NADPH-dependent Thioredoxin Reductase C Research Articles

Exogenous trehalose improves the survival of date palm suckers by enhancing thioredoxin-antioxidant systems activity

Trehalose, a disaccharide, regulates osmotic pressure, stabilizes proteins, and plays in the signaling of carbon allocation in response to abiotic stresses. This study was evaluated the effects of 100 mM pre-separation (7 days before de-suckering) and post-separation (immediately after planting) spraying of trehalose on the survival of ‘Mazafati’ date palm suckers from a molecular perspective. Our results showed a 42% increase in the survival rate of the suckers sprayed with trehalose. By triggering trehalose-phosphate synthase (TPS) gene expression, promoting endogenous trehalose accumulation in suckers sprayed with trehalose could be responsible for sufficient intracellular NADP+ supplying arising from enhancing NAD kinase (NADK) enzyme activity. In addition, higher 2-cysteine peroxiredoxin (2-Cys-Prx) gene expression and enzyme activity could be ascribed to triggering NADPH-dependent thioredoxin reductase C (NTRC) gene expression and enzyme activity, promoting thioredoxin (Trx) enzyme activity concomitant with triggering nucleoside diphosphate kinase (NDPK) gene expression. Lower H2O2 accumulation in suckers sprayed with trehalose could be responsible for protecting membrane integrity as shown by lower electrolyte leakage and malondialdehyde (MDA) accumulation. In addition to promoting antioxidant system activity, higher phenols and anthocyanins accumulation in date palm suckers sprayed with trehalose could be ascribed to enhancing shikimate-phenylpropanoid pathways activity by Trx system activity. The results indicate that the Trx system could be employed by endogenous trehalose accumulation for improving photosynthesis efficiency and enhancing the survival rate of the suckers.

2-Cys peroxiredoxins contribute to thylakoid lipid unsaturation by affecting ω-3 Fatty Acid Desaturase 8.

Fatty acid unsaturation levels affect chloroplast function and plant acclimation to environmental cues. However, the regulatory mechanism(s) controlling fatty acid unsaturation in thylakoid lipids is poorly understood. Here, we have investigated the connection between chloroplast redox homeostasis and lipid metabolism by focusing on 2-Cys peroxiredoxins (Prxs), which play a central role balancing the redox state within the organelle. The chloroplast redox network relies on NADPH-dependent thioredoxin reductase C (NTRC), which controls the redox balance of 2-Cys Prxs to maintain the reductive activity of redox-regulated enzymes. Our results show that Arabidopsis (Arabidopsis thaliana) mutants deficient in 2-Cys Prxs contain decreased levels of trienoic fatty acids, mainly in chloroplast lipids, indicating that these enzymes contribute to thylakoid membrane lipids unsaturation. This function of 2-Cys Prxs is independent of NTRC, the main reductant of these enzymes, hence 2-Cys Prxs operates beyond the classic chloroplast regulatory redox system. Moreover, the effect of 2-Cys Prxs on lipid metabolism is primarily exerted through the prokaryotic pathway of glycerolipid biosynthesis and Fatty Acid Desaturase 8 (FAD8). While 2-Cys Prxs and FAD8 interact in leaf membranes as components of a large protein complex, the levels of FAD8 were markedly decreased when FAD8 is overexpressed in 2-Cys Prxs-deficient mutant backgrounds. These findings reveal a function for 2-Cys Prxs, possibly acting as a scaffold protein, affecting the unsaturation degree of chloroplast membranes.

NTRC regulates CP12 to activate Calvin–Benson cycle during cold acclimation

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

The contribution of glutathione peroxidases to chloroplast redox homeostasis in Arabidopsis

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy.

Plastid 2-Cys peroxiredoxins are essential for embryogenesis in Arabidopsis

The redox couple formed by NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (Prxs) allows fine-tuning chloroplast performance in response to light intensity changes. Accordingly, the Arabidopsis 2cpab mutant lacking 2-Cys Prxs shows growth inhibition and sensitivity to light stress. However, this mutant also shows defective post-germinative growth, suggesting a relevant role of plastid redox systems in seed development, which is so far unknown. To address this issue, we first analyzed the pattern of expression of NTRC and 2-Cys Prxs in developing seeds. Transgenic lines expressing GFP fusions of these proteins showed their expression in developing embryos, which was low at the globular stage and increased at heart and torpedo stages, coincident with embryo chloroplast differentiation, and confirmed the plastid localization of these enzymes. The 2cpab mutant produced white and abortive seeds, which contained lower and altered composition of fatty acids, thus showing the relevance of 2-Cys Prxs in embryogenesis. Most embryos of white and abortive seeds of the 2cpab mutant were arrested at heart and torpedo stages of embryogenesis suggesting an essential function of 2-Cys Prxs in embryo chloroplast differentiation. This phenotype was not recovered by a mutant version of 2-Cys Prx A replacing the peroxidatic Cys by Ser. Neither the lack nor the overexpression of NTRC had any effect on seed development indicating that the function of 2-Cys Prxs at these early stages of development is independent of NTRC, in clear contrast with the operation of these regulatory redox systems in leaves chloroplasts.

