABA action, whereas conversely, an increase in ABA sensitivity would imply that ABI1 is a negative regulator of ABA responses.

The Intragenic Revertants Have Enhanced Sensitivity to Applied ABA

The abi1-1 mutant displays a lower sensitivity than the wild type to the inhibition of seed germination by exogenous ABA (Koornneef et al., 1984). Dose–response analyses showed that in agreement with the selection scheme used for mutant screening, this characteristic abi1-1 phenotype was suppressed in each of the abi1-1R1 to abi1-1R7 revertant alleles (Figure 4A and data not shown). Actually, as compared with the wild type, the intragenic revertants showed a phenotype opposite to that of abi1-1. Each of the abi1-1R1 to abi1-1R7 lines, indeed, was more sensitive than the wild type to the ABA inhibition of seed germination (Figure 4A and Table 3). In several independent experiments, the ABA dose required to achieve 50% inhibition of seed germination (ID50) was approximately two- to threefold lower for the revertants than for the wild type (Figure 4A and data not shown). Similarly, sensitivity of root growth to ABA inhibition was higher in each of the revertants than in the wild type (Figure 4B and Table 3). The ID50 for root growth inhibition was again approximately two- to threefold lower for the revertants (Figure 4B and data not shown). These results thus demonstrate that in these two bioassays, the seven distinct intragenic revertants all display a higher sensitivity to ABA than does the wild type.

Figure 3.
Figure 3.

In Vitro PP2C Activity of Recombinant Proteins.

(A) Coomassie blue staining of a 10% SDS–polyacrylamide gel loaded with the indicated affinity-purified GST fusion proteins. The aliquots analyzed on the gel were estimated to contain 300 ng of intact fusion protein (indicated by the arrow) compared with BSA standards (not shown).

(B) In vitro protein phosphatase activity. GST–ABI1 (0.5 ng; filled squares), 15 ng of GST–abi1-R1 (open triangles), and 60 ng of GST–abi1-R2 (open circles) fusion proteins were incubated with 32P-labeled casein at 30°C for the indicated times. Phosphatase activity is expressed in picomoles of 32Pi released. Values have been subtracted for the amount of 32Pi released in control samples that contained buffer only (always <0.1 pmol). Values shown are means of duplicate assays, and the range of values obtained was smaller than the symbol size.

If this increased ABA sensitivity actually is caused by the loss of ABI1 PP2C activity described above, one would expect the revertant alleles to be recessive to the wild-type ABI1 allele. We thus examined the dominance relationships between the wild-type ABI1 and the revertant abi1-1R alleles. Homozygous revertants were crossed to wild-type Ler plants. The F1 progeny and self-fertilized seed of each parent were tested for their ability to germinate in the presence of ABA. For each of the four independent revertants analyzed, the germination rate of the heterozygous F1 progeny was not significantly different from that of wild-type Ler seed (Table 4). These results indicate that each of the revertant alleles tested is recessive to the wild-type ABI1 allele in enhancing the sensitivity of seed germination to applied ABA. These genetic data show that the increased ABA sensitivity of the revertant alleles is due to a loss of function in ABI1, and the data correlate well with the changes in ABI1 PP2C activity observed in vitro.

The Intragenic Revertants Have Enhanced Seed Dormancy and Drought Adaptive Responses

The dose–response analyses described above showed that the intragenic revertants are more sensitive than the wild type to applied ABA. We then investigated whether these mutants also display enhanced responsiveness to endogenous ABA under physiological conditions. Because ABA plays important roles in the promotion of seed dormancy and in the regulation of plant water status, we analyzed whether these processes are affected in the intragenic revertants.

The seeds of ABA-deficient mutants and of ABA-resistant mutants, including abi1-1, are less dormant than wild-type seeds (Karssen et al., 1983; Koornneef et al., 1984, 1989b; Léon-Kloosterziel et al., 1996). In contrast, each of the abi11R1 to abi1-1R7 revertants displayed an increased seed dormancy compared with the wild type (Table 3). This result indicates that the seeds of the revertants have an enhanced responsiveness to endogenous ABA.

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Table 2.

In Vitro Protein Phosphatase Activities

Figure 4.
Figure 4.

Sensitivity to Applied ABA.

