Abstract
      The plant hormone abscisic acid (ABA) plays a key role in regulation of plant development and coordination of the adaptive responses under environmental stress conditions. To obtain further insights into ABA signalling, we have characterized a new family of interacting partners of the clade A PP2C HAB1. These interacting partners belong to a 14-member subfamily of the Betv1-like superfamily and they have been named PYR/PYL/RCARs (Pyrabactin resistance/pyrabactin resistance-like/regulatory components of ABA receptor). Interaction between HAB1 and one of the PYR/PYL/RCAR members, PYL5, was confirmed by yeast-two-hybrid, BiFC and co-immunoprecipitation assays. PYL5 was localized both in nucleus and cytosol. Due to the large number of family members, an over-expression approach was carried out to study the role of these proteins in ABA signalling. PYL5 over-expression led to a globally hypersensitive response to ABA, which contrasted with the opposite phenotype reported for HAB1 over-expressing plants, indicating that this new family played a positive role in ABA signalling. Furthermore, PYL5 over-expressing plants, showed an enhanced resistance to drought. F2 plants that over-expressed both HAB1 and PYL5 exhibited hypersensitivity to the hormone, indicating that PYL5 was antagonizing HAB1 function. Moreover, PYL5 and other members of its family were able to inhibit HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner, which indicated that the PYR/PYL/RCAR members were exerting their positive role in ABA signalling by inhibiting the negative regulators of the pathway. In addition, Isothermal titration calorymetry assays revealed that PYL5 was able to bind (+)ABA, in a saturable and specific manner, with Kd values of 1.1 µM or 38 nM in the absence or presence of the PP2Ccatalytic core of HAB1, respectively. PYL5 also presented partial stereoespecificity, being able to bind the non-natural form of (-)ABA with  lower affinity values (Kd=19 µM). All these data suggested that these new family might be an intracellular family of ABA receptors. To further confirm that, we carried out structural studies and as a result we obtained the crystal structure of the Arabidopsis thaliana PYR1 member. The PYR1 structure consisted of a dimer in which one of the subunits was bound to ABA. The monomeric subunits of this receptor protein were structurally arranged to form a hydrophobic central cavity where the hormone accommodates. Comparison of both, the free-ABA and the ABA-bound subunits, allowed studying the conformational changes induced upon binding of the hormone. In the ligand-bound subunit it could be appreciated that the loops surrounding the entry to the binding cavity fold over the ABA molecule, burying it inside, whereas in the empty subunit they were forming a passage, leaving an open access to the cavity. This was indicating that the conformational changes in these loops had a critical role in the stabilization of the hormone-receptor complex. In addition, a mutational analysis has also revealed that these loops and their conformational change upon ABA binding also played a critical role on the interaction with the PP2C. Further characterization of this receptors family, has revealed that they can be divided in monomeric and dimeric receptors in their unactivated apo form. We have characterized a key residue in PYR1, His60, that is variable between family members and that played a critical role in determining oligomeric state. The formation of homodimers has been seen to be disfavourable for ABA and PP2C binding. This different oligomeric state confers them different properties, which suggests that the PYR/PYL/RCAR members might have differential cellular responses over the physiological range of ABA concentrations. 
      Moreover, we have seen that the ABA-PYR/PYL/RCAR-PP2C signalling pathway is conserved in cultivated plants, in particular in rice. This provides a new framework in ABA signalling susceptible of modification, as well as for the design or identification of new ABA activators that would allow the manipulation of ABA responses in plants, in order to improve crop yield under drought contidions.