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication.

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH-dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC-mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2-Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC-mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.

NTRC Effects on Non-Photochemical Quenching Depends on PGR5.

Non-photochemical quenching (NPQ) protects plants from the detrimental effects of excess light. NPQ is rapidly induced by the trans-thylakoid proton gradient during photosynthesis, which in turn requires PGR5/PGRL1-dependent cyclic electron flow (CEF). Thus, Arabidopsis thaliana plants lacking either protein cannot induce transient NPQ and die under fluctuating light conditions. Conversely, the NADPH-dependent thioredoxin reductase C (NTRC) is required for efficient energy utilization and plant growth, and in its absence, transient and steady-state NPQ is drastically increased. How NTRC influences NPQ and functionally interacts with CEF is unclear. Therefore, we generated the A. thaliana line pgr5 ntrc, and found that the inactivation of PGR5 suppresses the high transient and steady-state NPQ and impaired growth phenotypes observed in the ntrc mutant under short-day conditions. This implies that NTRC negatively influences PGR5 activity and, accordingly, the lack of NTRC is associated with decreased levels of PGR5, possibly pointing to a mechanism to restrict upregulation of PGR5 activity in the absence of NTRC. When exposed to high light intensities, pgr5 ntrc plants display extremely impaired photosynthesis and growth, indicating additive effects of lack of both proteins. Taken together, these findings suggest that the interplay between NTRC and PGR5 is relevant for photoprotection and that NTRC might regulate PGR5 activity.

Arabidopsis thimet oligopeptidases are redox-sensitive enzymes active in the local and systemic plant immune response

Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity.

Genome-wide identification, characterization and expression profile of glutaredoxin gene family in relation to fruit ripening and response to abiotic and biotic stresses in banana (Musa acuminata)

Glutaredoxins (GRXs) are disulfide oxidoreductases that are involved in various biological processes. However, little information on the role of GRXs in the regulation of fruit ripening and the response to stress is available. In this study, we isolated 64 GRX genes from banana genome. Their encoded GRX proteins could be classified into four classes: CC, CGFS, CPYC and GRL types. The distribution and synteny of these GRXs on chromosomes, the gene structures, the promoter sequences, and the possible protein subcellular localizations were characterized. Molecular interaction network analysis suggested that MaGRX might interact with glutathione reductase (GR), sulfiredoxin, peroxiredoxin (Prx), and NADPH-dependent thioredoxin reductase C (NTRC), contributing to the antioxidative defense of banana fruit. MicroRNA prediction showed that MaGRX genes might be targeted by different miRNAs. Transcriptome analysis characterized the expression profiles of different MaGRX genes during banana fruit ripening, and in response to different storage stresses. The results suggested that CC-type, CPYC-type and GRL-type MaGRXs might be more active than CGFS-type MaGRXs during banana fruit ripening and the response to stress. Moreover, MaGRX6/7/9/11/17/23/28 and MaGRL3/16/19 might play important roles in regulating fruit ripening or in response to low and high temperature, or Fusarium proliferatum infection.

Differential carbohydrate dynamics in Arabidopsis wild-type and ntrc mutant after trehalose feeding

The trehalose sugar plays pivotal roles in plants. In Arabidopsis, trehalose supplied to the medium leads to starch accumulation through redox activation of the ADP-glucose pyrophosphorylase (AGPase) by the NADPH-dependent thioredoxin reductase C (NTRC). To gain further insight on the regulation of carbohydrate metabolism by trehalose and NTRC, Arabidopsis wild-type (wt) and ntrc plants were raised in the MS medium supplemented with or without 100-mM sucrose or trehalose and alterations in sugar and starch contents as well as the activities and expressions of some carbohydrate metabolizing enzymes were investigated. The ntrc plants displayed lower growth rate compared to wt by sugar feeding. Under trehalose treatment, the total soluble, reducing and non-reducing sugars of wt and ntrc plants decreased between end of the day and end of the night, but starch in the ntrc plants remained unaltered during the aforementioned period. The obtained data showed that trehalase activity and gene expression display diurnal rhythms. Meanwhile, under darkness, trehalase gene expression required functional NTRC. Trehalose feeding induced NTRC expression during the day, but suppressed it during the night. Moreover, AGPase expression found to be jointly regulated by carbon nutrition and the light–dark period in both wt and mutant plants. The current data also revealed that AMY3 (α-amylase 3) gene expression depends on the presence of functional NTRC in plants. It seems that cross-talk between trehalose and NTRC regulates diurnal carbohydrate metabolism in plants.