(A) ABA dose response for germination inhibition. Seeds of Ler wild type (filled squares), abi1-1 (open diamonds), abi1-1R1 (open circles), and abi1-1R5 (open triangles) were plated on medium supplemented with the indicated concentrations of ABA, chilled for 4 days at 4°C in darkness, and incubated for 2 days at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) was expressed as the percentage of the total number of seeds plated (100 to 200). For 0.1, 0.3, 0.6, and 1 μM ABA, the germination rates of the abi1-1R1 and abi1-1R5 revertants were lower than that of Ler (P < 0.001; chi square test of independence).

(B) ABA dose response for root growth inhibition. Seeds of Ler wild type (filled squares), abi1-1 (open diamonds), abi1-1R2 (open circles), and abi1-1R3 (open triangles) were germinated and grown for 6 days on ABA-free medium. These seedlings then were incubated vertically on medium supplemented with the indicated concentrations of ABA, and their root growth was scored after 5 days. Root growth of ABA-treated seedlings is expressed as a percentage relative to controls incubated on ABA-free medium. Values shown are mean ±se from samples composed of 20 to 30 seedlings each. In all cases, the error bars were smaller than the symbol size.

Another characteristic shared by the ABA-deficient and the abi1-1 mutants is a defect in the regulation of stomatal aperture that leads to disturbed water relations (Koornneef et al., 1982, 1984; Léon-Kloosterziel et al., 1996). In isolated epidermal strips, stomata of abi1-1 are more open than are those of the wild type (Leung et al., 1994; Roelfsema and Prins, 1995; Pei et al., 1997). In contrast, stomatal apertures were 15 to 20% smaller in the intragenic revertants tested (abi1-1R1 and abi1-1R5) than in the wild type (e.g., 4.7 ± 0.1 μm in abi1-1R1 versus 5.8 ± 0.1 μm; n = 120).

Water loss in excised leaves or aerial parts is markedly higher in abi1-1 than in the wild type (Koornneef et al., 1984; Finkelstein, 1994b; Leung et al., 1997). The kinetics of water loss in detached leaves of each of the abi1-1R1 to abi1-1R7 revertants was similar to that in wild-type leaves (data not shown). The phenotype of the abi1-1 mutant thus was fully suppressed in the intragenic revertants, but this assay on detached leaves did not permit us to distinguish the revertants from the wild type.

However, when whole plants were subjected to a progressive drought stress, the intragenic revertants tested (abi1-1R1 and abi1-1R3) displayed an enhanced drought tolerance compared with the wild type, whereas in contrast, abi1-1 was highly drought sensitive. When the soil water content had decreased to 35%, rosette leaves of abi1-1 were permanently wilted, whereas wild-type plants only showed signs of withering (Figures 5A and 5B). When the soil water content had dropped further to 15%, the abi1-1R3 plants still were flowering and had turgid rosette leaves, whereas wild-type plants already were completely wilted (Figures 5C and 5D).

Under these conditions of progressive drought stress, wild-type plants also develop an adaptive response at the root level, designated as drought rhizogenesis (Vartanian et al., 1994). This response results in the formation, from a threshold water deficit, of short, tuberized, and hairless roots that remain quiescent under prolonged drought stress but are able to rapidly recover elongation and hair formation upon rehydration. Endogenous ABA plays a promotive role in this process because the production of drought-induced short roots is reduced in the ABA-deficient aba-1 mutant (Vartanian et al., 1994). Accordingly, the ABA-resistant abi1-1 mutant also displayed a lower response than the wild type (Table 5; Vartanian et al., 1994). In contrast, the intensity of drought rhizogenesis was enhanced in the two revertants tested (abi1-1R1 and abi1-1R3) relative to the wild type (Table 5). These results thus indicate that the intragenic revertants are more responsive to endogenous ABA levels under physiological conditions.

DISCUSSION

The only mutant allele of ABI1 that has been analyzed thus far is the abi1-1 mutant, which displays a marked reduction in ABA sensitivity. However, because the abi1-1 mutation is dominant, it was impossible to conclude whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or a negative regulator of ABA action. We describe here the characterization of seven novel alleles of the ABI1 gene (abi1-1R1 to abi1-1R7). These mutants initially were selected as showing a reversion of the resistance of abi1-1 to ABA inhibition of seed germination and seedling root growth. Actually, these mutants displayed a phenotype opposite that of abi1-1. Quantitative dose–response analyses revealed that in the two bioassays mentioned above, the abi1-1R1 to abi1-1R7 mutants are more sensitive to applied ABA than is the wild type. In addition, these mutants displayed increases in seed dormancy, whole-plant drought tolerance, and drought rhizogenesis intensity, which are indicative of increased responsiveness to the endogenous ABA levels that control these physiological processes.

