RESUMO
Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.
Assuntos
Antinematódeos , Tylenchoidea , Animais , Humanos , Antinematódeos/química , Antinematódeos/metabolismo , Antinematódeos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Tylenchoidea/efeitos dos fármacos , Tylenchoidea/metabolismo , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/parasitologia , Doenças das Plantas , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Dimerização , Ligantes , Proteínas de Membrana Transportadoras/químicaRESUMO
CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.
RESUMO
Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.
Assuntos
Kluyveromyces , Kluyveromyces/genética , Kluyveromyces/metabolismo , Acetatos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/genética , Engenharia Metabólica , Regulação Fúngica da Expressão Gênica , Ácido Abscísico/metabolismoRESUMO
Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an â¼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from â¼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.
Assuntos
Ácido Abscísico/antagonistas & inibidores , Quinolinas/síntese química , Triazóis/síntese química , Ácido Abscísico/agonistas , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Benzamidas/síntese química , Benzamidas/química , Proteínas de Transporte/metabolismo , Química Click/métodos , Cicloexanos/síntese química , Cicloexanos/química , Expressão Gênica , Germinação , Modelos Moleculares , Reguladores de Crescimento de Plantas/metabolismo , Quinolinas/farmacologia , Sementes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico , Triazóis/farmacologiaRESUMO
Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally in plants and involve a hormone receptor that binds a hormone, triggering a conformational change in the receptor that enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are characterized well by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio of the hormone receptor to the binding protein. Thus, the same molecular ratchet can have a pico- or micromolar EC50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of the receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including enzyme-linked immunosorbent assay-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation.
Assuntos
Proteínas de Transporte , Proteínas , Dimerização , Ligantes , Proteínas/metabolismo , Proteínas de Transporte/metabolismo , HormôniosRESUMO
Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas bpm3 bpm5 plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.
Assuntos
Ácido Abscísico/farmacocinética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Culina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteólise , Motivos de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Culina/genética , Fosfoproteínas Fosfatases/genéticaRESUMO
Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.
Assuntos
Ácido Abscísico/metabolismo , Agroquímicos/farmacologia , Amidas/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Carboxílicos/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Plantas/efeitos dos fármacos , Plantas/metabolismo , Água/metabolismo , Aclimatação/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Secas , Engenharia Genética , Genótipo , Ligantes , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Modelos Moleculares , Transpiração Vegetal/efeitos dos fármacos , Plantas/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 µm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein-metabolite interaction and better understand protein-ligand interactions in plants.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Ácido Graxo/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligantes , Ácidos Linolênicos/química , Ácidos Linolênicos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Transdução de SinaisRESUMO
Abscisic acid (ABA) is a drought stress signaling molecule, and simple methods for detecting its levels could benefit agriculture. Here, we present proof-of-concept detection for ABA in aqueous solutions by the use of a mixture of Cyanine 5.5 (Cy5.5) fluorophore- and BHQ3 quencher-conjugated endogenous ABA receptor pyrabactin resistance 1 like proteins (PYL3). These dye-conjugated PYL3 protein form dimers in solutions without ABA and monomerize upon ABA binding. When they are in dimers, fluorescence of Cy5.5 is either nearly completely quenched by the BHQ3 or 20% quenched by another Cy5.5. Consequently, mixtures of equal amounts of the two protein conjugates were used to detect ABA in aqueous solution. As the ABA concentration increased from <1 µM to 1 mM, the intensity of fluorescence detected at around 680 nm from the mixture was more than doubled as a result of ABA-induced monomerization, which leads to halt of quenching and recovery of fluorescence of Cy5.5 in monomers. Kinetic modeling was used to simulate the fluorescence response from the mixture and the results generally agree with the experimentally observed trend. This work demonstrates that fluorescence measurements of a single dissociation reaction in one spectral region are adequate to assess the ABA concentration of a solution.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Secas , Receptores de Superfície Celular/metabolismo , Estresse Fisiológico , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Fluorescência , Reguladores de Crescimento de Plantas/farmacologia , Receptores de Superfície Celular/genética , Transdução de SinaisRESUMO
The phytohormone ABA mediates many physiological and developmental responses, and its key role in plant water relations has fueled efforts to improve crop water productivity by manipulating ABA responses. ABA's core signaling components are encoded by large gene families, which has hampered functional studies using classical genetic approaches due to redundancy. Chemical approaches can complement genetic approaches and have the advantage of delivering both biological probes and potential agrochemical leads; these benefits have spawned the discovery and design of new chemical modulators of ABA signaling and biosynthesis, which have contributed to the identification of ABA receptors and helped to define PYR1 and related subfamily III receptors as key cellular targets for chemically manipulating water productivity. In this review, we provide an overview of small molecules that have helped dissect both ABA signaling and metabolic pathways. We further discuss how the insights gleaned using ABA probe molecules might be translated to improvements in crop water productivity and future opportunities for development of small molecules that affect ABA metabolism and signaling.
