RESUMO
Upon recognition of microbes, pattern recognition receptors (PRRs) activate pattern-triggered immunity. FLAGELLIN SENSING2 (FLS2) and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) form a typical PRR complex that senses bacteria. Here, we report that the kinase activity of the malectin-like receptor-like kinase STRESS INDUCED FACTOR 2 (SIF2) is critical for Arabidopsis (Arabidopsis thaliana) resistance to bacteria by regulating stomatal immunity. SIF2 physically associates with the FLS2-BAK1 PRR complex and interacts with and phosphorylates the guard cell SLOW ANION CHANNEL1 (SLAC1), which is necessary for abscisic acid (ABA)-mediated stomatal closure. SIF2 is also required for the activation of ABA-induced S-type anion currents in Arabidopsis protoplasts, and SIF2 is sufficient to activate SLAC1 anion channels in Xenopus oocytes. SIF2-mediated activation of SLAC1 depends on specific phosphorylation of Ser 65. This work reveals that SIF2 functions between the FLS2-BAK1 initial immunity receptor complex and the final actuator SLAC1 in stomatal immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Histona Desacetilases/metabolismo , Proteínas de Membrana/metabolismo , Estômatos de Plantas/imunologia , Proteínas Repressoras/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Animais , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Resistência à Doença/fisiologia , Feminino , Histona Desacetilases/genética , Histona Desacetilases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mutação , Oócitos/fisiologia , Fosforilação , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Serina/metabolismo , XenopusRESUMO
Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ â 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.
Assuntos
Acetatos/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Estômatos de Plantas/fisiologia , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Peróxido de Hidrogênio/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Nicotiana/metabolismo , Nicotiana/fisiologiaRESUMO
Allyl isothiocyanate (AITC) induces stomatal closure accompanied by reactive oxygen species (ROS) production and glutathione (GSH) depletion in Arabidopsis thaliana. In this study, stomatal responses to three other isothiocyanates (ITCs), benzyl isothiocyanate (BITC), sulforaphane (SFN), and phenethyl isothiocyanate (PEITC), were investigated in A. thaliana. All these ITCs significantly induced stomatal closure, where PEITC and BITC were most effective. The selected ITCs also induced ROS accumulation, cytosolic alkalization, and GSH depletion in guard cells. Moreover, all ITCs increased the frequency of cytosolic free calcium ([Ca2+]cyt) spikes (transient elevation), while PEITC and BITC showed the highest frequency. There was a strong positive correlation between the number of [Ca2+]cyt spikes per guard cell and the decrease in stomatal aperture. Both cytosolic alkalization and GSH content have a positive correlation with the decrease in stomatal aperture, but ROS production did not have a significant correlation with the decrease in stomatal apertures. These results indicate that the molecules with a functional ITC group induce stomatal closure that is accompanied by GSH depletion, cytosolic alkalization, [Ca2+]cyt spikes, and ROS production, and that the former three cellular events, rather than ROS production, are highly correlated with the decrease in stomatal aperture.
Assuntos
Arabidopsis , Citosol , Isotiocianatos/farmacologia , Estômatos de Plantas , Espécies Reativas de OxigênioRESUMO
The glucosinolate-myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.
Assuntos
Arabidopsis , Estômatos de Plantas , Cálcio , Isotiocianatos/farmacologiaRESUMO
Selenium (Se) causes oxidative damage to plants. Proline is accumulated as a compatible solute in plants under stress conditions and mitigates stresses. Selenate at 250 µM increased cell death and inhibited the growth of tobacco BY-2 cells while exogenous proline at 10 mM did not mitigate the inhibition by selenate. Selenate increased accumulation of Se and ROS and activities of antioxidant enzymes but not lipid peroxidation in the BY-2 cells. Proline increased Se accumulation and antioxidant enzyme activities but not either ROS accumulation or lipid peroxidation in the selenate-stressed cells. Glutathione (GSH) rather than ascorbic acid (AsA) mitigated the growth inhibition although both reduced the accumulation of ROS induced by selenate. These results indicate that proline increases both antioxidant enzyme activities and Se accumulation, which overall fails to ameliorate the growth inhibition by selenate and that the growth inhibition is not accounted for only by ROS accumulation. Abbreviations: APX: ascorbate peroxidase; AsA: ascorbic acid; BY-2: Bright Yellow-2; CAT: catalase; DAI: days after inoculation; DW: dry weight; FW: fresh weight; GSH: glutathione; ROS: reactive oxygen species.
