Molecular and genetic analyses of the PP2C-ABA receptor interaction in the abscisic acid signaling pathway

La fitohormona ácido abscísico (ABA) juega un papel crucial en el control de la respuesta a estrés y en la regulación del crecimiento y desarrollo de la planta. La unión del ABA a los receptores intracelulares PYR/PYL/RCAR conlleva la inhibición de las PP2Cs del clado A tales como ABI1 o HAB1, causando la activación de la ruta de señalización del ABA. Para obtener más información en la señalización del ABA nos hemos centrado en la caracterización de miembros de estas dos familias proteicas. Hemos generado una versión mutada de HAB1 que contiene una mutación en el Trp-385, residuo clave para la interacción con los receptores y con la molécula de ABA. Como resultado, hab1W385A se mostró refractaria a la inhibición por los receptores PYR/PYL/RCAR. Así, en ensayos de actividad quinasa in vitro encontramos que hab1W385A era capaz de desfosforilar a OST1 incluso en presencia de ABA y de los receptores. hab1W385A y hab1G246D pueden ser clasificadas como mutaciones dominantes hipermórficas. Mientras que hab1G246D posee una actividad fosfatasa reducida, el nuevo alelo dominante muestra una actividad idéntica al genotipo salvaje. Líneas transgénicas de Arabidopsis sobreexpresando hab1W385A mostraron una fuerte insensibilidad al ABA. También hemos analizado el papel de las PP2Cs del clado A pertenecientes a la rama representada por PP2CA. La generación de un mutante doble pp2ca-1hai1-1, que muestra mayor sensibilidad a la hormona en comparación con el genotipo salvaje y con los mutantes sencillos, reveló que HAI1 es un regulador negativo de la ruta de señalización del ABA. El análisis de la localización subcelular mostró que tanto HAI1 como PP2CA se localizan en el núcleo, aunque también están presentes en el citosol y en la fracción microsomal. Tres miembros de la rama de PP2CA i.e.: PP2CA, AHG1 y HAI1, mostraron una inhibición selectiva por los receptores PYR/PYL/RCAR. Estos resultados sugieren que estos receptores pueden discriminar entre miembros del clado A de las PP2Cs. pyl8 es el único mutante sencillo que muestra sensibilidad reducida al ABA en ensayos de crecimiento de raíz. Análisis usando el gen reportero GUS mostraron que PYL8 estaba presente en la estela, en la epidermis de la raíz y en la caliptra, y la cuantificación de la actividad beta-glucuronidasa en raíz mostró que PYL8 es uno de los receptores con mayor nivel de expresión. La caliptra juega un papel crucial en la respuesta hidrotrópica. El estudio de esta respuesta en mutantes múltiples de las PP2Cs y de los PYR/PYL/RCAR reforzó la idea de que el ABA regula este proceso. Así, mientras el mutante séxtuple pyr/pyl112458 presentó una curvatura menor al aplicársele un gradiente de humedad, el mutante cuádruple de las PP2Cs (Qabi2-2) mostró una curvatura más pronunciada en estas condiciones, evitando las zonas con menor potencial hídrico. Finalmente, en la última parte de este trabajo se utilizaron abordajes genético-químicos para aumentar la resistencia a la sequía. Hemos llevado a cabo un rastreo con compuestos químicos para aislar nuevos agonistas del ABA. Basado en datos estructurales de los receptores, se seleccionaron 500 compuestos que fueron ensayados en Arabidopsis. De estos, el compuesto 2C06 inhibió el crecimiento de raíz en plantas salvajes más que en mutantes pyr/pyl/rcar insensibles a ABA y produjo resultados prometedores in vitro al inhibir a las PP2Cs e interaccionar con éstas en ensayos de doble híbrido.Antoni Alandes, R. (2013). Molecular and genetic analyses of the PP2C-ABA receptor interaction in the abscisic acid signaling pathway [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29756TESISPremiad

Selective Inhibition of Clade A Phosphatases Type 2C by PYR/PYL/RCAR Abscisic Acid Receptors

[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.).Antoni-Alandes, R.; González Guzmán, M.; Rodriguez, L.; Rodrigues, A.; Pizzio Bianchi, GA.; Rodríguez Egea, PL. (2012). Selective Inhibition of Clade A Phosphatases Type 2C by PYR/PYL/RCAR Abscisic Acid Receptors. Plant Physiology. 158(2):970-980. https://doi.org/10.1104/pp.111.188623S970980158

The PYL4 A194T mutant uncovers a key role of PYL4-PP2CA interaction for ABA signaling and plant drought resistance

