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1.
Plant J ; 114(4): 836-854, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36883867

RESUMEN

Arabidopsis histone deacetylase HDA19 is required for gene expression programs of a large spectrum of plant developmental and stress-responsive pathways. How this enzyme senses cellular environment to control its activity remains unclear. In this work, we show that HDA19 is post-translationally modified by S-nitrosylation at 4 Cysteine (Cys) residues. HDA19 S-nitrosylation depends on the cellular nitric oxide level, which is enhanced under oxidative stress. We find that HDA19 is required for cellular redox homeostasis and plant tolerance to oxidative stress, which in turn stimulates its nuclear enrichment, S-nitrosylation and epigenetic functions including binding to genomic targets, histone deacetylation and gene repression. The Cys137 of the protein is involved in basal and stress-induced S-nitrosylation, and is required for HDA19 functions in developmental, stress-responsive and epigenetic controls. Together, these results indicate that S-nitrosylation regulates HDA19 activity and is a mechanism of redox-sensing for chromatin regulation of plant tolerance to stress.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Cromatina/metabolismo , Óxido Nítrico/metabolismo
2.
J Exp Bot ; 75(14): 4287-4299, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-38787597

RESUMEN

Land plants have to face an oxidizing, heterogeneous, and fast changing environment. Redox-dependent post-translational modifications emerge as a critical component of plant responses to stresses. Among the thiol oxidoreductase superfamily, class III CC-type glutaredoxins (called ROXYs) are land plant specific, and their evolutionary history is highly dynamic. Angiosperms encode many isoforms, classified into five subgroups (Aα, Aß, Bα, Bß, Bγ) that probably evolved from five common ancestral ROXYs, with higher evolutionary dynamics in the Bγ subgroup compared with the other subgroups. ROXYs can modulate the transcriptional activity of TGA transcription factor target genes, although their biochemical function is still debated. ROXYs participate in the control of proper plant development and reproduction, and are mainly negative regulators of plant responses to biotic and abiotic stresses. This suggests that most ROXYs could play essential and conserved functions in resetting redox-dependent changes in transcriptional activity upon stress signaling to ensure the responsiveness of the system and/or avoid exaggerated responses that could lead to major defects in plant growth and reproduction. In Arabidopsis Bγ members acquired important functions in responses to nitrogen availability and endogenous status, but the rapid and independent evolution of this subclass might suggest that this function results from neofunctionalization, specifically observed in core eudicots.


Asunto(s)
Glutarredoxinas , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Desarrollo de la Planta/genética , Adaptación Fisiológica , Evolución Biológica , Evolución Molecular , Regulación de la Expresión Génica de las Plantas
3.
J Exp Bot ; 75(15): 4459-4475, 2024 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-38642408

RESUMEN

Developmental and environmental constraints influence genome expression through complex networks of regulatory mechanisms. Epigenetic modifications and remodelling of chromatin are some of the major actors regulating the dynamic of gene expression. Unravelling the factors relaying environmental signals that induce gene expression reprogramming under stress conditions is an important and fundamental question. Indeed, many enzymes involved in epigenetic and chromatin modifications are regulated by redox pathways, through post-translational modifications of proteins or by modifications of the flux of metabolic intermediates. Such modifications are potential hubs to relay developmental and environmental changes for gene expression reprogramming. In this review, we provide an update on the interaction between major redox mediators, such as reactive oxygen and nitrogen species and antioxidants, and epigenetic changes in plants. We detail how redox status alters post-translational modifications of proteins, intracellular epigenetic and epitranscriptional modifications, and how redox regulation interplays with DNA methylation, histone acetylation and methylation, miRNA biogenesis, and chromatin structure and remodelling to reprogram genome expression under environmental constraints.


Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Oxidación-Reducción , Plantas , Plantas/genética , Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Transcriptoma , Redes Reguladoras de Genes
4.
Plant Cell Environ ; 46(8): 2337-2357, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37267089

RESUMEN

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , NADP/metabolismo , Dióxido de Carbono/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Arabidopsis/metabolismo , Fotosíntesis/fisiología , Cloroplastos/metabolismo , Oxidación-Reducción , Hojas de la Planta/metabolismo , Tiorredoxinas/metabolismo , Aclimatación
5.
J Exp Bot ; 74(8): 2489-2507, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-36794477

RESUMEN

As sessile organisms, plants are particularly affected by climate change and will face more frequent and extreme temperature variations in the future. Plants have developed a diverse range of mechanisms allowing them to perceive and respond to these environmental constraints, which requires sophisticated signalling mechanisms. Reactive oxygen species (ROS) are generated in plants exposed to various stress conditions including high temperatures and are presumed to be involved in stress response reactions. The diversity of ROS-generating pathways and the ability of ROS to propagate from cell to cell and to diffuse through cellular compartments and even across membranes between subcellular compartments put them at the centre of signalling pathways. In addition, their capacity to modify the cellular redox status and to modulate functions of target proteins, notably through cysteine oxidation, show their involvement in major stress response transduction pathways. ROS scavenging and thiol reductase systems also participate in the transmission of oxidation-dependent stress signals. In this review, we summarize current knowledge on the functions of ROS and oxidoreductase systems in integrating high temperature signals, towards the activation of stress responses and developmental acclimation mechanisms.


