ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

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.     

Maternal Effects Govern Variable Dominance of Two Abscisic Acid Response Mutations in Arabidopsis thaliana

Three abscisic acid (ABA)-controlled responses (seed dormancy, inhibition of germination by applied ABA, and stomatal closure) were compared in wild-type versus homo- and heterozygotes of two Arabidopsis thaliana ABA-insensitive mutants, abi1 and abi2. We found that sensitivity of seeds to applied ABA is partially maternally controlled but that seed dormancy is determined by the embryonic genotype. The effects of the abi1 and abi2 mutations on ABA sensitivity of seed germination ranged from recessive to nearly fully dominant, depending on the parental source of the mutant allele. This maternal effect disappeared during vegetative growth. Stomatal regulation in heterozygotes showed substantial variability, but the average water loss was intermediate between that of homozygous mutants and wild type.     

Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.     

Molecular character of a phosphatase 2C (PP2C) gene relation to stress tolerance in Arabidopsis thaliana

Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. HAI-1 gene had been shown to affect both seed and vegetative responses to ABA, which is one of PP2Cs clade A in Arabidopsis thaliana. Transgenic plants containing pHAI-1::GUS (β-glucuronidase) displayed GUS activity existing in the vascular system of leave veins, stems and petioles. Green fluorescent protein fused HAI-1 (HAI-1-GFP) was found in the nucleus through transient transformation assays with onion epidermal cells. The water-loss assays indicated the loss-of-function mutants did not show symptoms of wilting and they had still turgid green rosette leaves. The assays of seed germination by exogenous ABA and NaCl manifested that the loss-of-function mutants displayed higher insensitivity than wild-type plants. Taken together, the final results suggest that the HAI-1 (AT5G59220) encoded a nuclear protein and it can be highly induced by ABA and wound in Arabidposis, the stress-tolerance phenotype showed a slightly improvement when HAI-1 gene was disrupted.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified 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 first 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 wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function 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.     

Disruption of a guard cell-expressed protein phosphatase 2A regulatory subunit,RCN1, confers abscisic acid insensitivity in Arabidopsis

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell-expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca(2+) increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling.     

An Arabidopsis homolog of importin β1 is required for ABA response and drought tolerance

The import of proteins into the nucleus in response to drought is critical for mediating the reprogramming of gene expression that leads to drought tolerance. However, regulatory mechanisms involved in nuclear protein import remain largely unknown. Here, we have identified an Arabidopsis gene (AtKPNB1) as a homolog of human KPNB1 (importin β1). AtKPNB1 was expressed in multiple organs, and the protein was localized in the cytoplasm and nucleus. AtKPNB1 was able to facilitate nuclear import of a model protein. Null mutation of AtKPNB1 delayed development under normal growth conditions and increased sensitivity to abscisic acid (ABA) during seed germination and cotyledon development. Inactivation of AtKPNB1 increased stomatal closure in response to ABA, reduced the rate of water loss, and substantially enhanced drought tolerance. AtKPNB1 interacted with several importin α proteins, nucleoporin AtNUP62, and the Arabidopsis Ran proteins. Inactivation of AtKPNB1 did not affect the ABA responsiveness or the expression level or subcellular localization of ABI1, ABI2 or ABI5, key regulators of the ABA signaling pathway. Moreover, phenotypic analysis of epistasis revealed that AtKPNB1 modulates the ABA response and drought tolerance through a pathway that is independent of ABI1 and ABI5. Collectively, our results show that AtKPNB1 is an Arabidopsis importin β that functions in ABA signaling.     

The abi1-1 mutation blocks ABA signaling downstream of cADPR action

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.     

The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction.

Abscisic acid (ABA) mediates seed maturation and adaptive responses to environmental stress. In Arabidopsis, the ABA-INSENSITIVE1 (ABI1) protein phosphatase 2C is required for proper ABA responsiveness both in seeds and in vegetative tissues. To determine whether the lack of recessive alleles at the corresponding locus could be explained by the existence of redundant genes, we initiated a search for ABI1 homologs. One such homolog turned out to be the ABI2 locus, whose abi2-1 mutation was previously known to decrease ABA sensitivity. Whereas abi1-1 is (semi)dominant, abi2-1 has been described as recessive and maternally controlled at the germination stage. Unexpectedly, the sequence of the abi2-1 mutation showed that it converts Gly-168 to Asp, which is precisely the same amino acid substitution found in abi1-1 and at the coincidental position within the ABI1 phosphatase domain (Gly-180 to Asp). In vitro assays and functional complementation studies in yeast confirmed that the ABI2 protein is an active protein phosphatase 2C and that the abi2-1 mutation reduced phosphatase activity as well as affinity to Mg2+. Although a number of differences between the two mutants in adaptive responses to stress have been reported, quantitative comparisons of other major phenotypes showed that the effects of both abi1-1 and abi2-1 on these processes are nearly indistinguishable. Thus, the homologous ABI1 and ABI2 phosphatases appear to assume partially redundant functions in ABA signaling, which may provide a mechanism to maintain informational homeostasis.