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[EN] Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana. This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA. Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling, Work in P.L.R.'s laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (Grant BIO2014-52537-R). Work in R.T.M.'s laboratory was supported by a grant from the Natural Sciences and Engineering Research Council of Canada and a University of Guelph Research Chair. Work in J.L.'s laboratory was supported by the Ministerio de Ciencia e Innovacion and Fondo Europeo de Desarrollo Regional Grants BIO2011-27526 and BIO2014-56067-P L.R. was supported by a FPI fellowship and M.G.-G. by a JAE-DOC research contract. Work in L.J.'s laboratory was supported by grants from the Research Grants Council of Hong Kong (C4011-14R and AoE/M-05/12). We thank S.Y. Bednarek for the ProCLC2: CLC2-mOrange construct.

15 pags, 8 figs, 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 PYL4A194T 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: PYL4A194T 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:PYL4A194T 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. 2013 American Society of Plant Biologists. All Rights Reserved., This work was supported by the Ministerio de Ciencia e Innovación, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Científicas (grant nos. BIO2011–23446 to P.L.R and BFU2011–25384 to A.A., fellowships to R.A. and L.R., and a Juan de la Cierva contract to M.G.-G.) and the Estonian Ministry of Science and Education and European Regional Fund (IUT2–21 and Center of Excellence Environ to E.M. and H.K.).

[EN] Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally, the phosphomimetic BRMS1760D (S1762D) mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants., Work in Dr. Rodriguez's laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (grant BIO2014-52537-R). M.P.-L. and L.R. were supported by FPI fellowships, and M.G.-G. by a JAE-DOC research contract. Funding of chromatin research in the Wagner laboratory is supported by National Science Foundation grant MCB-0925071.

[EN] Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 mu M ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone., We thank Joseph Ecker and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, the Arabidopsis Biological Resource Center/Nottingham Arabidopsis Stock Centre for distributing these seeds, and Sean Cutler for the pyr1-1, 114 and 1124 mutants. This study was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant BIO2011-23446 to P. L. R; fellowship to R. A.; Juan de la Cierva contract to M. G.-G.), by the Estonian Ministry of Science and Education (ETF7763 and ETF9208, SF0180071s07), by the European Regional Fund (Center of Excellence in Environmental Adaptation), and by a Marie Curie Incoming International Fellowship to G. W. B.

[EN] Clade A protein phosphatases type 2C (PP2Cs) are negative regulators of abscisic acid (ABA) signaling that are inhibited in an ABA-dependent manner by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) intracellular receptors. We provide genetic evidence that a previously uncharacterized member of this PP2C family in Arabidopsis (Arabidopsis thaliana), At5g59220, is a negative regulator of osmotic stress and ABA signaling and that this function was only apparent when double loss-of-function mutants with pp2ca-1/ahg3 were generated. At5g59220-green fluorescent protein and its close relative PP2CA-green fluorescent protein showed a predominant nuclear localization; however, hemagglutinin-tagged versions were also localized to cytosol and microsomal pellets. At5g59220 was selectively inhibited by some PYR/PYL ABA receptors, and close relatives of this PP2C, such as PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (AHG3) and AHG1, showed a contrasting sensitivity to PYR/PYL inhibition. Interestingly, AHG1 was resistant to inhibition by the PYR/PYL receptors tested, which suggests that this seed-specific phosphatase is still able to regulate ABA signaling in the presence of ABA and PYR/PYL receptors and therefore to control the highly active ABA signaling pathway that operates during seed development. Moreover, the differential sensitivity of the phosphatases At5g59220 and PP2CA to inhibition by ABA receptors reveals a functional specialization of PYR/PYL ABA receptors to preferentially inhibit certain PP2Cs., This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant nos. BIO2008-00221 and BIO2011-23446 to P. L. R; fellowships to R.A. and L.R.; Juan de la Cierva contract to M.G.-G.).

The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1W385A, which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1W385A caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1W385A qualifies as a hypermorphic mutation. Expression of hab1W385A in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.

The stress hormone abscisic acid (ABA) induces expression of defense genes in many organs, modulates ion homeostasis and metabolism in guard cells and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H+ efflux (wat1-1D, over-expression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than wild type. This suggested that ABA could inhibit H+ efflux (H+-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibits the root plasma membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involves the core ABA signaling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1) and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicate that ABA, acting through the PYR/PYL/RCAR receptors, induces membrane hyperpolarization (due to K+ efflux through GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots are different from those in guard cells. ABA does not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+-ATPase in roots and SnRK2.2 phosphorylates in vitro the C-terminal regulatory domain of H+-ATPase while the guard cell kinase SnRK2.6/OST1 does not., This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish ‘Ministerio de Economía y Competitividad’, Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the ‘Generalitat Valenciana’, Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish ‘Consejo Superior de Investigaciones Científicas’, Madrid, Spain.