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Table 3.

Phenotypic Analysis of Intragenic Revertants

The seven suppressor mutations (R1 to R7) are missense mutations in conserved regions of the PP2C domain of ABI1. In the absence and in the presence of the abi1-1 mutation, these suppressor mutations severely reduced the protein phosphatase activity of the ABI1 protein in our in vitro assay. In particular, the abi1-1R1 to abi1-1R7 proteins all lacked detectable PP2C activity, indicating that the specific activities of these mutant proteins was at least 1000-fold lower than that of the wild-type ABI1 protein. Moreover, the enhanced ABA sensitivities of the corresponding revertant plants appear to result from losses of function in ABI1 because, in germination assays on ABA, the revertant alleles were recessive to both the abi1-1 and the wild-type ABI1 alleles. These combined genetic and molecular characteristics thus indicate that the ABA supersensitivity of the various revertants analyzed here is due to a loss of ABI1 phosphatase activity. It would be of clear interest to analyze the effect on ABA sensitivity of the suppressor mutations alone (i.e., in the absence of abi1-1), and currently, we are attempting to isolate such plants. However, the observation that the ABA-supersensitive phenotype of the revertant alleles is recessive to the wild-type ABI1 allele, whereas the abi1-1 mutation alone leads to a dominant ABA-resistance phenotype, makes it unlikely that the abi1-1R1 to abi1-1R7 revertant alleles interfere with ABA sensitivity simply because of the presence of the abi1-1 mutation. Hence, the present data indicate that a loss of ABI1 phosphatase activity leads to an enhanced responsiveness to ABA. This study thus provides genetic evidence that ABI1 does regulate ABA sensitivity and, more specifically, that the ABI1 phosphatase is a negative regulator of ABA responses.

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Table 4.

Germination on ABA of Parental and F1 Seeds

Figure 5.
Figure 5.

Differential Phenotypes of Plants Subjected to Progressive Drought Stress.

Plants were grown in a microphytotron under well-watered conditions for 1 month and then subjected to drought stress by withholding water. The soil surface was protected from evaporation by a sheet of Parafilm (American National Can, Chicago, IL).

(A) and (B) abi1-1 and Ler wild-type plants, respectively, were photographed when the soil water content had decreased to 35% (20 days after the onset of the progressive drought stress).

(C) and (D) Ler wild-type and abi1-1R3 plants, respectively, were photographed when the soil water content had decreased to 15%. This value of soil water content was reached 32 and 42 days after the onset of the drought stress for Ler and abi1-1R3, respectively.

The ABA-supersensitive phenotype of the abi1-1R1 to abi1-1R7 revertants readily explains why recessive loss-of-function mutants of ABI1 have not been isolated in screens based on ABA-resistant seed germination (Koornneef et al., 1984; Finkelstein, 1994a). Our results indicate that recessive mutants of ABI1 should rather be identifiable in screens for seeds with increased sensitivity to the ABA inhibition of germination. Thus far, this type of screening process has led to the isolation of three classes of ABA-supersensitive mutants, which were designated as era1 to era3 (Cutler et al., 1996). The era1 mutants have several phenotypes in common with the revertant alleles of ABI1 described here (Cutler et al., 1996; Pei et al., 1998). However, the ERA1 gene encodes the β subunit of a protein farnesyl transferase and thus clearly is distinct from ABI1 (Cutler et al., 1996). Likewise, the era3 mutation has been mapped to a chromosome other than ABI1 (P. McCourt, personal communication). It remains to be investigated whether the era2 mutant is allelic to ABI1.

