RESUMEN
Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estrés Fisiológico/genética , Zinc/metabolismo , Defensinas/genética , Defensinas/metabolismo , Defensinas/farmacología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genéticaRESUMEN
Calcium and nitric oxide (NO) are two important biological messengers. Increasing evidence indicates that Ca(2+) and NO work together in mediating responses to pathogenic microorganisms and microbe-associated molecular patterns. Ca(2+) fluxes were recognized to account for NO production, whereas evidence gathered from a number of studies highlights that NO is one of the key messengers mediating Ca(2+) signaling. Here, we present a concise description of the current understanding of the molecular mechanisms underlying the cross talk between Ca(2+) and NO in plant cells exposed to biotic stress. Particular attention will be given to the involvement of cyclic nucleotide-gated ion channels and Ca(2+) sensors. Notably, we provide new evidence that calmodulin might be regulated at the posttranslational level by NO through S-nitrosylation. Furthermore, we report original transcriptomic data showing that NO produced in response to oligogalacturonide regulates the expression of genes related to Ca(2+) signaling. Deeper insight into the molecules involved in the interplay between Ca(2+) and NO not only permits a better characterization of the Ca(2+) signaling system but also allows us to further understand how plants respond to pathogen attack.
Asunto(s)
Señalización del Calcio , Óxido Nítrico/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Inmunidad de la Planta/inmunologíaRESUMEN
Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H(2)O(2) and O(2) (-), are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H(2)O(2) was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.
Asunto(s)
Arabidopsis , Botrytis/inmunología , Peróxido de Hidrógeno/inmunología , Enfermedades de las Plantas , Inmunidad de la Planta/fisiología , Hojas de la Planta , Superóxidos/inmunología , Ácido Abscísico/genética , Ácido Abscísico/inmunología , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Carboxiliasas/genética , Carboxiliasas/inmunología , Coenzima A Ligasas/genética , Coenzima A Ligasas/inmunología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lípidos de la Membrana/genética , Lípidos de la Membrana/inmunología , Mutación/inmunología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/microbiología , Trametes/genéticaRESUMEN
NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys(110), Cys(526) and Cys(664) as a targets for S-nitrosylation. Cys(526) is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys(526). Similarly, substitution of Cys(526) by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys(526) in the regulation of CDC48 by oxidative/nitrosative agents.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Óxido Nítrico/farmacología , Proteínas de Plantas/efectos de los fármacos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Nicotiana/metabolismo , Proteína que Contiene ValosinaRESUMEN
In animals, nitric oxide (NO) functions as a ubiquitous signaling molecule involved in diverse physiological processes such as immunity. Recent studies provided evidence that plants challenged by pathogenic microorganisms also produce NO. The emerging picture is that NO functions as a signal in plant immunity and executes part of its effects through posttranslational protein modifications. Notably, the characterization of S-nitrosylated proteins provided insights into the molecular mechanisms by which NO exerts its activities. Based on these findings, it appears that NO is involved in both the activation and the negative control of the signaling pathways related to plant immunity.
Asunto(s)
Óxido Nítrico/fisiología , Inmunidad de la Planta/fisiología , Proteínas de Arabidopsis , NADPH Oxidasas , Óxido Nítrico/biosíntesis , Proteínas de Plantas , Plantas/metabolismo , Transducción de SeñalRESUMEN
Under agroforestry practices, inter-specific facilitation between tree rows and cultivated alleys occurs when plants increase the growth of their neighbors especially under nutrient limitation. Owing to a coarse root architecture limiting soil inorganic phosphate (Pi) uptake, walnut trees (Juglans spp.) exhibit dependency on soil-borne symbiotic arbuscular mycorrhizal fungi that extend extra-radical hyphae beyond the root Pi depletion zone. To investigate the benefits of mycorrhizal walnuts in alley cropping, we experimentally simulated an agroforestry system in which walnut rootstocks RX1 (J. regia x J. microcarpa) were connected or not by a common mycelial network (CMN) to maize plants grown under two contrasting Pi levels. Mycorrhizal colonization parameters showed that the inoculum reservoir formed by inoculated walnut donor saplings allowed the mycorrhization of maize recipient roots. Relative to non-mycorrhizal plants and whatever the Pi supply, CMN enabled walnut saplings to access maize Pi fertilization residues according to significant increases in biomass, stem diameter, and expression of JrPHT1;1 and JrPHT1;2, two mycorrhiza-inducible phosphate transporter candidates here identified by phylogenic inference of orthologs. In the lowest Pi supply, stem height, leaf Pi concentration, and biomass of RX1 were significantly higher than in non-mycorrhizal controls, showing that mycorrhizal connections between walnut and maize roots alleviated Pi deficiency in the mycorrhizal RX1 donor plant. Under Pi limitation, maize recipient plants also benefited from mycorrhization relative to controls, as inferred from larger stem diameter and height, biomass, leaf number, N content, and Pi concentration. Mycorrhization-induced Pi uptake generated a higher carbon cost for donor walnut plants than for maize plants by increasing walnut plant photosynthesis to provide the AM fungus with carbon assimilate. Here, we show that CMN alleviates Pi deficiency in co-cultivated walnut and maize plants, and may therefore contribute to limit the use of chemical P fertilizers in agroforestry systems.
