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Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Inmunidad de la Planta , Estomas de Plantas/inmunología , Estomas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Filogenia , Zinc/metabolismo , Metales/metabolismo , Plantas/metabolismo , Defensinas/genética , Defensinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética , Raíces de Plantas/metabolismoRESUMEN
Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , NADPH Oxidasas/química , NADPH Oxidasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Arabidopsis/efectos de los fármacos , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Secuencia Conservada , Citosol/efectos de los fármacos , Citosol/metabolismo , Resistencia a la Enfermedad , Flagelina/farmacología , Células HEK293 , Humanos , Modelos Biológicos , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Desarrollo de la Planta/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Pseudomonas syringae/patogenicidad , Pseudomonas syringae/fisiología , Virulencia/efectos de los fármacosRESUMEN
Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.
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Arabidopsis/enzimología , Arabidopsis/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Células HEK293 , Humanos , Mutación , NADPH Oxidasas/clasificación , NADPH Oxidasas/genética , Fosforilación , Raíces de Plantas/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrolloRESUMEN
Plant phenotypes caused by mineral deficiencies differ depending on growth conditions. We recently reported that the growth of Arabidopsis thaliana was severely inhibited on MGRL-based zinc (Zn)-deficient medium but not on Murashige-Skoog-based Zn-deficient medium. Here, we explored the underlying reason for the phenotypic differences in Arabidopsis grown on the different media. The root growth and chlorophyll contents reduced by Zn deficiency were rescued by the addition of extra manganese (Mn) during short-term growth (10 or 14 d). However, this treatment did not affect the growth recovery after long-term growth (38 d). To investigate the reason for plant recovery from Zn deficiency, we performed the RNA-seq analysis of the roots grown on the Zn-basal medium and the Zn-depleted medium with/without additional Mn. Principal component analysis of the RNA-seq data showed that the gene expression patterns of plants on the Zn-basal medium were similar to those on the Zn-depleted medium with Mn, whereas those on the Zn-depleted medium without Mn were different from the others. The expression of several transcription factors and reactive oxygen species (ROS)-related genes was upregulated in only plants on the Zn-depleted medium without Mn. Consistent with the gene expression data, ROS accumulation in the roots grown on this medium was higher than those grown in other conditions. These results suggest that plants accumulate ROS and reduce their biomass under undesirable growth conditions, such as Zn depletion. Taken together, this study shows that the addition of extra Mn to the Zn-depleted medium induces transcriptional changes in ROS-related genes, thereby alleviating short-term growth inhibition due to Zn deficiency.
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Manganeso/farmacología , Plantones/metabolismo , Zinc/deficiencia , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Transcriptoma/efectos de los fármacos , Zinc/metabolismoRESUMEN
High salinity is an increasingly prevalent source of stress to which plants must adapt. The receptor-like protein kinases, including members of the Cys-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants that are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses; however, their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis (Arabidopsis thaliana) and also modulates root length. We established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a role for callose deposition in response to increased salinity and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins, we observed specific changes in the subcellular localization of CRK2 in response to various stress treatments. Many of CRK2's cellular functions were dependent on phospholipase D activity, as were the subcellular localization changes. Thus, we propose that CRK2 acts downstream of phospholipase D during salt stress, promoting callose deposition and regulating plasmodesmal permeability, and that CRK2 adopts specific stress-dependent subcellular localization patterns that allow it to carry out its functions.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Germinación/efectos de los fármacos , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tolerancia a la Sal , Estrés Fisiológico/genética , Estrés Fisiológico/fisiologíaRESUMEN
In plants, receptor-like kinases (RLKs) and extracellular reactive oxygen species (ROS) contribute to the communication between the environment and the interior of the cell. Apoplastic ROS production is a frequent result of RLK signaling in a multitude of cellular processes; thus, by their nature, these two signaling components are inherently linked. However, it is as yet unclear how ROS signaling downstream of receptor activation is executed. In this review, we provide a broad view of the intricate connections between RLKs and ROS signaling and describe the regulatory events that control and coordinate extracellular ROS production. We propose that concurrent initiation of ROS-dependent and -independent signaling linked to RLKs might be a critical element in establishing cellular responses. Furthermore, we discuss the possible ROS sensing mechanisms in the context of the biochemical environment in the apoplast. We suggest that RLK-dependent modulation of apoplastic and intracellular conditions facilitates ROS perception and signaling. Based on data from plant and animal models, we argue that specific RLKs could be components of the ROS sensing machinery or ROS sensors. The importance of the crosstalk between RLK and ROS signaling is discussed in the context of stomatal immunity. Finally, we highlight challenges in the understanding of these signaling processes and provide perspectives for future research.
