SummaryThe Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identi®ed by the dominant mutations abi1±1 and abi2±1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the ®rst reduction-of-function allele of ABI2, abi2±1R1. The in vitro phosphatase activity of the abi2±1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2±1R1 plants displayed a wildtype ABA sensitivity. However, doubly mutant plants combining the abi2±1R1 allele and a loss-offunction allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.

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. INTRODUCTIONAbscisic 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, 1...

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethyleneinsensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1 , CTR1 , and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene-and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth. INTRODUCTIONAbscisic acid (ABA) regulates various aspects of plant growth and development, including seed maturation and dormancy, as well as adaptation to abiotic environmental stresses (Zeevaart and Creelman, 1988; Davies and Jones, 1991). Substantial progress has been made in the characterization of ABA signaling pathways (Busk and Pagès, 1997; Bonetta and McCourt, 1998;Leung and Giraudat, 1998;MacRobbie, 1998). In particular, mutational analyses in Arabidopsis have led to the identification of several genes that control ABA responsiveness. These genetic screens were based primarily on the inhibition of seed germination by applied ABA. The ABA-insensitive ( abi ) mutants abi1 to abi5 are able to germinate in the presence of ABA concentrations that are inhibitory to the wild type (Koornneef et al., 1984;Ooms et al., 1993; Finkelstein, 1994;Nambara et al., 1995). In contrast, germination of the era1 (enhanced response to ABA) to era3 mutant seed is prevented by low concentrations of ABA that ordinarily permit germination of wild-type seed (Cutler et al., 1996). As judged from their impact on seed dormancy, these two sets of mutations also alter the regulation of seed germination by endogenous ABA. Like ABA-deficient mutants (Karssen et al., 1983;Léon-Kloosterziel et al., 1996a), the ABA-insensitive mutants abi1 to abi3 display marked reductions in seed dormancy (Koornneef et al., 1984;Ooms et al., 1993;Nambara et al., 1995). Conversely, the ABA-supersensitive era1 mutation confers enhanced seed dormancy (Cutler et al., 1996).The abi3 , abi4, and abi5 mutants exhibit additional defects in various aspects of seed maturation (Koornneef et al., 1984; Finkelstein and Somerville, 1990;Ooms et al., 1993; Finkelstein, 1994;Parcy et al., 1994;Nambara et al., 1995). The ABI3 and ABI4 genes have been cloned and encode putative transcriptional regulators. ABI3 is orthologous to the maize Viviparous 1 protein (McCarty et al., 1991;Giraudat e...

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 veg-etative 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 rever-tants 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.