Abscisic acid (ABA) is a plant hormone that plays a crucial role in adaptive responses of plants to drought stress, salinity or cold, in addition to regulating important processes of plant development such as embryo development, dormancy, germination, vegetative growth, organogenesis and flowering. In the field of ABA signaling, there are many evidences showing that protein phosphatase 2C (PP2Cs) are key players in the processes that mediates ABA. Among PP2C phosphatases from Arabidopsis thaliana, the protein ABI1 and ABI2 (ABA-INSENSITIVE) act as negative regulators of signal transduction pathway of ABA. Our work focuses on the PP2C HAB1 (HYPERSENSITIVE TO ABA), whose genome sequence was cloned by homology to ABI1 and ABI2. To study and characterize this new PP2C, we found that HAB1 is ubiquitously expressed at important sites of action of ABA as guard cells or seeds and its expression is inducible by ABA. 
We performed a reverse genetic approach, new in the field of ABA signalling, with the isolation and characterization of the loss of function allele hab1-1 and, with the generation and study of lines that overexpress HAB1. The hypersensitivity showed by hab1-1 mutant and the insensitivity of plants 35S: HAB1 provide a new genetic evidence of the role of PP2C HAB1 as negative regulator of ABA signaling. In order to improve the knoledge and to go deeper into the molecular details of HAB1 function in ABA-signaling pathway, we performed a yeast two-hybrid screening for potential interaction targets of HAB1 using a cDNA library of Arabidopsis. In this screening we detected that HAB1 interacts with the protein SWI3B, a homolog of the SWI3B subunit of the chromatin remodeling complex SWI/SNF in yeast. The interaction mapped to the middle of the N-terminal domain of AtSWI3B, is HAB1 specific and requires an intact catalytic domain of the phosphatase. We confirmed the nuclear interaction between SWI3B and HAB1 by assays of protein-protein interaction of bimolecular fluorescent complementation (BIFC) in Nicotiana benthamiana and in coimmunoprecipitation experiments. Swi3b mutants show reduced sensitivity to ABA in inhibition of germination and growth assays, as well as a reduced expression of ABA-response genes RAB18 and RD29B. Chromatin immunoprecipitation experiments (ChIP) demonstrate that the presence of HAB1 in the promoters of the marker genes RAB18 and RD29B is suppressed by the presence of ABA, which directly involves HAB1 in the regulation of transcription in response to ABA. Additionally, these results show the role of SWI3B as a new positive regulator of ABA signaling, being HAB1 a modulator of the response to ABA through a putative chromatin remodeling complex type SWI / SNF.
Because clade A PP2Cs family of proteins constituted by HAB1, ABI1, ABI2 and PP2CA act as key negative regulators of ABA signaling pathway, we isolated and characterized single mutants subsequently to generate different combinations of double and triple mutant null for these PP2Cs. Previous studies to this thesis have not analyzed single, double and triple knockout mutants in plant PP2Cs. The objective was to determine its contribution to the ABA signaling pathway and unravel possible interactions between genes and possible functional redundancy between PP2Cs. The comparison of responses to ABA of different pp2cs mutants showed a progressive increase in ABA sensitivity obtained through the combined inactivation of these PP2Cs. These results indicate that the response to ABA is finely regulated by the integrated action of these genes, which is required to prevent constitutive response to endogenous ABA which could have deleterious effects on growth and development in the absence of environmental stress. Abscisic acid has an essential role in the response to drought. Despite of the fact that it has been described many mutants that are hypersensitive to ABA, few of them show tolerance to drought.
In this thesis we have generated hypersensitive to ABA mutants that are drought tolerant by the combined inactivation of the PP2Cs HAB1 and ABI1. The double mutants hab1-1abi1-2 and hab1-1abi1-3 show enhanced responses to ABA in both seed and vegetative tissue, they are also particularly sensitive to inhibition of seed germination mediated by ABA showing hypersensitivity to ABA in growth stomatal closure and genes induction of ABA-response assays, compared with single mutants. In experiments of water loss through transpiration under drought hab1-1abi1-2 and hab1-1abi1-3 showed a significant reduction in the loss of water compared to single parental mutants. These results show that combined inactivation of specific PP2Cs involved in ABA signaling may be a biotechnological tool in the improvement of drought-tolerant crops.