Asunto(s)
Estrés Oxidativo , Plantas , Especies Reactivas de Oxígeno/metabolismo , Temperatura , Plantas/metabolismo , Oxidación-Reducción
6.
J Exp Bot ; 74(8): 2707-2725, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-36715641

RESUMEN

In the context of climate change, the global rise of temperature and intense heat waves affect plant development and productivity. Among the molecular perturbations that high temperature induces in living cells is the accumulation of reactive oxygen species (ROS), which perturbs the cellular redox state. In plants, the dynamics of the cellular and subcellular redox state have been poorly investigated under high temperature. Glutathione plays a major role in maintaining the cellular redox state. We investigated its contribution in adaptation of Arabidopsis thaliana to contrasting high temperature regimes: high ambient temperature inducing thermomorphogenesis and heat stress affecting plant viability. Using the genetically encoded redox marker roGFP2, we show that high temperature regimes lead to cytoplasmic and nuclear oxidation and impact the glutathione pool. This pool is restored within a few hours, which probably contributes to plant adaptation to high temperatures. Moreover, low glutathione mutants fail to adapt to heat stress and to induce thermomorphogenesis, suggesting that glutathione is involved in both heat adaptation mechanisms. We also evaluate the transcriptomic signature in the two high temperature regimes and identified gene expression deviations in low glutathione mutants, which might contribute to their sensitivity to high temperature. Thus, we define glutathione as a major player in the adaptation of Arabidopsis to contrasting high temperature regimes.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Glutatión/metabolismo , Proteínas de Arabidopsis/metabolismo , Oxidación-Reducción , Respuesta al Choque Térmico , Regulación de la Expresión Génica de las Plantas
7.
J Exp Bot ; 74(15): 4384-4400, 2023 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-37179467

RESUMEN

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.


Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , ADN Ribosómico/metabolismo , Metilación , Hierro/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo
8.
Plant Physiol ; 184(2): 676-692, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32826321

RESUMEN

Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glutarredoxinas/metabolismo , Respuesta al Choque Térmico , Estrés Oxidativo , Termotolerancia , Arabidopsis , Proteínas de Arabidopsis/genética , Glutarredoxinas/genética , Polimerizacion
9.
Plant Cell Environ ; 44(5): 1417-1435, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33537988

RESUMEN

Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to post-translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol-reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry-based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH-thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2-CysPRXs or type-II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning.


Asunto(s)
Arabidopsis/enzimología , Arabidopsis/fisiología , Citosol/metabolismo , Oxidorreductasas/metabolismo , Estomas de Plantas/fisiología , Plastidios/metabolismo , Ácido Abscísico/metabolismo , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Peróxido de Hidrógeno/metabolismo , Modelos Biológicos , Mutación/genética , Fenotipo , Estomas de Plantas/citología , Transcriptoma/genética
10.
Semin Cell Dev Biol ; 80: 3-12, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-28733165

RESUMEN

As sessile oxygenic organisms with a plastic developmental programme, plants are uniquely positioned to exploit reactive oxygen species (ROS) as powerful signals. Plants harbor numerous ROS-generating pathways, and these oxidants and related redox-active compounds have become tightly embedded into plant function and development during the course of evolution. One dominant view of ROS-removing systems sees them as beneficial antioxidants battling to keep damaging ROS below dangerous levels. However, it is now established that ROS are a necessary part of subcellular and intercellular communication in plants and that some of their signaling functions require ROS-metabolizing systems. For these reasons, it is suggested that "ROS processing systems" would be a more accurate term than "antioxidative systems" to describe cellular components that are most likely to interact with ROS and, in doing so, transmit oxidative signals. Within this framework, our update provides an overview of the complexity and compartmentation of ROS production and removal. We place particular emphasis on the importance of ROS-interacting systems such as the complex cellular thiol network in the redox regulation of phytohormone signaling pathways that are crucial for plant development and defense against external threats.


