RESUMEN
Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Ligandos , Sequías , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Plant adaptation to environmental stress generated by low water availability requires continuous search for moisture niches in the soil. Thus, roots have evolved a hydrotropic response to sense differences in water potential of the soil, and through asymmetric growth, roots can bend to avoid lower water potential sites. Different experimental systems have been devised for hydrotropism assays, which usually rely on air moisture or split agar assays. This latter system uses plates containing an osmolyte only in a region of the plate in order to generate a water potential gradient. Seedlings are placed on the agar plate containing normal medium (NM) so that their root tips are near the junction between NM and the region supplemented with the osmolyte. As a result, a hydrotropic response is elicited to avoid the low water potential medium, which is reflected in the root curvature angle.
Asunto(s)
Arabidopsis , Agar , Raíces de Plantas , Suelo , AguaRESUMEN
ABA receptor agonists capable of improving plant performance under drought conditions have been described during the last years. However, monocot and eudicot plant species respond differently to various agonists. Here, we provide a detailed methodology to evaluate the anti-transpirant activity of ABA receptor agonists in both monocot and eudicot plant species using infrared imaging and image data analysis. Plant growth conditions, chemical application, and infrared image analysis are explained in detail to evaluate the anti-transpirant activity of ABA receptor agonists in the eudicot model Arabidopsis thaliana and in the C4-monocot model Setaria viridis.
Asunto(s)
Arabidopsis , Setaria (Planta) , Ácido Abscísico/farmacología , SequíasRESUMEN
Roots are composed of different root types and, in the dicotyledonous Arabidopsis, typically consist of a primary root that branches into lateral roots. Adventitious roots emerge from non-root tissue and are formed upon wounding or other types of abiotic stress. Here, we investigated adventitious root (AR) formation in Arabidopsis hypocotyls under conditions of altered abscisic acid (ABA) signaling. Exogenously applied ABA suppressed AR formation at 0.25 µM or higher doses. AR formation was less sensitive to the synthetic ABA analog pyrabactin (PB). However, PB was a more potent inhibitor at concentrations above 1 µM, suggesting that it was more selective in triggering a root inhibition response. Analysis of a series of phosphonamide and phosphonate pyrabactin analogs suggested that adventitious root formation and lateral root branching are differentially regulated by ABA signaling. ABA biosynthesis and signaling mutants affirmed a general inhibitory role of ABA and point to PYL1 and PYL2 as candidate ABA receptors that regulate AR inhibition.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal , Arabidopsis/metabolismo , Hipocótilo/crecimiento & desarrollo , Raíces de Plantas/metabolismoRESUMEN
The hab1-1abi1-2abi2-2pp2ca-1 quadruple mutant (Qabi2-2) seedlings lacking key negative regulators of ABA signaling, namely, clade A protein phosphatases type 2C (PP2Cs), show more apoplastic H+ efflux in roots and display an enhanced root growth under normal medium or water stress medium compared to the wild type. The presence of low ABA concentration (0.1 micromolar), inhibiting PP2C activity via monomeric ABA receptors, enhances root apoplastic H+ efflux and growth of the wild type, resembling the Qabi2-2 phenotype in normal medium. Qabi2-2 seedlings also demonstrate increased hydrotropism compared to the wild type in obliquely-oriented hydrotropic experimental system, and asymmetric H+ efflux in root elongation zone is crucial for root hydrotropism. Moreover, we reveal that Arabidopsis ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of Arabidopsis plasma membrane H+-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr947), whose dephosphorylation negatively regulates AHA2.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , PlantonesRESUMEN
The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Phoeniceae , Ácido Abscísico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas , Phoeniceae/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés FisiológicoRESUMEN
Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.
Asunto(s)
Ácido Abscísico/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores de Superficie Celular/agonistas , Bibliotecas de Moléculas Pequeñas/análisis , Proteínas de Arabidopsis/aislamiento & purificación , Pruebas de Enzimas , Proteína Fosfatasa 2C/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especially in the adaptation to drought. During the last years, chemical genomics has gained attention as an alternative approach to decipher complex traits. Additionally, chemical-based strategies have been very useful to untangle genetic redundancy, which is hard to address by other approaches such as classical genetics. Here, we describe the use of an ABA-inducible luciferase (LUC) reporter line for the high-throughput identification of chemical activators of the ABA signaling pathway. In this assay, seven-day-old pMAPKKK18-LUC+ seedlings are grown on 96-well plates and treated with test compounds. Next, the activity of the LUC reporter is quantified semiautomatically by image analysis. Candidate compounds able to activate the reporter are thus identified and subjected to a secondary screen by analyzing their effect on ABA-related phenotypes (e.g., inhibition of seed germination). This assay is fast, high-throughput, nondestructive, semiquantitative and can be applied to any other luciferase reporter lines, making it ideal for forward chemical genetic screenings.
Asunto(s)
Ácido Abscísico/metabolismo , Genes Reporteros , Luciferasas/metabolismo , Transducción de Señal , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Procesamiento de Imagen Asistido por Computador , Plantas Modificadas Genéticamente , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Following virtual screening and structure-based ligand optimization, researchers have developed opabactin (OP), an abscisic acid (ABA)-receptor agonist with tenfold greater in vivo activity than ABA. This new ligand surpasses previous agonists for its potency and bioactivity on staple crops. OP leads a new class of agrochemicals designed to protect crops from drought.