Our screen clearly was not at saturation because only one suppressor mutation was isolated twice independently (in abi1-1R5 and abi1-1R8). It is nevertheless intriguing that we did not recover any suppressor mutation introducing, for instance, a premature stop codon in the ABI1 gene. Similarly, the five independent intragenic suppressors of abi1-1 that were isolated by Steber et al. (1998) all contain missense mutations in the ABI1 gene (F. Gosti, D. Guerrier, J. Giraudat, and P. McCourt, unpublished observations). It is thus likely that true molecular null alleles of ABI1 were counterselected in the suppressor screens, although such alleles would be predicted to have lost the ABA-resistant phenotype of abi1-1. Among other possibilities, it is conceivable that molecular null alleles of ABI1 might have a highly increased degree of seed dormancy and hence were not retained in the screens because of their poor germination rate, even in the absence of added ABA. In any case, characterizing the phenotype of knockout mutants would be instrumental in obtaining further insights into the physiological roles of ABI1 in the plant. In principle at least, such mutants should be present as heterozygotes in collections of insertion mutants and can be sought for by molecular techniques.

As summarized above, this study provides genetic evidence that the phosphatase activity of the wild-type ABI1 protein negatively regulates ABA signaling. This conclusion is consistent with the recent report that constitutive overexpression of ABI1 inhibited ABA action in maize protoplasts (Sheen, 1998). The molecular details of ABI1 action in the plant, and in particular the nature of the endogenous substrate of this PP2C, are not known. Nevertheless, Figure 6 shows two simple models of the possible mode of action of ABI1 as a negative regulator of ABA signaling. These models postulate that the catalytic activity of the ABI1 phosphatase is not (Figure 6A) or is (Figure 6B) regulated by ABA, respectively. In the model in Figure 6A, the ABI1 protein belongs to a regulatory branch that attenuates ABA signaling. In the model in Figure 6B, ABI1 is a core element of the ABA transduction cascade, and ABA triggers physiological responses by inhibiting the catalytic activity of the ABI1 protein. Discriminating between these models most likely will require monitoring in the plant of the time course of the changes in ABI1 activity after the application of an ABA stimulus. The abundance of the ABI1 mRNA is enhanced by ABA treatment, and this induction is inhibited in the abi1-1 mutant (Leung et al., 1997). In both models discussed above, transcriptional induction of the ABI1 gene thus could create a negative feedback regulatory loop. This loop might be responsible for resetting the ABA signaling cascade, thereby allowing the cell to continuously monitor the presence or absence of ABA. This scenario is analogous to the proposed role of other PP2Cs in the regulation of stress-induced pathways (Gaits et al., 1997; Meskiene et al., 1998).

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Table 5.

Drought Rhizogenesis

The dominant abi1-1 mutation converts Gly-180 to Asp in the vicinity of the PP2C active site (Leung et al., 1994; Meyer et al., 1994; Das et al., 1996) and leads to a decrease in ABA responsiveness (Koornneef et al., 1984). Our results indicate that the wild-type ABI1 phosphatase is a negative regulator of ABA signaling. Hence, a simple explanation of the abi1-1 phenotype would be that this mutant protein has enhanced phosphatase activity. This possibility is, however, hard to reconcile with the observation that the mutant abi1-1 protein had a lower PP2C activity than did the wild-type ABI1 in both in vitro enzymatic assays and yeast mutant complementation studies (Bertauche et al., 1996; Leube et al., 1998). Nevertheless, in the model in Figure 6B, the phenotype of the abi1-1 mutant could be explained by hypothesizing that the residual PP2C activity of this mutant protein is no longer regulated by ABA and that this constitutive activity is sufficient to inhibit ABA responsiveness. In the intragenic revertants, the loss of the residual PP2C activity of abi1-1 then would readily account for the suppression of the abi1-1 phenotypes. Alternatively, in the model depicted in Figure 6A, one could hypothesize that the mutant abi1-1 protein has dominant negative effects because, as a result of conformational alterations in the catalytic site, it traps its endogenous substrate into a poison complex. This substrate then no longer would be able to play its role in relaying the ABA signal within the transduction cascade. The intragenic suppressor mutations, which all map to highly conserved domains in proximity to the ABI1 PP2C catalytic site, would disrupt the formation of the dysfunctional protein complex between abi1-1 and its substrate. The various hypotheses envisaged above can be experimentally tested with confidence only once the endogenous substrate of ABI1 has been identified.

