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.     

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.     

Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance

To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.     

The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination

Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed.     

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.     

Abscisic acid inhibition of radicle emergence but not seedling growth is suppressed by sugars

Low concentrations of sugars altered the sensitivity of seed germination to inhibition by exogenous abscisic acid (ABA). Germination of wild-type and ABA-insensitive (abi) Arabidopsis seeds was assayed on media containing ABA and a variety of sugars and sugar alcohols. The inhibitory effects of ABA were strongly repressed in the presence of 15 to 90 mM glucose (Glc), sucrose, or fructose, but not by comparable concentrations of sorbitol or mannitol. Several features of the response to Glc are inconsistent with a purely nutritional effect: The optimal sugar concentration is low and differs between the wild type and the abi mutants. Furthermore, Glc suppression of ABA inhibition is light dependent and limited to the process of radicle emergence.     

Interactions between abscisic acid and ethylene signaling cascades

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 ethylene-insensitive 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.     

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.     

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.