Plant NADPH-dependent thioredoxin reductases are crucial for the metabolism of sink leaves and plant acclimation to elevated CO2.

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.

Glutathione-mediated thermomorphogenesis and heat stress responses in Arabidopsis thaliana.

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.

Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress.

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.

A glutathione-dependent control of the indole butyric acid pathway supports Arabidopsis root system adaptation to phosphate deprivation.

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.

Cytosolic Isocitrate Dehydrogenase from Arabidopsis thaliana Is Regulated by Glutathionylation.

NADP-dependent (Nicotinamide Adénine Dinucléotide Phosphate-dependent) isocitrate dehydrogenases (NADP-ICDH) are metabolic enzymes involved in 2-oxoglutarate biosynthesis, but they also supply cells with NADPH. Different NADP-ICDH genes are found in Arabidopsis among which a single gene encodes for a cytosolic ICDH (cICDH) isoform. Here, we show that cICDH is susceptible to oxidation and that several cysteine (Cys) residues are prone to S-nitrosylation upon nitrosoglutathione (GSNO) treatment. Moreover, we identified a single S-glutathionylated cysteine Cys363 by mass-spectrometry analyses. Modeling analyses suggest that Cys363 is not located in the close proximity of the cICDH active site. In addition, mutation of Cys363 consistently does not modify the activity of cICDH. However, it does affect the sensitivity of the enzyme to GSNO, indicating that S-glutathionylation of Cys363 is involved in the inhibition of cICDH activity upon GSNO treatments. We also show that glutaredoxin are able to rescue the GSNO-dependent inhibition of cICDH activity, suggesting that they act as a deglutathionylation system in vitro. The glutaredoxin system, conversely to the thioredoxin system, is able to remove S-nitrosothiol adducts from cICDH. Finally, NADP-ICDH activities were decreased both in a catalase2 mutant and in mutants affected in thiol reduction systems, suggesting a role of the thiol reduction systems to protect NADP-ICDH activities in planta. In line with our observations in Arabidopsis, we found that the human recombinant NADP-ICDH activity is also sensitive to oxidation in vitro, suggesting that this redox mechanism might be shared by other ICDH isoforms.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis.

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.

Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria.

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.

NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.