The ABI2 gene is closely related to ABI1. Furthermore, the dominant abi1-1 and abi2-1 mutations translate into precisely the same amino acid change in the PP2C domain of the corresponding protein and lead to largely similar phenotypes of ABA insensitivity (Koornneef et al., 1984; Leung et al., 1997; Rodriguez et al., 1998a). Our results thus suggest that by homology to ABI1, the wild-type ABI2 phosphatase as well would be a negative regulator of ABA action. These two homologous phosphatases are unlikely to have completely redundant roles because the revertant alleles of ABI1 displayed a detectable ABA-supersensitive phenotype in various assays. Nevertheless, it would be informative to test whether simultaneous disruption of the ABI1 and ABI2 PP2C activities leads to a further increase in ABA responsiveness. Moreover, although corresponding mutants have not been isolated thus far, two additional Arabidopsis PP2Cs have been proposed to regulate ABA signal transduction (Rodriguez et al., 1998b; Sheen, 1998) and also may function as redundant negative regulators. The signaling pathways of various plant hormones recently have been shown to involve homologous and redundant negative regulators. ETR1-related ethylene receptors act as redundant negative regulators of ethylene responses in Arabidopsis (Hua and Meyerowitz, 1998). Likewise, available evidence suggests that the putative transcription factor GAI (for gibberellin insensitive) (Peng et al., 1997) and its homolog RGA (for repressor of the gal-3 mutant (Silverstone et al., 1998) are redundant negative regulators of gibberellin action in Arabidopsis. Our findings suggest that ABA signaling also may be controlled by homologous and redundant negative regulators, in this case PP2Cs.

Figure 6.
Figure 6.

Hypothetical Models for the Role of ABI1 in ABA Signaling.

(A) The catalytic activity of the ABI1 protein is not regulated by ABA. Perception of the ABA signal stimulates the transduction cascade, leading to the activation of the physiological response. The ABI1 phosphatase attenuates the transduction cascade by dephosphorylating one of the signaling proteins. In the abi1-1R1 to abi1-1R7 revertant alleles (abi1-1Ri), the loss of ABI1 PP2C activity (indicated by the X) leads to enhanced ABA responsiveness. The ABA-induced elevation of ABI1 mRNA amount creates a negative feedback loop (indicated by the dotted line).

(B) ABA inhibits the catalytic activity of the ABI1 protein. In the absence of ABA, the phosphatase activity of ABI1 represses the response. ABA (or an ABA-derived signal) inhibits the PP2C activity of ABI1, thereby releasing the repression and triggering the physiological response. In the revertant alleles, the loss of ABI1 PP2C activity (indicated by the X) enhances the response. The ABA-induced elevation of ABI1 mRNA level creates a negative feedback loop (indicated by the dotted line).

METHODS

Plant Material and Growth Conditions

The abi1-1 line used in this work carries the linked visible marker eceriferum4 (cer4) that was used to easily distinguish wild-type individuals (CER+) from abscisic acid (ABA)–sensitive revertants derived from our mutagenized abi1-1 population (cer–). The recessive cer4 mutation reduces the amount of epicuticular wax on the stem, which leads to a characteristic bright green color (Koornneef et al., 1989a). We verified that the cer4 mutant does not significantly differ from the wild type in the various assays used in this study to analyze sensitivity to applied or endogenous ABA. For instance, seed germination rates were 88.4% for Landsberg erecta (Ler) and 87.7% for cer4 on 0.3 μM ABA, and 13.9% for Ler and 18.3% for cer4 on 0.6 μM ABA. Seedling root growth (expressed as a percentage relative to controls incubated on ABA-free medium) was 58.34 ± 0.98 for Ler and 55.68 ± 1.27 for cer4 on 0.6 μM ABA, and 10.54 ± 0.37 for Ler and 11.83 ± 0.69 for cer4 on 3 μM ABA. Water loss from detached aerial parts (expressed as a percentage of initial fresh weight) was 16.99 ± 1.39 for Ler and 16.07 ± 3.06 for cer4 after 20 min, and 22.64 ± 1.54 for Ler and 21.57 ± 4.12 for cer4 after 40 min. Stomatal apertures in isolated epidermal strips were 4.8 ± 0.1 μm (n = 120) for both Ler and cer4. Finally, drought rhizogenesis intensity (number of short roots produced per milligram of total root biomass) was 13.48 ± 0.76 for Ler and 13.24 ± 0.52 for cer4.

The abi1-1 (Koornneef et al., 1984) and cer4 (Koornneef et al., 1989a) mutants are both in the Arabidopsis thaliana ecotype Ler, and the abi1-1 cer4 double mutant was selected in the F2 progeny of a cross between homozygous abi1-1 and cer4 single mutants.