Assuntos
Antioxidantes/metabolismo , Nicotiana/citologia , Estresse Oxidativo/efeitos dos fármacos , Prolina/farmacologia , Ácido Selênico/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Nicotiana/enzimologia , Nicotiana/metabolismoRESUMO
We have demonstrated that reactive carbonyl species (RCS) function as an intermediate downstream of hydrogen peroxide (H2O2) production in abscisic acid (ABA) signaling for stomatal closure in guard cells using transgenic tobacco plants overexpressing alkenal reductase. We investigated the conversion of the RCS production into downstream signaling events in the guard cells. Both ABA and H2O2 induced production of the RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), in epidermal tissues of wild-type Arabidopsis thaliana plants. Application of the RCS scavengers, carnosine and pyridoxamine, did not affect the ABA-induced H2O2 production but inhibited the ABA- and H2O2-induced stomatal closure. Both acrolein and HNE induced stomatal closure in a plasma membrane NAD(P)H oxidase mutant atrbohD atrbohF as well as in the wild type, but not in a calcium-dependent kinase mutant cpk6. Acrolein activated plasma membrane Ca2+-permeable cation channels, triggered cytosolic free Ca2+ concentration ([Ca2+]cyt) elevation, and induced stomatal closure accompanied by depletion of glutathione in the guard cells. These results suggest that RCS production is a signaling event between the ROS production and [Ca2+]cyt elevation during guard cell ABA signaling.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Peróxido de Hidrogênio/metabolismo , Fitocromo/metabolismo , Transdução de SinaisRESUMO
Stomatal movements regulate gas exchange, thus directly affecting the efficiency of photosynthesis and the sensitivity of plants to air pollutants such as ozone. The GARP family transcription factors GOLDEN 2-LIKE1 (GLK1) and GLK2 have known functions in chloroplast development. Here, we show that Arabidopsis thaliana (A. thaliana) plants expressing the chimeric repressors for GLK1 and -2 (GLK1/2-SRDX) exhibited a closed-stomata phenotype and strong tolerance to ozone. By contrast, plants that overexpress GLK1/2 exhibited an open-stomata phenotype and higher sensitivity to ozone. The plants expressing GLK1-SRDX had reduced expression of the genes for inwardly rectifying K(+) (K(+) in) channels and reduced K(+) in channel activity. Abscisic acid treatment did not affect the stomatal phenotype of 35S:GLK1/2-SRDX plants or the transcriptional activity for K(+) in channel gene, indicating that GLK1/2 act independently of abscisic acid signaling. Our results indicate that GLK1/2 positively regulate the expression of genes for K(+) in channels and promote stomatal opening. Because the chimeric GLK1-SRDX repressor driven by a guard cell-specific promoter induced a closed-stomata phenotype without affecting chloroplast development in mesophyll cells, modulating GLK1/2 activity may provide an effective tool to control stomatal movements and thus to confer resistance to air pollutants.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/efeitos dos fármacos , Ozônio/toxicidade , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Fatores de Transcrição/fisiologia , Poluentes Atmosféricos/toxicidade , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Cloroplastos/fisiologia , Regulação da Expressão Gênica de Plantas , Herbicidas/toxicidade , Oxidantes/toxicidade , Paraquat/toxicidade , Estômatos de Plantas/efeitos dos fármacos , Transpiração Vegetal , Plantas Geneticamente Modificadas/fisiologia , Canais de Potássio/fisiologia , Dióxido de Enxofre/toxicidade , Fatores de Transcrição/genéticaRESUMO
Stomatal immunity restricts bacterial entry to leaves through the recognition of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs) and downstream abscisic acid and salicylic acid signaling. Through a reverse genetics approach, we characterized the function of the L-type lectin receptor kinase-V.2 (LecRK-V.2) and -VII.1 (LecRK-VII.1). Analyses of interactions with the PRR FLAGELLIN SENSING2 (FLS2) were performed by co-immunoprecipitation and bimolecular fluorescence complementation and whole-cell patch-clamp analyses were used to evaluate guard cell Ca2+ -permeable cation channels. The Arabidopsis thaliana LecRK-V.2 and LecRK-VII.1 and notably their kinase activities were required for full activation of stomatal immunity. Knockout lecrk-V.2 and lecrk-VII.1 mutants were hyper-susceptible to Pseudomonas syringae infection and showed defective stomatal closure in response to bacteria or to the MAMPs flagellin and EF-Tu. By contrast, Arabidopsis over-expressing LecRK-V.2 or LecRK-VII.1 demonstrated a potentiated stomatal immunity. LecRK-V.2 and LecRK-VII.1 are shown to be part of the FLS2 PRR complex. In addition, LecRK-V.2 and LecRK-VII.1 were critical for methyl jasmonate (MeJA)-mediated stomatal closure, notably for MeJA-induced activation of guard cell Ca2+ -permeable cation channels. This study highlights the role of LecRK-V.2 and LecRK-VII.1 in stomatal immunity at the FLS2 PRR complex and in MeJA-mediated stomatal closure.