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Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
During plant vascular development, xylem tracheary elements (TEs) form water-conducting, empty pipes by genetically regulated cell death. Cell death is prevented from spreading to non-TEs by unidentified intercellular mechanisms, downstream of METACASPASE9 (MC9)-mediated regulation of autophagy in TEs. Here, we identified differentially abundant extracellular peptides in vascular-differentiating wild-type and MC9-down-regulated Arabidopsis cell suspensions. A peptide named Kratos rescued the abnormally high ectopic non-TE death resulting from either MC9 knockout or TE-specific overexpression of the ATG5 autophagy protein during experimentally induced vascular differentiation in Arabidopsis cotyledons. Kratos also reduced cell death following mechanical damage and extracellular ROS production in Arabidopsis leaves. Stress-induced but not vascular non-TE cell death was enhanced by another identified peptide, named Bia. Bia is therefore reminiscent of several known plant cell death-inducing peptides acting as damage-associated molecular patterns. In contrast, Kratos plays a novel extracellular cell survival role in the context of development and during stress response.
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Apoptosis/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Unión al ARN/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Caspasas/genética , Caspasas/metabolismo , Regulación hacia Abajo/fisiología , Hojas de la Planta/fisiología , Proteínas de Unión al ARN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Xilema/fisiologíaRESUMEN
Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.
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Adaptación Fisiológica/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Estrés Oxidativo/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Ascomicetos/inmunología , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas Serina-Treonina Quinasas/genética , Pseudomonas syringae/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Xantina Oxidasa/metabolismoRESUMEN
In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Calcio/metabolismo , NADPH Oxidasas/fisiología , Tubo Polínico/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Ionomicina/farmacología , Toxinas Marinas , Datos de Secuencia Molecular , Mutación , NADPH Oxidasas/química , NADPH Oxidasas/genética , Oxazoles/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Frío , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Calcio/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , NADPH Oxidasas/química , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The equilibrium properties of a HIV-1-protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native-like, but their binding can be non-native.
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Precursores Enzimáticos/química , Proteasa del VIH/química , VIH-1/enzimología , Multimerización de Proteína/fisiología , Calorimetría , Precursores Enzimáticos/metabolismo , Proteasa del VIH/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Calcio/metabolismo , Retroalimentación Fisiológica , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Ionóforos de Calcio/metabolismo , Inhibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Ionomicina/metabolismo , Toxinas Marinas , NADPH Oxidasas/química , NADPH Oxidasas/genética , Oxazoles/metabolismo , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales de Calcio/metabolismo , Genes de Plantas , Membranas Intracelulares/metabolismo , Poaceae/metabolismo , Vacuolas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Canales de Calcio/genética , Señalización del Calcio , Cationes/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación , Oryza/genética , Oryza/metabolismo , Técnicas de Placa-Clamp , Poaceae/genética , Triticum/genética , Triticum/metabolismoRESUMEN
Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.
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Regulación de la Expresión Génica de las Plantas , NADPH Oxidasas , Células HEK293 , Humanos , Riñón/metabolismo , NADPH Oxidasas/metabolismo , Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain.
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Arabidopsis/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Estrés Fisiológico , Arabidopsis/enzimologíaRESUMEN
Advanced-phase chronic myeloid leukemia patients treated with imatinib often relapse due to point mutations in the Abl kinase domain. We herein examine the in vitro and in vivo effects of a Bcr-Abl/Lyn dual tyrosine kinase inhibitor, NS-187, on seven mutated Bcr-Abl proteins. NS-187 inhibited both Tyr393-phosphorylated and Tyr393-unphosphorylated Abl, resulting in significant in vitro growth inhibition of cells expressing six of seven mutated Bcr-Abl kinases, though not T315I. Furthermore, NS-187 prolonged the survival of mice injected with leukemic cells expressing all mutated Bcr-Abl tested except T315I, and its efficacy correlated well with its in vitro effects.
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Proteínas de Fusión bcr-abl/efectos de los fármacos , Proteínas de Fusión bcr-abl/genética , Leucemia/tratamiento farmacológico , Pirimidinas/administración & dosificación , Familia-src Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Benzamidas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Leucemia/genética , Leucemia/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Fosforilación , Piperazinas/administración & dosificación , Piperazinas/farmacología , Pirimidinas/farmacología , Relación Estructura-Actividad , Tasa de Supervivencia , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes. In Arabidopsis, NADPH oxidases RbohH and RbohJ are localized at the plasma membrane of pollen tube tip and produce ROS in a Ca(2+)-dependent manner. The ROS produced by Rbohs and Ca(2+) presumably play a critical role in the positive feedback regulation that maintains the tip growth. Ultrastructural cytochemical analysis revealed ROS accumulation in the apoplast/cell wall of the pollen grains on the stigmatic papillae in the wild type, but not in the rbohH rbohJ double mutant, suggesting that apoplastic ROS derived from RbohH and RbohJ are involved in pollen tube elongation into the stigmatic papillae by affecting the cell wall metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , NADPH Oxidasas/metabolismo , Polinización , Especies Reactivas de Oxígeno/metabolismo , Cerio/metabolismo , Retroalimentación , Modelos Biológicos , Tubo Polínico/citología , Tubo Polínico/metabolismoRESUMEN
The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.