RESUMO
The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas Serina-Treonina Quinases , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Corrente Citoplasmática , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/metabolismo , Mutação , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Translocadoras de Prótons/metabolismoRESUMO
Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.
Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Prótons , Transdução de Sinais , Álcalis , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Ativação Enzimática , Proteínas F-Box/metabolismo , Concentração de Íons de Hidrogênio , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
In Arabidopsis, the receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown. Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM. During this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid-ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid-binding ability and breaking the feedback regulation at later time points. The formation of the flg22-induced FLS2-BAK1 immune complex, which depends on the integrity of FER-containing nanodomains, is impaired in fer and pentuple car14569 mutant. Together, we propose that the FER-CAR module controls the formation of PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedback.
Assuntos
Arabidopsis , Transdução de Sinais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Fosfotransferases/metabolismo , Desenvolvimento Vegetal , Transdução de Sinais/genética , Estresse Fisiológico/genética , Imunidade Vegetal/genéticaRESUMO
Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Epistasia Genética/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Nicotiana/metabolismo , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Endocitose/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Arabidopsis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Ácidos Naftalenoacéticos/farmacologia , Transporte Proteico , Rede trans-Golgi/efeitos dos fármacosRESUMO
The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Estômatos de Plantas/genética , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Estômatos de Plantas/química , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/metabolismo , Ligação Proteica/genética , Transporte Proteico/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Vacúolos/genética , Vacúolos/metabolismo , Água/metabolismoRESUMO
The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Núcleo Celular/metabolismo , Modelos Biológicos , Mutação/genética , Células Vegetais/metabolismo , Proteólise , Frações Subcelulares/metabolismo , UbiquitinaçãoRESUMO
The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL85KR mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Abscísico/agonistas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Genes de Plantas , Ligantes , Meristema/metabolismo , Mutação , Reguladores de Crescimento de Plantas/agonistas , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Estabilidade Proteica/efeitos dos fármacos , Quinolonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , UbiquitinaçãoRESUMO
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.
RESUMO
Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Transdução de Sinais , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/química , Sítios de Ligação , Calorimetria , Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Modelos Biológicos , Fenótipo , Fosfolipídeos/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Soluções , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Membrane-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 Ca(2+)-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Receptores de Superfície Celular/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Modelos Moleculares , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteólise , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Mutação/genética , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/efeitos dos fármacos , UbiquitinaçãoRESUMO
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.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Meia-Vida , Proteínas de Membrana Transportadoras/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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
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.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/farmacologia , Proteínas de Arabidopsis/fisiologia , Mutação , Fosfoproteínas Fosfatases/fisiologia , Estresse Fisiológico/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Mapas de Interação de Proteínas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Transdução de Sinais/genética , Água/metabolismoRESUMO
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.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Immunoblotting , Espectrometria de Massas , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Proteoma/genética , Proteoma/metabolismo , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/metabolismo , Água/metabolismo , Água/farmacologiaRESUMO
Abscisic acid (ABA) plays a crucial role in the plant's response to both biotic and abiotic stress. Sustainable production of food faces several key challenges, particularly the generation of new varieties with improved water use efficiency and drought tolerance. Different studies have shown the potential applications of Arabidopsis PYR/PYL/RCAR ABA receptors to enhance plant drought resistance. Consequently the functional characterization of orthologous genes in crops holds promise for agriculture. The full set of tomato (Solanum lycopersicum) PYR/PYL/RCAR ABA receptors have been identified here. From the 15 putative tomato ABA receptors, 14 of them could be grouped in three subfamilies that correlated well with corresponding Arabidopsis subfamilies. High levels of expression of PYR/PYL/RCAR genes was found in tomato root, and some genes showed predominant expression in leaf and fruit tissues. Functional characterization of tomato receptors was performed through interaction assays with Arabidopsis and tomato clade A protein phosphatase type 2Cs (PP2Cs) as well as phosphatase inhibition studies. Tomato receptors were able to inhibit the activity of clade A PP2Cs differentially in an ABA-dependent manner, and at least three receptors were sensitive to the ABA agonist quinabactin, which inhibited tomato seed germination. Indeed, the chemical activation of ABA signalling induced by quinabactin was able to activate stress-responsive genes. Both dimeric and monomeric tomato receptors were functional in Arabidopsis plant cells, but only overexpression of monomeric-type receptors conferred enhanced drought resistance. In summary, gene expression analyses, and chemical and transgenic approaches revealed distinct properties of tomato PYR/PYL/RCAR ABA receptors that might have biotechnological implications.