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/fisiologia , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Estômatos de Plantas/imunologia , Estômatos de Plantas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Resistência à Doença/efeitos dos fármacos , Flagelina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Estômatos de Plantas/citologia , Estômatos de Plantas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Stomata within the plant epidermis regulate CO2 uptake for photosynthesis and water loss through transpiration. Stomatal opening in Arabidopsis thaliana is determined by various factors, including blue light as a signal and multiple phytohormones. Plasma membrane transporters, including H+-ATPase, K+ channels and anion channels in guard cells, mediate these processes, and the activities and expression levels of these components determine stomatal aperture. However, the regulatory mechanisms involved in these processes are not fully understood. In this study, we used infrared thermography to isolate a mutant defective in stomatal opening in response to light. The causative mutation was identified as an allele of the brassinosteroid (BR) biosynthetic mutant dwarf5. Guard cells from this mutant exhibited normal H+-ATPase activity in response to blue light, but showed reduced K+ accumulation and inward-rectifying K+ (K+in) channel activity as a consequence of decreased expression of major K+in channel genes. Consistent with these results, another BR biosynthetic mutant, det2-1, and a BR receptor mutant, bri1-6, exhibited reduced blue light-dependent stomatal opening. Furthermore, application of BR to the hydroponic culture medium completely restored stomatal opening in dwarf5 and det2-1 but not in bri1-6. However, application of BR to the epidermis of dwarf5 did not restore stomatal response. From these results, we conclude that endogenous BR acts in a long-term manner and is required in guard cells with the ability to open stomata in response to light, probably through regulation of K+in channel activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Estômatos de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Estômatos de Plantas/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismoRESUMO
Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.
Assuntos
Arabidopsis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Estômatos de Plantas/efeitos dos fármacos , Ácido Salicílico/farmacologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Acetatos/metabolismo , Acetatos/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Flavonoides/farmacologia , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Transdução de Sinais , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Drought is responsible for a massive reduction in crop yields. In response to drought, plants synthesize the hormone ABA, which induces stomatal closure, thus reducing water loss. In guard cells, ABA triggers production of reactive oxygen species (ROS), which is mediated by NAD(P)H oxidases. The production of ROS is a key factor for ABA-induced stomatal closure, but it remains to be clarified how the production of ROS is transduced into downstream signaling components in guard cells. We investigated roles of reactive carbonyl species (RCS) in ABA-induced stomatal closure using transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis 2-alkenal reductase (AER-OE), which scavenges RCS. ABA and hydrogen peroxide (H2O2) induced accumulation of RCS including acrolein and 4-hydroxy-(E)-2-nonenal in wild-type tobacco but not in AER-OE. Stomatal closure and RCS accumulation in response to ABA and H2O2 were inhibited in AER-OE unlike in the wild type, while ABA-induced H2O2 production in guard cells was observed in AER-OE as well as in the wild type. Moreover, ABA inhibited inward-rectifying K+ channels in wild-type guard cells but not in AER-OE guard cells. These results suggest that RCS is involved in ABA-induced stomatal closure and functions downstream of H2O2 production in the ABA signaling pathway in guard cells.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/genética , Radicais Livres/metabolismo , Nicotiana/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Proteínas de Arabidopsis/genética , Secas , Radicais Livres/análise , Expressão Gênica , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
BACKGROUND: Na(+) exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K(+) transporter (HKT) family have been demonstrated to mediate leaf blade-Na(+) exclusion upon salinity stress via Na(+)-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na(+) exclusion mechanism in rice remain to be elucidated. RESULTS: Here, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na(+) transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na(+) selectivity among cations tested, including Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4 (+), in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na(+) overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, (22)Na(+) tracer experiments using peduncles of RNAi and WT plants suggested xylem Na(+) unloading by OsHKT1;4. CONCLUSIONS: Taken together, our results indicate a newly recognized function of OsHKT1;4 in Na(+) exclusion in stems together with leaf sheaths, thus excluding Na(+) from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Simportadores/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Perfilação da Expressão Gênica , Genes de Plantas , Transporte de Íons , Oócitos , Fenótipo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Caules de Planta/metabolismo , Protoplastos/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Cloreto de Sódio/metabolismo , Estresse Fisiológico , Simportadores/genética , Xenopus laevis/genéticaRESUMO
Isothiocyanates are enzymatically produced from glucosinolates in plants, and allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis thaliana. In this study, we investigated stomatal responses to AITC in Vicia faba. AITC-induced stomatal closure accompanied by reactive oxygen species (ROS) and NO production, cytosolic alkalization and glutathione (GSH) depletion in V. faba. GSH monoethyl ester induced stomatal reopening and suppressed AITC-induced GSH depletion in guard cells. Exogenous catalase and a peroxidase inhibitor, salicylhydroxamic acid, inhibited AITC-induced stomatal closure, unlike an NAD(P)H oxidase inhibitor, diphenylene iodonium chloride. The peroxidase inhibitor also abolished the AITC-induced ROS production, NO production, and cytosolic alkalization. AITC-induced stomatal closure was suppressed by an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and an agent to acidify cytosol, butyrate. These results indicate that AITC-induced stomatal closure in V. faba as well as in A. thaliana and suggest that AITC signaling in guard cells is conserved in both plants.