RESUMEN
Botrytis cinerea is a major pre- and post-harvest necrotrophic pathogen with a broad host range that causes substantial crop losses. The plant hormone jasmonic acid (JA) is involved in the basal resistance against this fungus. Despite basal resistance, virulent strains of B. cinerea can cause disease on Arabidopsis thaliana and virulent pathogens can interfere with the metabolism of the host in a way to facilitate infection of the plant. However, plant genes that are required by the pathogen for infection remain poorly described. To find such genes, we have compared the changes in gene expression induced in A. thaliana by JA with those induced after B. cinerea using genome-wide microarrays. We have identified genes that are repressed by JA but that are induced by B. cinerea. In this study, we describe one candidate gene, ATGRXS13, that encodes for a putative glutaredoxin and that exhibits such a crossed expression. In plants that are infected by this necrotrophic fungus, ATGRXS13 expression was negatively controlled by JA and TGA transcription factors but also through a JA-salicylic acid (SA) cross-talk mechanism as B. cinerea induced SA production that positively controlled ATGRXS13 expression. Furthermore, plants impaired in ATGRXS13 exhibited resistance to B. cinerea. Finally, we present a model whereby B. cinerea takes advantage of defence signalling pathways of the plant to help the colonization of its host.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Botrytis/patogenicidad , Glutarredoxinas/metabolismo , Enfermedades de las Plantas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Clonación Molecular , Ciclopentanos/metabolismo , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Glutarredoxinas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxilipinas/metabolismo , Enfermedades de las Plantas/microbiología , ARN de Planta/genética , Ácido Salicílico/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Calcium is a ubiquitous second messenger regulating numbers of cellular processes in living organisms. It encodes and transmits information perceived by cells to downstream sensors, including calmodulin (CaM), that initiate cellular responses. In plants, CaM has been involved in the regulation of plant responses to biotic and abiotic environmental cues. Plant CaMs possess a cysteine residue in their first calcium-binding motif EF-hand, which is not conserved in other eucaryotic organisms. In this work, we report the near-complete backbone chemical shift assignment of tobacco CaM2 with calcium. These results will be useful to study the impact of this particular EF-hand domain regarding CaM interaction with partners involved in stress responses.
Asunto(s)
Calmodulina , Nicotiana , Calcio/metabolismo , Calmodulina/metabolismo , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Nicotiana/metabolismoRESUMEN
The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans-activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1) or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses.
Asunto(s)
Arabidopsis/genética , Ciclopentanos/farmacología , Etilenos/farmacología , Oxilipinas/farmacología , Transactivadores/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Botrytis/crecimiento & desarrollo , Defensinas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Inmunidad Innata/genética , Modelos Genéticos , Mutación , Proteínas Nucleares/genética , Factores de Terminación de Péptidos/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Among Plant Protection Products (PPP), a new emerging category of pesticides act by stimulating plant defense in order to improve plant resistance against microbial pathogens. Given that these compounds, the so-called Plant Defense Stimulators (PDS) act on innate immunity, we tested, using an in vitro approach on human mononuclear leucocytes (PBMC), the potential toxicity (XTT assay) and inflammatory effects (production of IL-1ß) of 4 PPP belonging to different chemical families. We found that two products (LBG-01F34® and Regalis®) did not induce any cytotoxicity or IL-1 ß production. The product BION-50 WG®, that contains Acibenzolar-S-methyl (ASM) and silica particles did not present any cytotoxicity but induced a significant increase in the production of the inflammatory cytokine IL-1 ß. Finally, Vacciplant® that contains laminarin, was highly cytotoxic and pro-inflammatory. It induced a strong production of IL-1 ß when used at a concentration in the culture medium, as low as 0.02 mg/mL. We also tested the potential toxic effect of these 4 PPP on 4 days old zebra fish larvae. After 24 h of exposure, our results indicate that Vacciplant® induced zebra fish larvae mortality at concentration of 20 µg/mL. LBG did not induced significant mortality at concentrations up to 1 mg/mL whereas Regalis was lethal for 0,3 mg/mL concentrations and BION-50 WG began to induce mortality at 2,5 mg/mL. Our results indicate possible effects of PDS on IL-1ß production in human cells and fish survival, calling for more studies on the potential noxious side effects of these compounds.
RESUMEN
SUMMARY: Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea, the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea, further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in gamma-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione-S-transferase 1 was primed by wounding in leaves inoculated with B. cinerea. In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B. cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea, a process that involves camalexin and glutathione.