Assuntos
Ácido Abscísico/biossíntese , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Criptocromos/genética , Indazóis/farmacologia , Luz , Plântula/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/genética , Hipocótilo/metabolismo , Immunoblotting , Indazóis/química , Indazóis/metabolismo , Transdução de Sinal Luminoso/efeitos dos fármacos , Transdução de Sinal Luminoso/genética , Transdução de Sinal Luminoso/efeitos da radiação , Estrutura Molecular , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Morfogênese/efeitos da radiação , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/crescimento & desenvolvimento , Plântula/metabolismoRESUMO
The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.
Assuntos
Ácido Abscísico/análogos & derivados , Proteínas de Arabidopsis/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Reguladores de Crescimento de Plantas , Ácido Abscísico/síntese química , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Germinação/efeitos dos fármacos , Lactuca/efeitos dos fármacos , Lactuca/metabolismo , Modelos Moleculares , Fosfoproteínas Fosfatases/metabolismo , Reguladores de Crescimento de Plantas/síntese química , Reguladores de Crescimento de Plantas/farmacologia , Fenômenos Fisiológicos Vegetais , Ligação Proteica , Raphanus/efeitos dos fármacos , Raphanus/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo , Relação Estrutura-AtividadeRESUMO
Agricultural productivity is dictated by water availability and consequently drought is the major source of crop losses worldwide. The phytohormone abscisic acid (ABA) is elevated in response to water deficit and modulates drought tolerance by reducing water consumption and inducing other drought-protective responses. The recent identification of ABA receptors, elucidation of their structures and understanding of the core ABA signaling network has created new opportunities for agrochemical development. An unusually large gene family encodes ABA receptors and, until recently, it was unclear if selective or pan-agonists would be necessary for modulating water use. The recent identification of the selective agonist quinabactin has resolved this issue and defined Pyrabactin Resistance 1 (PYR1) and its close relatives as key targets for water use control. This review provides an overview of the structure and function of ABA receptors, progress in the development of synthetic agonists, and the use of orthogonal receptors to enable agrochemical control in transgenic plants.
Assuntos
Ácido Abscísico/metabolismo , Agroquímicos/farmacologia , Plantas/efeitos dos fármacos , Plantas/metabolismo , Quinolonas/farmacologia , Sulfonamidas/farmacologia , Água/metabolismo , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/agonistas , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.
Assuntos
Aclimatação/fisiologia , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Modelos Moleculares , Folhas de Planta/citologia , Ácido Abscísico/agonistas , Aclimatação/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Cristalografia por Raios X , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Estrutura Molecular , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/fisiologia , Quinolonas/farmacologia , Sulfonamidas/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR)/PYR1-like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracellular receptors promotes the formation of stable complexes with certain protein phosphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest plant hormone receptor family known; however, it is unclear what functional differentiation exists among receptors. Here, we identify two distinct classes of receptors, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms. Moreover, we find a residue in PYR1, H60, that is variable between family members and plays a key role in determining oligomeric state. In silico modelling of the ABA activation pathway reveals that monomeric receptors have a competitive advantage for binding to ABA and PP2Cs. This work illustrates how receptor oligomerization can modulate hormonal responses and more generally, the sensitivity of a ligand-dependent signalling system.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Proteína Fosfatase 2C , Receptores de Superfície Celular/metabolismo , TermodinâmicaRESUMO
The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.
Assuntos
Ácido Abscísico/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Estresse Fisiológico/fisiologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutação , Fenótipo , Fosforilação , Protoplastos/fisiologiaRESUMO
The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone-receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Arabidopsis , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved beta-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling.