title: The PYL4 A194T mutant uncovers a key role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA interaction for abscisic acid signaling and plant drought resistance.
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.
authors.affiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, ES-46022 Valencia, Spain.
authors.forename: Gaston A
authors.initials: GA
authors.lastname: Pizzio
authors.forename: Lesia
authors.initials: L
authors.lastname: Rodriguez
authors.forename: Regina
authors.initials: R
authors.lastname: Antoni
authors.forename: Miguel
authors.initials: M
authors.lastname: Gonzalez-Guzman
authors.forename: Cristina
authors.initials: C
authors.lastname: Yunta
authors.forename: Ebe
authors.initials: E
authors.lastname: Merilo
authors.forename: Hannes
authors.initials: H
authors.lastname: Kollist
authors.forename: Armando
authors.initials: A
authors.lastname: Albert
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Rodriguez
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology*; D055864:Droughts; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D030821:Plants, Genetically Modified / Q000502:physiology; D060066:Protein Interaction Maps; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics*; D014867:Water / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C587488:PYL4 protein, Arabidopsis; D011956:Receptors, Cell Surface; D014867:Water; D000040:Abscisic Acid; C463465:AHG3 protein, Arabidopsis; D010749:Phosphoprotein Phosphatases