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

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. 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...

Role of Abscisic Acid in Seed Dormancy

“…The ABI1 gene of Arabidopsis, encodes a member of the 2C class of serine/threonine protein phosphatases (PP2Cs), and its activity is regulated by proton and Mg 2+ ions, but not by Ca 2+ ions (Leube and others 1998). The ABI2 gene of Arabidopsis also encodes a 2C class protein phosphatase, and it is likely that a family of these phosphatases acts as redundant negative regulators of ABA signaling (Gosti and others 1999), a contention supported by analysis of the Arabidopsis genome. There are about 70 proteins with homology to members of the family in Arabidopsis, several of which are related to ABI1 and ABI2 proteins (The Arabidopsis Genome Initiative 2000).…”

“…Even though the abi1 mutant is characterized by reduced sensitivity to ABA, ABI1 is indeed a negative regulator of ABA signaling (Gosti and others 1999). Protein phosphatase 2C activity is absent in intragenic revertants of abi1 and abi2 mutants, and the revertants are characterized by ABA hypersensitivity (Gosti and others 1999;Merlot and others 2001).…”

“…Protein phosphatase 2C activity is absent in intragenic revertants of abi1 and abi2 mutants, and the revertants are characterized by ABA hypersensitivity (Gosti and others 1999;Merlot and others 2001). Overexpression of ABI1 in maize protoplasts decreases the ABA-mediated activation of a chimeric gene driven by an ABA-inducible promoter and this, along with other evidence (Tahtiharju and Palva 2001), is consistent with the role of ABI1 as a negative regulator of ABA signaling (Sheen 1998).…”

Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs

“…These mutations were isolated 25-years ago by genetic screenings in Arabidopsis, aimed to the identification of ABAinsensitive mutants [27] and the cloning of the mutant loci provided pioneering insights into ABA signaling [28,29,30,31]. Both abi1-1D and abi2-1D are strong ABA-insensitive mutants that show diminished response to ABA in seeds and vegetative tissues, however, analysis of lossof-function alleles indicates that ABI1 and ABI2 gene products, as well as other clade A PP2Cs, are negative regulators of ABA signaling [32,33,34,35]. Since the phenotype of the abi1-1D and abi2-1D dominant alleles is just the opposite of loss-of-function alleles, they can´t represent dominant negative alleles, as it was accurately noted by Robert et al, [36].…”

Functional analysis of the ABA-responsive protein family in ABA and stress signal transduction in Arabidopsis

“…The phytohormone ABA plays a key role in regulating different aspects of plant growth and development, as well as the plant response to biotic and abiotic stress [1,2]. Therefore, it is of great interest to reveal the molecular mechanism of the function of ABA in signal transduction.…”