Asunto(s)
Antioxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Oxígeno/metabolismo , Fenómenos Fisiológicos de las Plantas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Estrés Oxidativo/fisiología
11.
Plant Physiol ; 181(2): 442-457, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31413204

RESUMEN

Photorespiration sustains photosynthesis in the presence of oxygen due to rapid metabolization of 2-phosphoglycolate, the major side-product of the oxygenase activity of Rubisco that also directly impedes carbon assimilation and allocation. Despite the fact that both the biochemical reactions and the underlying genetics are well characterized, information concerning the regulatory mechanisms that adjust photorespiratory flux is rare. Here, we studied the impact of mitochondrial-localized thioredoxin o1 (TRXo1) on photorespiratory metabolism. The characterization of an Arabidopsis (Arabidopsis thaliana) transfer DNA insertional line (trxo1-1) revealed an increase in the stoichiometry of photorespiratory CO2 release and impaired Gly-to-Ser turnover after a shift from high-to-low CO2 without changes in Gly decarboxylase (GDC) gene or protein expression. These effects were distinctly pronounced in a double mutant, where the TRXo1 mutation was combined with strongly reduced GDC T-protein expression. The double mutant (TxGT) showed reduced growth in air but not in high CO2, decreased photosynthesis, and up to 54-fold more Gly alongside several redox-stress-related metabolites. Given that GDC proteins are potential targets for redox-regulation, we also examined the in vitro properties of recombinant GDC l-proteins (lipoamide dehydrogenase) from plants and the cyanobacterium Synechocystis species strain PCC6803 and observed a redox-dependent inhibition by either artificial reducing agents or TRXo1 itself. Collectively, our results demonstrate that TRXo1 potentially adjusts photorespiration via redox-regulation of GDC in response to environmental changes.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Mitocondrias/metabolismo , Fotosíntesis , Tiorredoxinas/metabolismo , Adaptación Fisiológica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Respiración de la Célula , Glicina-Deshidrogenasa (Descarboxilante)/genética , Oxidación-Reducción , Pisum sativum , Synechocystis , Tiorredoxinas/genética
12.
J Exp Bot ; 71(16): 4843-4857, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32309856

RESUMEN

Root system architecture results from a highly plastic developmental process to adapt to environmental conditions. In particular, the development of lateral roots and root hair growth are constantly optimized to the rhizosphere properties, including biotic and abiotic constraints. The development of the root system is tightly controlled by auxin, the driving morphogenic hormone in plants. Glutathione, a major thiol redox regulator, is also critical for root development but its interplay with auxin is scarcely understood. Previous work showed that glutathione deficiency does not alter root responses to indole acetic acid (IAA), the main active auxin in plants. Because indole butyric acid (IBA), another endogenous auxinic compound, is an important source of IAA for the control of root development, we investigated the crosstalk between glutathione and IBA during root development. We show that glutathione deficiency alters lateral roots and root hair responses to exogenous IBA but not IAA. Detailed genetic analyses suggest that glutathione regulates IBA homeostasis or conversion to IAA in the root cap. Finally, we show that both glutathione and IBA are required to trigger the root hair response to phosphate deprivation, suggesting an important role for this glutathione-dependent regulation of the auxin pathway in plant developmental adaptation to its environment.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Ácido Butírico , Glutatión , Ácidos Indolacéticos , Indoles , Fosfatos , Raíces de Plantas
13.
Proc Natl Acad Sci U S A ; 114(31): 8414-8419, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28724723

RESUMEN

Cellular accumulation of reactive oxygen species (ROS) is associated with a wide range of developmental and stress responses. Although cells have evolved to use ROS as signaling molecules, their chemically reactive nature also poses a threat. Antioxidant systems are required to detoxify ROS and prevent cellular damage, but little is known about how these systems manage to function in hostile, ROS-rich environments. Here we show that during oxidative stress in plant cells, the pathogen-inducible oxidoreductase Nucleoredoxin 1 (NRX1) targets enzymes of major hydrogen peroxide (H2O2)-scavenging pathways, including catalases. Mutant nrx1 plants displayed reduced catalase activity and were hypersensitive to oxidative stress. Remarkably, catalase was maintained in a reduced state by substrate-interaction with NRX1, a process necessary for its H2O2-scavenging activity. These data suggest that unexpectedly H2O2-scavenging enzymes experience oxidative distress in ROS-rich environments and require reductive protection from NRX1 for optimal activity.

14.
Plant Cell Physiol ; 60(11): 2369-2381, 2019 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-31318380

RESUMEN

The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.