Plants were routinely grown in a greenhouse (22°C with a 16-hr photoperiod) on soil irrigated with mineral nutrients. For aseptic growth, seeds were surface-sterilized and plated on a medium containing the inorganic salts of Murashige and Skoog (1962) at half concentrations, 100 mg/L myoinositol, 1 mg/L thiamine-HCl, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, 1% sucrose (unless otherwise indicated), 0.8% agar, and 2.5 mM (2-[N-morpholino] ethanesulfonic acid)–KOH, pH 5.7. ABA (mixed isomers; Sigma) was diluted from 10-mM stock solutions prepared in methanol; equivalent volumes of methanol were included in the ABA-free controls.

Mutagenesis and Mutant Screening

Approximately 4000 abi1-1 cer4 seeds were mutagenized by imbibition in 0.4% (v/v) ethyl methanesulfonate (EMS; Sigma) for 8 hr at room temperature. After extensive washes with water to remove residual EMS, the mutagenized seeds (M1) were sown and grown under standard greenhouse conditions. The M2 seeds were harvested as 185 independent pools (each pool corresponding to ∼15 viable M1 plants).

To screen for revertants of abi1-1, we surface-sterilized ∼750 seeds from each M2 pool and plated the seeds on medium (without sucrose) supplemented with 3 μM ABA. Plates were kept in the dark at 4°C for 3 days to break seed dormancy and then were incubated at 21°C with a 16-hr-light photoperiod. After 4 days at 21°C, the seeds that displayed an appearance similar to that of wild-type controls (absence of radicle emergence or emerged-but-growth-arrested seedling; see Figure 1A) were transferred to agar plates without ABA. The seedlings that germinated and developed well after 7 days on the ABA-free medium were retained as candidate suppressors. These M2 candidates then were examined for their sensitivity to the inhibition of root growth by ABA. The seedlings were incubated vertically on agar plates supplemented with 10 μM ABA, and their root growth was analyzed after 3 days. Finally, the selected plantlets were propagated in soil, and their phenotype was retested in the next (M3) generation. Revertants originating from the same pool of M2 seeds were assumed to be siblings, and only one representative was retained for further analysis.

Phenotypic Analyses

Detailed phenotypic characterization of the intragenic revertants was performed with lines that had been backcrossed at least once to abi1-1 cer4. Seeds used for comparative phenotypic studies were derived from maternal plants grown and harvested simultaneously. The kinetics of water loss from detached rosette leaves were analyzed as previously described (Leung et al., 1997). Stomatal aperture measurements on leaf epidermal strips were performed according to Webb and Hetherington (1997). To examine drought responses of whole plants, we subjected adult Arabidopsis to progressive drought stress as described previously (Vartanian et al., 1994). In brief, plants were grown for 1 month under well-watered conditions and then subjected to a progressive drought stress by withholding watering under controlled conditions in a microphytotron (temperature of 22°C, relative atmospheric humidity of 56%, and photon flux density of 200 μmol m–2 sec–1 during an 8-hr-light photoperiod). To determine the drought rhizogenesis intensity, we counted the number of drought-induced short roots on plants harvested after the complete decline in the transpiration rate and when the soil moisture had decreased below 0.8% (Vartanian et al., 1994).

DNA Sequencing

Overlapping fragments encompassing the entire ABI1 gene were obtained from plant genomic DNA (Konieczny and Ausubel, 1993) with polymerase chain reaction (PCR) by using specific primers. The amplified products were sequenced directly on both strands by using appropriate primers. Double-stranded DNA was sequenced on an Applied Biosystems (Foster City, CA) automated DNA sequencer (model 373A). Sequence analyses and alignments were performed using programs of the Genetics Computer Group (Madison, WI) software package (Devereux et al., 1984).

