Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABAhypersensitive 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.

To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35STcDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 mM ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F 2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35STcDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35STAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABAhypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35STAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.