Abstract
Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. PYRABACTIN RESISTANCE1/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS ABA receptors interact with high affinity and inhibit clade A phosphatases type-2C (PP2Cs) in an ABA-dependent manner. We generated an allele library composed of 10,000 mutant clones of Arabidopsis (Arabidopsis thaliana) PYL4 and selected mutations that promoted ABA-independent interaction with PP2CA/ABA-HYPERSENSITIVE3. In vitro protein-protein interaction assays and size exclusion chromatography confirmed that PYL4(A194T) was able to form stable complexes with PP2CA in the absence of ABA, in contrast to PYL4. This interaction did not lead to significant inhibition of PP2CA in the absence of ABA; however, it improved ABA-dependent inhibition of PP2CA. As a result, 35S:PYL4(A194T) plants showed enhanced sensitivity to ABA-mediated inhibition of germination and seedling establishment compared with 35S:PYL4 plants. Additionally, at basal endogenous ABA levels, whole-rosette gas exchange measurements revealed reduced stomatal conductance and enhanced water use efficiency compared with nontransformed or 35S:PYL4 plants and partial up-regulation of two ABA-responsive genes. Finally, 35S:PYL4(A194T) plants showed enhanced drought and dehydration resistance compared with nontransformed or 35S:PYL4 plants. Thus, we describe a novel approach to enhance plant drought resistance through allele library generation and engineering of a PYL4 mutation that enhances interaction with PP2CA.
Highlights
Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors
Monomeric ABA receptors are able to interact in the absence of ABA to some extent with the catalytic core of ABI1/ABI2/HYPERSENSITIVE TO ABA1 (HAB1)/PP2CA phosphatases, less stable complexes are formed compared with ternary complexes with ABA (Dupeux et al, 2011a; Hao et al, 2011; Antoni et al, 2013)
PYL4 interacts in an ABA-dependent manner with PP2CA in Y2H assays (Lackman et al, 2011; Fig. 1A)
Summary
Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. Monomeric ABA receptors are able to interact in the absence of ABA to some extent with the catalytic core of ABI1/ABI2/HAB1/PP2CA phosphatases, less stable complexes are formed compared with ternary complexes with ABA (Dupeux et al, 2011a; Hao et al, 2011; Antoni et al, 2013) Boosting of such interaction might lead to faster association kinetics with PP2Cs or faster response to low-intermediate ABA levels. Y2H interactions of PYR/PYLs and PP2Cs that are dependent on exogenous ABA offer the possibility to set up screenings involving the generation of allele libraries and growth tests aimed to identify mutations that render ABAindependent interactions Such mutations might lead in the plant cell to receptors that (1) show enhanced association kinetics with PP2Cs by generating additional contact points and (2) interfere with PP2C function by steric hindrance or, when several mutations are combined, to CA receptors that inhibit PP2Cs in the absence of ABA. PP2CA is a physiologically relevant target to design PYR/ PYL receptors that show enhanced interaction with the phosphatase, affecting ABA signaling and plant stress response
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