Asunto(s)
Arabidopsis/metabolismo , Carbono/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Oxidación-Reducción , Oxidorreductasas/genética , Fotosíntesis/genética , Fotosíntesis/fisiología , Proteínas de Plantas/genética
15.
Plant Cell Physiol ; 60(1): 213-229, 2019 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-30329109

RESUMEN

Thioredoxins (Trxs) modulate metabolic responses during stress conditions; however, the mechanisms governing the responses of plants subjected to multiple drought events and the role of Trxs under these conditions are not well understood. Here we explored the significance of the mitochondrial Trx system in Arabidopsis following exposure to single and repeated drought events. We analyzed the previously characterized NADPH-dependent Trx reductase A and B double mutant (ntra ntrb) and two independent mitochondrial thioredoxin o1 (trxo1) mutant lines. Following similar reductions in relative water content (∼50%), Trx mutants subjected to two drought cycles displayed a significantly higher maximum quantum efficiency (Fv/Fm) and were less sensitive to drought than their wild-type counterparts and than all genotypes subjected to a single drought event. Trx mutant plants displayed a faster recovery after two cycles of drought, as observed by the higher accumulation of secondary metabolites and higher stomatal conductance. Our results indicate that plants exposed to multiple drought cycles are able to modulate their subsequent metabolic and physiological response, suggesting the occurrence of an exquisite acclimation in stressed Arabidopsis plants. Moreover, this differential acclimation involves the participation of a set of metabolic changes as well as redox poise alteration following stress recovery.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sequías , Mitocondrias/metabolismo , Tiorredoxinas/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Metaboloma , Mutación/genética , Nucleótidos/metabolismo , Oxidación-Reducción , Estomas de Plantas/fisiología , Análisis de Componente Principal , Estrés Fisiológico , Agua
16.
New Phytol ; 224(4): 1569-1584, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31372999

RESUMEN

A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Plastidios/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Prueba de Complementación Genética , Glutatión Reductasa/genética , Mitocondrias/metabolismo , Mutación , Oxidación-Reducción , Plantas Modificadas Genéticamente , Plastidios/genética , Semillas/genética
17.
Nucleic Acids Res ; 45(20): 11891-11907, 2017 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-28981840

RESUMEN

RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Cisteína/metabolismo , ARN Bicatenario/metabolismo , ARN de Planta/metabolismo , Proteínas Represoras/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Cisteína/genética , Glutatión/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Oxidación-Reducción , Dominios Proteicos , División del ARN , ARN Bicatenario/química , ARN Bicatenario/genética , ARN de Planta/química , ARN de Planta/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Motivos de Unión al ARN/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Homología de Secuencia de Ácido Nucleico
18.
J Exp Bot ; 69(14): 3491-3505, 2018 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-29194485

RESUMEN

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.


Asunto(s)
Arabidopsis/metabolismo , Malato Deshidrogenasa/metabolismo , Estrés Oxidativo , Arabidopsis/enzimología , Citosol/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción
19.
Proc Natl Acad Sci U S A ; 112(11): E1392-400, 2015 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-25646482

RESUMEN

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: "What regulates flux through this pathway in vivo?" Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when (13)C-glucose, (13)C-malate, or (13)C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.


Asunto(s)
Ciclo del Ácido Cítrico , Mitocondrias/metabolismo , Tiorredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Isótopos de Carbono , Citratos/metabolismo , Genes de Plantas , Prueba de Complementación Genética , Metabolómica , Modelos Biológicos , Mutación/genética , Hojas de la Planta/enzimología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plastidios/metabolismo , Reproducibilidad de los Resultados , Semillas/crecimiento & desarrollo , Semillas/metabolismo
20.
Annu Rev Genet ; 43: 335-67, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19691428

RESUMEN

Since their discovery as a substrate for ribonucleotide reductase (RNR), the role of thioredoxin (Trx) and glutaredoxin (Grx) has been largely extended through their regulatory function. Both proteins act by changing the structure and activity of a broad spectrum of target proteins, typically by modifying redox status. Trx and Grx are members of families with multiple and partially redundant genes. The number of genes clearly increased with the appearance of multicellular organisms, in part because of new types of Trx and Grx with orthologs throughout the animal and plant kingdoms. The function of Trx and Grx also broadened as cells achieved increased complexity, especially in the regulation arena. In view of these progressive changes, the ubiquitous distribution of Trx and the wide occurrence of Grx enable these proteins to serve as indicators of the evolutionary history of redox regulation. In so doing, they add a unifying element that links the diverse forms of life to one another in an uninterrupted continuum. It is anticipated that future research will embellish this continuum and further elucidate the properties of these proteins and their impact on biology. The new information will be important not only to our understanding of the role of Trx and Grx in fundamental cell processes but also to future societal benefits as the proteins find new applications in a range of fields.


Asunto(s)
Glutarredoxinas/metabolismo , Tiorredoxinas/metabolismo , Animales , Escherichia coli/enzimología , Humanos , Mamíferos/metabolismo , Oxidación-Reducción , Saccharomyces cerevisiae/enzimología
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