Assuntos
Isotiocianatos/farmacologia , Estômatos de Plantas/efeitos dos fármacos , Transdução de Sinais , Vicia faba/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Benzoatos/farmacologia , Ácido Butírico/farmacologia , Catalase/antagonistas & inibidores , Catalase/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Sequestradores de Radicais Livres/farmacologia , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Imidazóis/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Oniocompostos/farmacologia , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Estômatos de Plantas/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Salicilamidas/farmacologia , Vicia faba/metabolismoRESUMO
Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1-mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA-dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA-dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA-responsive genes, such as PR1 and PR2. Furthermore, other SA-accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.
Assuntos
Arabidopsis/fisiologia , Ligases/deficiência , Reguladores de Crescimento de Plantas/fisiologia , Estômatos de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Desidratação/fisiopatologia , Ligases/genética , Ligases/fisiologia , Dados de Sequência MolecularRESUMO
Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Equorina/genética , Equorina/metabolismo , Canais de Cálcio/genética , Membrana Celular/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Citosol/metabolismo , Endo-1,4-beta-Xilanases/farmacologia , Proteínas Fúngicas/farmacologia , Células HEK293 , Humanos , Oryza/citologia , Oryza/genética , Oryza/microbiologia , Células Vegetais , Proteínas de Plantas/genética , FitoalexinasRESUMO
Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K(+) channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfolipase D/metabolismo , Estômatos de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Citosol/efeitos dos fármacos , Citosol/metabolismo , Teste de Complementação Genética , Germinação/efeitos dos fármacos , Mutação , Óxido Nítrico/metabolismo , Fenótipo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sementes/efeitos dos fármacos , Sementes/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Salicylic acid (SA), yeast elicitor (YEL), and chitosan (CHT) induced stomatal closure in Arabidopsis wild-type and aba2-2 plants, induced stomatal closure in fluridon-treated wild-type plants, and induced stomatal closure in aos mutants. These results suggest that neither endogenous abscisic acid nor endogenous jasmonic acid is involved in SA-, YEL-, or CHT-induced stomatal closure.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Quitosana/farmacologia , Ciclopentanos/metabolismo , Glucosamina/farmacologia , Oxilipinas/metabolismo , Estômatos de Plantas/anatomia & histologia , Salicilatos/farmacologia , Arabidopsis/anatomia & histologia , Arabidopsis/efeitos dos fármacos , Estômatos de Plantas/efeitos dos fármacosRESUMO
Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.
Assuntos
Arabidopsis/efeitos dos fármacos , Glucosinolatos/metabolismo , Isotiocianatos/farmacologia , Nitrilas/farmacologia , Estômatos de Plantas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiocianatos/farmacologia , Arabidopsis/anatomia & histologia , Arabidopsis/citologia , Arabidopsis/metabolismo , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Isotiocianatos/metabolismo , Óxido Nítrico/biossíntese , Nitrilas/metabolismo , Peroxidase/metabolismo , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Estômatos de Plantas/anatomia & histologia , Tiocianatos/metabolismoRESUMO
An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.
Assuntos
Ácido Abscísico/farmacologia , Vicia faba/metabolismo , Acetatos/farmacologia , Ciclopentanos/farmacologia , Óxido Nítrico/farmacologia , Oxilipinas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Vicia faba/efeitos dos fármacos , Vicia faba/genéticaRESUMO
Methylglyoxal (MG) is a reactive aldehyde derived by glycolysis. In Arabidopsis, MG inhibited light-induced stomatal opening in a dose-dependent manner. It significantly inhibited both inward-rectifying potassium (K(in)) channels in guard-cell protoplasts and an Arabidopsis K(in) channel, KAT1, heterologously expressed in Xenopus oocytes. Thus it appears that MG inhibition of stomatal opening involves MG inhibition of K(+) influx into guard cells.