PYR/PYL/RCAR蛋白介导植物ABA的信号转导

脱落酸(ABA)在各个植物生长发育阶段以及植物对生物与非生物胁迫的响应过程中都发挥着重要的作用。最近研究表明, 在ABA信号转导途径中有3种核心组份：ABA受体PYR/PYL/RCAR蛋白、负调控因子2C类蛋白磷酸酶(PP2C)和正调控因子SNF1相关的蛋白激酶2(SnRK2), 它们共同组成了一个双重负调控系统-- PYR/PYL/RCAR-| PP2C-| SnRK2来调控ABA信号转导及其下游反应, 且3种核心组份在植物体内的结合方式受时空和生化等因素的影响, 通过特定组合形成的ABA信号转导复合体介导特定的ABA信号反应。文章就PYR/PYL/RCAR蛋白介导的植物ABA信号识别与转导途径的分子基础及其调控机制, 以及PYR/PYL/RCAR-PP2C-SnRK2参与的ABA信号调控网络等研究进展做一概述, 并对该领域今后的研究进行了展望。     

ABA信号通路的起源与进化

植物激素脱落酸（abscisic acid,ABA）在植物的生长、发育和胁迫反应方面起重要的调控作用,其信号转导通路由4个核心组分共同组成一个双重负调控系统（PYR/PYL/RCAR—| PP2C—| SnRK2—ABF/AREB）,调控ABA应答反应。本文在综述和分析ABA信号通路4个核心组分的起源与进化的基础上,初步提出ABA信号通路的起源与进化路径：A类PP2C、第Ⅲ亚类SnRK2以及转录因子AREB/ABF在水生植物轮藻中已经进化产生,当陆生植物进化产生ABA受体PYR/PYL/RCAR后,即与其它3个组分形成完整的ABA信号通路。在植物进化过程中,ABA信号通路4个核心组分各家族成员的数量（亚类）呈递增趋势。     

The PP2C-SnRK2 complex: The central regulator of an abscisic acid signaling pathway

The phytohormone abscisic acid (ABA), an important bioactive compound in plants, is implicated in several essential processes such as development and the abiotic stress response. Many components have been reported to have roles in these processes. Although 2C-type protein phosphatases (PP2C) and SNF1-related protein kinases2 (SnRK2) family are known to be important signal mediators, the molecular mechanisms by which these components regulate the ABA signaling pathway have not been elucidated. Recent identification of soluble ABA receptors, PYR/PYL/RCAR, has provided a major breakthrough in understanding the signaling mechanisms of ABA and revealed the importance of PP2Cs. In addition, the physical, biochemical and physiological connections between PP2C and SnRK2 have been clearly demonstrated. Taken together, the molecular basis of the major ABA signaling pathway has been established, from perception to gene expression. In this addendum, we discuss this emerging ABA signaling pathway, which has a conventional protein phosphorylation/dephosphorylation regulatory circuit and consider its physiological and functional relevance.Key words: ABA receptor, abscisic acid, PP2C, signal transduction, SnRK2, plant hormone, phosphoarylation     

PYR/PYL/RCAR蛋白介导植物ABA的信号转导

脱落酸(ABA)在各个植物生长发育阶段以及植物对生物与非生物胁迫的响应过程中都发挥着重要的作用。最近研究表明,在ABA信号转导途径中有3种核心组份:ABA受体PYR/PYL/RCAR蛋白、负调控因子2C类蛋白磷酸酶(PP2C)和正调控因子SNF1相关的蛋白激酶2(SnRK2),它们共同组成了一个双重负调控系统——PYR/PYL/RCAR—|PP2C—|SnRK2来调控ABA信号转导及其下游反应,且3种核心组份在植物体内的结合方式受时空和生化等因素的影响,通过特定组合形成的ABA信号转导复合体介导特定的ABA信号反应。文章就PYR/PYL/RCAR蛋白介导的植物ABA信号识别与转导途径的分子基础及其调控机制,以及PYR/PYL/RCAR—PP2C—SnRK2参与的ABA信号调控网络等研究进展做一概述,并对该领域今后的研究进行了展望。     

ABA signal transduction from ABA receptors to ion channels

The plant hormone abscisic acid (ABA) is involved in regulating a number of major processes such as seed dormancy, seedling development, and biotic and abiotic stress responses. The function and effect of ABA on pathogens are still unclear, but the roles of ABA in seed germination and abiotic stress responses have been well characterized. Abiotic stresses elevate ABA levels and activate ABA signaling; thus, inducing a variety of responses, including the expression of stress-related genes and stomatal closure. The past decade has witnessed many significant advances in our understanding of ABA signal transduction due to application of a combination of approaches including genetics, biochemistry, electrophysiology, and chemical genetics. A number of proteins associated with the ABA signal transduction pathway such as PYR/PYL/RCAR family of START proteins, have been identified. These ABA receptors bind to ABA and positively regulate ABA signaling via inactivation of PP2C phosphatase activity, which inhibits SnRK2-type kinases by direct interaction and dephosphorylation. Additionally, SnRK2-type kinases and PP2Cs interact with one another and with other components of ABA signaling and function as positive and negative ABA regulators, respectively. In this review, we focus on ABA function to abiotic stresses and highlight each component in relation to ABA and its interactions.     