Asunto(s)
Arabidopsis/microbiología , Botrytis/patogenicidad , Inmunidad Innata , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Antiinfecciosos/uso terapéutico , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Botrytis/efectos de los fármacos , Glutatión/aislamiento & purificación , Glutatión/metabolismo , Indoles/uso terapéutico , Tiazoles/uso terapéuticoRESUMEN
Nitric oxide is regarded as a key signaling messenger in several organisms. Its physiological relevance is partly due to its capacity to induce posttranslational modifications of proteins through its direct or indirect reaction with specific amino acid residues. Among them, S-nitrosylation has been shown to be involved in a broad range of cellular signaling pathways both in animals and plants. The identification of S-nitrosylated proteins has been made possible by the development of the Biotin-Switch Technique (BST) in the early 2000s. Here, we describe the BST protocol we routinely use to check in vitro S-nitrosylation of recombinant proteins induced by NO donors.
Asunto(s)
Biotina , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Cisteína/química , Cisteína/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitrosación , Oxidación-Reducción , Conformación ProteicaRESUMEN
Type-2 HDACs (HD2s) are plant-specific histone deacetylases that play diverse roles during development and in responses to biotic and abiotic stresses. In this study we characterized the six tobacco genes encoding HD2s that mainly differ by the presence or the absence of a typical zinc finger in their C-terminal part. Of particular interest, these HD2 genes exhibit a highly conserved intron/exon structure. We then further investigated the phylogenetic relationships among the HD2 gene family, and proposed a model of the genetic events that led to the organization of the HD2 family in Solanaceae. Absolute quantification of HD2 mRNAs in N. tabacum and in its precursors, N. tomentosiformis and N. sylvestris, did not reveal any pseudogenization of any of the HD2 genes, but rather specific regulation of HD2 expression in these three species. Functional complementation approaches in Arabidopsis thaliana demonstrated that the four zinc finger-containing HD2 proteins exhibit the same biological function in response to salt stress, whereas the two HD2 proteins without zinc finger have different biological function.
Asunto(s)
Evolución Molecular , Nicotiana/fisiología , Proteínas de Plantas/genética , Tolerancia a la Sal/genética , Secuencia de Aminoácidos , Modelos Genéticos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Nicotiana/genética , Dedos de Zinc/fisiologíaRESUMEN
Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03-1 mg mL-1) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1ß inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1ß. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1ß and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection.
RESUMEN
Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.
Asunto(s)
Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transducción de Señal/fisiología , Plantas/microbiologíaRESUMEN
In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Óxido Nítrico/metabolismo , Solanaceae/metabolismo , Calcio/química , Cationes Bivalentes/química , Línea Celular , Membrana Celular/metabolismo , Peso Molecular , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the beta-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca2+](nuc) elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca2+](nuc) elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca2+](nuc) rise depends on free cytosolic calcium, IP3, and active oxygen species (AOS) but is independent of nitric oxide.
Asunto(s)
Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Nicotiana/metabolismo , Oligosacáridos/fisiología , Proteínas de Plantas/fisiología , Señalización del Calcio/genética , Núcleo Celular/genética , Células Cultivadas , Marcación de Gen , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Mutantes Quiméricas/fisiología , Oligosacáridos/metabolismo , Fosforilación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/citología , Nicotiana/genéticaRESUMEN
Nitric oxide (NO) is a free radical gas involved in a myriad of plant physiological processes including immune responses. How NO mediates its biological effects in plant facing microbial pathogen attack is an unresolved question. Insights into the molecular mechanisms by which it propagates signals reveal the contribution of this simple gas in complex signaling pathways shared with reactive oxygen species (ROS) and the second messenger Ca(2+). Understanding of the subtle cross-talks operating between these signals was greatly improved by the recent identification and the functional analysis of proteins regulated through S-nitrosylation, a major NO-dependent post-translational protein modification. Overall, these findings suggest that NO is probably an important component of the mechanism coordinating and regulating Ca(2+) and ROS signaling in plant immunity.
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Óxido Nítrico/metabolismo , Inmunidad de la Planta , Transducción de Señal/inmunología , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.
RESUMEN
The nicotianamine synthase (NAS) enzymes catalyze the formation of nicotianamine (NA), a non-proteinogenic amino acid involved in iron homeostasis. We undertook the functional characterization of AtNAS4, the fourth member of the Arabidopsis thaliana NAS gene family. A mutant carrying a T-DNA insertion in AtNAS4 (atnas4), as well as lines overexpressing AtNAS4 both in the atnas4 and the wild-type genetic backgrounds, were used to decipher the role of AtNAS4 in NA synthesis, iron homeostasis and the plant response to iron deficiency or cadmium supply. We showed that AtNAS4 is an important source for NA. Whereas atnas4 had normal growth in iron-sufficient medium, it displayed a reduced accumulation of ferritins and exhibited a hypersensitivity to iron deficiency. This phenotype was rescued in the complemented lines. Under iron deficiency, atnas4 displayed a lower expression of the iron uptake-related genes IRT1 and FRO2 as well as a reduced ferric reductase activity. Atnas4 plants also showed an enhanced sensitivity to cadmium while the transgenic plants overexpressing AtNAS4 were more tolerant. Collectively, our data, together with recent studies, support the hypothesis that AtNAS4 displays an important role in iron distribution and is required for proper response to iron deficiency and to cadmium supply.