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...

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.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Four clones corresponding to Arabidopsis thaliana transcripts regulated by progressive drought stress were isolated. Abundance of the AtDi8, AtDi19 and AtDi21 mRNAs increased in both roots and leaves during progressive drought. The AtDr4 mRNA was expressed in a root-specific manner in regularly watered plants, and became undetectable under drought conditions. In all cases, the drought-induced modifications of mRNA abundance could be reversed by subsequent rehydration. The predicted AtDr4 protein displays extensive similarity to various members of the Künitz protein family, suggesting that AtDr4 might be a root-specific protease inhibitor. Of these four genes, only AtDi8 and AtDi21 responded to an exogenous supply of abscisic acid (ABA). Analysis of the ABA-deficient aba mutant demonstrated that endogenous ABA indeed participates in the drought regulation of these two transcripts. This ABA-dependent response was differentially affected in the various classes of ABA-insensitive Arabidopsis mutants. The AtDi19 and AtDr4 mRNAs both responded to drought in an ABA-independent manner, but at distinct thresholds of the progressive drought stress. Regulation of these four target genes by progressive drought stress thus appears to be mediated by at least three distinct signals, only one of which is ABA.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.