PYR/PYL/RCAR family members are major in‐vivo ABI1 protein phosphatase 2C‐interacting proteins in Arabidopsis

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group‐A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP‐tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1‐interacting proteins by mass‐spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA‐signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1‐interacting proteins in all LC‐MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA‐binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co‐immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA‐induced stomatal closure and ABA‐inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase‐PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR‐mediated ABA signalling.     

Selective inhibition of clade A phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors

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.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant- specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co- immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.     

Characterization of potential ABA receptors in Vitis vinifera

Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. The phytohormone abscisic acid (ABA) is a key endogenous messenger in a plant’s response to such stresses. A novel ABA binding mechanism which plays a key role in plant cell signaling cascades has recently been uncovered. In the absence of ABA, a type 2C protein phosphatase (PP2C) interacts and inhibits the kinase SnRK2. Binding of ABA to the PYR/PYLs receptors enables interaction between the ABA receptor and the PP2C protein, and abrogates the SnRK2 inactivation. The active SnRK2 is then free to activate the ABA-responsive element Binding Factors which target ABA-dependent gene expression. We used the grape as a model to study the ABA perception mechanism in fruit trees. The grape ABA signaling cascade consists of at least seven ABA receptors and six PP2Cs. We used a yeast two-hybrid system to examine physical interaction in vitro between the grape ABA receptors and their interacting partners, and found that twenty-two receptor-PP2C interactions can occur. Moreover, quantifying these affinities by the use of the LacZ reporter enables us to show that VvPP2C4 and VvPP2C9 are the major binding partners of the ABA receptor. We also tested in vivo the root and leaf gene expression of the various ABA receptors and PP2Cs in the presence of exogenic ABA and under different abiotic stresses such as high salt concentration, cold and drought, and found that many of these genes are regulated by such abiotic environmental factors. Our results indicate organ specificity in the ABA receptor genes and stress specificity in the VvPP2Cs. We suggest that VvPP2C4 is the major PP2C involved in ABA perception in leaves and roots, and VvRCAR6 and VvRCAR5 respectively, are the major receptors involved in ABA perception in these organs. Identification, characterization and manipulation of the central players in the ABA signaling cascades in fruit trees is likely to prove essential for improving their performance in the future.     

Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases

Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6–HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C–SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.     

Signaling mechanisms in abscisic acid-mediated stomatal closure

The plant hormone abscisic acid (ABA) plays a central role in the regulation of stomatal movements under water-deficit conditions. The identification of ABA receptors and the ABA signaling core consisting of PYR/PYL/RCAR ABA receptors, PP2C protein phosphatases and SnRK2 protein kinases has led to studies that have greatly advanced our knowledge of the molecular mechanisms mediating ABA-induced stomatal closure in the past decade. This review focuses on recent progress in illuminating the regulatory mechanisms of ABA signal transduction, and the physiological importance of basal ABA signaling in stomatal regulation by CO₂ and, as hypothesized here, vapor-pressure deficit. Furthermore, advances in understanding the interactions of ABA and other stomatal signaling pathways are reviewed here. We also review recent studies investigating the use of ABA signaling mechanisms for the manipulation of stomatal conductance and the enhancement of drought tolerance and water-use efficiency of plants.     

保卫细胞中ABA信号调控机制研究进展

脱落酸(ABA)具有调节植物快速响应逆境的重要功能。植物细胞中ABA核心信号通路由ABA受体PYR1/PYLs/RCARs、A类碱性蛋白磷酸酶PP2Cs和Snf1相关蛋白激酶SnRK2s组成。活性氧(ROS)和Ca²⁺是保卫细胞中的重要第二信使,调控ABA诱导的气孔关闭。该文对保卫细胞中核心ABA信号蛋白的调控以及ROS和Ca²⁺介导的ABA信号转导等最新研究成果进行综述,旨在阐明保卫细胞中ABA信号调控机制。     

Abscisic acid receptors: past, present and future

Jin-Gui Chen (Corresponding author) Abscisic acid (ABA) is the key plant stress hormone. Consistent with the earlier studies in support of the presence of both membrane- and cytoplasm-localized ABA receptors, recent studies have identified multiple ABA receptors located in various subcellular locations. These include a chloroplast envelope-localized receptor (the H subunit of Chloroplast Mg(2+) -chelatase/ABA Receptor), two plasma membrane-localized receptors (G-protein Coupled Receptor 2 and GPCR-type G proteins), and one cytosol/nucleus-localized Pyrabactin Resistant (PYR)/PYR-Like (PYL)/Regulatory Component of ABA Receptor 1 (RCAR). Although the downstream molecular events for most of the identified ABA receptors are currently unknown, one of them, PYR/PYL/RCAR was found to directly bind and regulate the activity of a long-known central regulator of ABA signaling, the A-group protein phosphatase 2C (PP2C). Together with the Sucrose Non-fermentation Kinase Subfamily 2 (SnRK2s) protein kinases, a central signaling complex (ABA-PYR-PP2Cs-SnRK2s) that is responsible for ABA signal perception and transduction is supported by abundant genetic, physiological, biochemical and structural evidence. The identification of multiple ABA receptors has advanced our understanding of ABA signal perception and transduction while adding an extra layer of complexity.