[EN] 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.Pizzio Bianchi, GA.; Rodriguez, L.; Antoni-Alandes, R.; Gonzalez Guzman, M.; Yunta, C.; Merilo, E.; Kollist, H.... (2013). The PYL4 A194T mutant uncovers a key role of PYL4-PP2CA interaction for ABA signaling and plant drought resistance. Plant Physiology. 163(1):441-455. doi:10.​1104/​pp.​113.​224162S441455163

PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root

[EN] Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-INSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. We also discovered that PYR/PYL receptors and clade A PP2Cs are crucial for the hydrotropic response that takes place to guide root growth far from regions with low water potential. Thus, an ABA-hypersensitive pp2c quadruple mutant showed enhanced hydrotropism, whereas an ABA-insensitive sextuple pyr/pyl mutant showed reduced hydrotropic response, indicating that ABA-dependent inhibition of PP2Cs by PYR/PYLs is required for the proper perception of a moisture gradient.This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant no. BIO2011-23446 to P. L. R.; fellowships to R. A., L. R., and M. P.-L.; Juan de la Cierva contract to M.G.-G.).Antoni-Alandes, R.; Gonzalez Guzman, M.; Rodriguez, L.; Peirats-Llobet, M.; Pizzio Bianchi, GA.; Fernández, MA.; De Winne, N.... (2013). PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiology. 161(2):931-941. doi:10.1104/pp.112.208678S931941161

C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis

Supplemental Data: http://www.plantcell.org/content/26/12/4802/suppl/DC1© 2014 American Society of Plant BiologistsMembrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.We thank Joerg Kudla (University of Munster) for kindly providing plasma membrane markers. This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (Grants BIO2011-23446 to P.L.R and BFU2011-25384 to A. A.; fellowships to L.R., R.A., and A.C.I.-G.; BES-2009- 016569; JAE-DOC contract to M.G.-G.) as well as the Senacyt-Ifarhu (Panama) (fellowship to M.D.).Rodriguez, L.; Gonzalez Guzman, M.; Díaz, M.; Rodrigues, A.; Izquierdo Garcia, AC.; Peirats-Llobet, M.; Fernández López, MA.... (2014). C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis. Plant Cell. 26(12):4802-4820. doi:10.1105/tpc.114.129973S48024820261

ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis

[EN] Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways.We thank Vera Nunes for plant management, Filip Rolland and Jorg Kudla for comments, and Julia Costa for help with the scintillation counter. The 35S:SnRK1.1-2 line was provided by Martine Thomas. E.B.-G. was supported by grants from Marie Curie IRG, the EMBO Installation program, Marie Curie Actions FP7-People-2010-ITN, the Fundacao para a Ciencia e a Tecnologia (FCT-PTDC/AGR-AAM/104939/2008), and the Portugal-Spain Bilateral Collaboration program Acoes integradas (Acao E-26/10). A.C. was supported by SFRH/BPD/47280/2008, C.M. was supported by SFRH/BD/33563/2008, L.M. was supported by SFRH/BD/51627/2011, and P.C. was supported by SFRH/BPD/79255/2011. A. Rabissi was supported by a Generalitat de Catalunya PhD grant (FI-AR067443). P.L.R. was supported by the Ministerio de Ciencia e Innovacion (grants BIO2011-23446 and PT2009-0155), R.A. was supported by the Junta para Ampliacion de Estudios e Investigaciones Cientificas-Consejo Superior de Investigaciones Cientificas fellowship, and M.G.-G. was supported by a Juan de la Cierva contract.Rodrigues, A.; Adamo, M.; Crozet, P.; Margalha, L.; Confraria, A.; Martinho, C.; Elias, A.... (2013). ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis. Plant Cell. 25(10):3871-3874. https://doi.org/10.​1105/​tpc.​113.​114066S38713874251

Selective single molecule sequencing and assembly of a human Y chromosome of African origin

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes

Selective single molecule sequencing and assembly of a human Y chromosome of African origin

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.This study was supported by the Spanish Ministry of Economy and Competitiveness with Proyectos de I+D “Excelencia” y Proyectos de I+D+I “Retos Investigación” BFU2014-55090-P awarded to T.M.-B. and O.F., Centro de Excelencia Severo Ochoa 2013–2017 and Centro de Excelencia Maria de Maeztu 2016–2019. We acknowledge the support from the CERCA Programme of the Generalitat de Catalunya, institutional support from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) through the Instituto de Salud Carlos III, from the Generalitat de Catalunya through the Departament de Salut and Departament d’Empresa i Coneixement, and co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program. L.F.K.K. is supported by an FPI fellowship associated with BFU2014-55090-P (MINECO/FEDER, UE). M.K. is supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship (KU 3467/1-1). T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya. D.J. is supported by a Juan de la Cierva fellowship (FJCI-2016-29558) from MICINN