PP2C Activities

Recombinant proteins that consisted of glutathione S-transferase (GST) fused to the wild type or various mutant forms of the full-length ABI1 protein were produced in Escherichia coli. Nucleotides 432 to 794 of the ABI1 cDNA (encoding the N-terminal part of ABI1) were amplified by PCR from pcABI1-C38 (GenBank accession number X77116; Leung et al., 1994) and cloned into the pGEX-2TK vector (Pharmacia Biotechnology) to create the pGEX-2TK/ABI1Nterm. The construct pGEX-2TK/ABI1 expressing a fusion between GST and the full-length wild-type ABI1 protein (GST–ABI1) then was generated by inserting into pGEX-2TK/ABI1Nterm the nucleotides 795 to 1926 of the ABI1 cDNA recovered as an EcoRI fragment from pcABI1/Sma (Bertauche et al., 1996). The construct pGEX-2TK/abi1-1 expressing GST fused to the mutant abi1-1 protein (GST–abi1-1) was obtained by the same procedure, except that the mutant pcabi1-1/Sma plasmid (Bertauche et al., 1996) was used instead of pcABI1/Sma. Because the R1 mutation destroys a unique SstI site in the ABI1 gene, cDNAs corresponding to the abi1-R1 and abi1-1R1 mutant genes were created by site-directed mutagenesis of the ABI1 and abi1-1 cDNAs, respectively (Bertauche et al., 1996). Nucleotides 795 to 1926 of the abi1-R1 and abi1-1R1 cDNAs then were inserted into pGEX-2TK/ABI1Nterm to generate fusions between GST and the abi1-R1 (GST–abi1-R1) and abi1-1R1 (GST–abi1-1R1) proteins, respectively. cDNA fragments for the abi1-1R2 to abi1-1R7 mutant genes were generated by reverse transcription (RT)–PCR, starting from total RNA of 6-day-old plantlets that had been treated with 10 μM ABA for 3 days. For the R2, R3, R5, and R6 suppressor mutations, the SstI-HindIII fragment comprising nucleotides 1341 to 1866 was excised from the RT-PCR product and cloned in place of the corresponding restriction fragment in pGEX-2TK/ABI1 or pGEX-2TK/abi1-1. For the R4 and R7 suppressor mutations, the BglII-SstI fragment comprising nucleotides 563 to 1341 was excised from the RT-PCR product and cloned in place of the corresponding restriction fragment in pGEX-2TK/abi1-1.

All constructs were verified by DNA sequencing. Recombinant proteins were expressed in the E. coli BL21 strain and were affinity purified on glutathione Sepharose 4B resin (Bertauche et al., 1996). Aliquots of the affinity-purified samples were analyzed by SDS-PAGE, and the amounts of recombinant proteins were quantified by scanning the Coomassie Brilliant Blue R 250–stained gel with BSA as a standard. Protein phosphatase activity was determined by standard procedures, using 32P-labeled casein (0.5 to 0.9 × 109 cpm/μmol Pi) as substrate (MacKintosh, 1993; Bertauche et al., 1996). All reactions were performed in duplicate. Specific enzymatic activity was determined by measuring the velocity of 32Pi release achieved with serial dilutions of the recombinant protein.

Acknowledgments

We thank Maarten Koornneef for providing the abi1-1 and cer4 mutants; Camille Steber and Peter McCourt for sharing their intragenic revertants of abi1-1; Eric Requis for help during the initial characterization of abi1-1R1; Marcel Jacquin, Lucette Marcotte, and Rémy Drouen for technical assistance with plant culture; Liliane Troussard for technical assistance with DNA sequencing; and Nathalie Mansion for photographic work. We also thank Alistair Hetherington and Gerd Jürgens for stimulating discussions, and Jeff Leung, Sylvain Merlot, and François Parcy for their continuous interest and valuable comments on this manuscript. This work was supported in part by the Centre National de la Recherche Scientifique, the International Human Frontier Science Program (Grant No. RG-303/95), and the European Community BIOTECH program (Grant No. BIO4-CT96-0062).

Footnotes

  • ↵1 Current address: Biochimie et Physiologie Moléculaire des Plantes, Institut National de la Recherche Agronomique, Place Viala, 34060 Montpellier Cedex 1, France.

  • ↵2 Current address: Department of Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, UK.

  • Received July 9, 1999.
  • Accepted August 11, 1999.
  • Published October 1, 1999.

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ABI1 Protein Phosphatase 2C Is a Negative Regulator of Abscisic Acid Signaling
Françoise Gosti, Nathalie Beaudoin, Carine Serizet, Alex A. R. Webb, Nicole Vartanian, Jérôme Giraudat
The Plant Cell Oct 1999, 11 (10) 1897-1909; DOI: 10.1105/tpc.11.10.1897
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