NTRC: A Key Regulatory Hub in Carbon Metabolism and Redox Balance in Developing Tomato Fruits.

By acting as a redox regulatory factor, NADPH-dependent thioredoxin reductase C (NTRC) is involved in many metabolic processes as well as plastid biogenesis. It is the only isoform, out of three, localized to chloroplasts. Arabidopsis ( Arabidopsis thaliana ) NTRC has been extensively studied in

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis.

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH-dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2-Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild-type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC-promoted growth, changes in root architecture, shifts in expression of VC-responsive CK- and ABA-regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc-Δ2cp plants with reduced 2-Cys peroxiredoxin expression. OxiTRAQ-based quantitative and site-specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis-related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.

Redox-control of chlorophyll biosynthesis mainly depends on thioredoxins.

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the palegreen phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes.

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway.

Overexpression of chloroplast-localized NADPH-dependent thioredoxin reductase C (NTRC) enhances tolerance to photo-oxidative and drought stresses in Arabidopsis thaliana

Chloroplast is a major organelle that conducts photosynthesis to produce ATP and NADPH via conversion of water to oxygen in plant. While the photosynthesis occurs, molecular oxygen easily changes to reactive oxygen species (ROS) consisting of toxic oxygen radicals resulting in oxidative stress. NADPH-dependent thioredoxin reductases (NTRs) play a pivotal role to regulate the redox state of the thioredoxin system providing reducing power to peroxidase. Here, we identify whether chloroplast NTRC confers stress tolerance through maintenance of ROS in Arabidopsis. NTRC transcripts were two-fold induced at 1 h treatment exposed to a photooxidative agent, methyl viologen (MV). The enhanced NTRC transcripts conferred oxidative stress tolerance displaying that NTRC overexpressing plants (NTRCOX) were tolerant compared to Col-0 and knock-out (ntrc-ko) plants on MVcontaining media. MV-mediated ROS induction was not detected in NTRCOX whereas that was highly accumulated in Col-0 and ntrc-ko. We further examined that NTRCOX showed extreme drought tolerance with lower water loss compared to Col-0 and ntrc-ko. Drought-responsive genes such as RD29A and DREB2A were enhanced in NTRCOX by drought compared to Col-0 and ntrc-ko. The results suggest that NTRC overexpression contributes to maintaining ROS homeostasis under stress conditions and confers the tolerance to photo-oxidative and drought stresses.

NADPH-dependent thioredoxin reductase C plays a role in nonhost disease resistance against Pseudomonas syringae pathogens by regulating chloroplast-generated reactive oxygen species.

Chloroplasts are cytoplasmic organelles for photosynthesis in eukaryotic cells. In addition, recent studies have shown that chloroplasts have a critical role in plant innate immunity against invading pathogens. Hydrogen peroxide is a toxic by-product from photosynthesis, which also functions as a signaling compound in plant innate immunity. Therefore, it is important to regulate the level of hydrogen peroxide in response to pathogens. Chloroplasts maintain components of the redox detoxification system including enzymes such as 2-Cys peroxiredoxins (2-Cys Prxs), and NADPH-dependent thioredoxin reductase C (NTRC). However, the significance of 2-Cys Prxs and NTRC in the molecular basis of nonhost disease resistance is largely unknown. We evaluated the roles of Prxs and NTRC using knock-out mutants of Arabidopsis in response to nonhost Pseudomonas syringae pathogens. Plants lacking functional NTRC showed localized cell death (LCD) accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, the Arabidopsis ntrc mutant showed enhanced bacterial growth and disease susceptibility of nonhost pathogens. Furthermore, the expression profiles of the salicylic acid (SA) and jasmonic acid (JA)-mediated signaling pathways and phytohormone analyses including SA and JA revealed that the Arabidopsis ntrc mutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhost P. syringae pathogens.

Arabidopsis Mg-Protoporphyrin IX Methyltransferase Activity and Redox Regulation Depend on Conserved Cysteines.

Redox regulation is an essential post-translational regulatory mechanism in prokaryotes and eukaryotes. The reversible oxidation and reduction of cysteine residues of proteins is also important in photosynthetic organisms to control enzymatic activities, protein stability and the interaction with other proteins of chloroplast-localized proteins. Several enzymes of the plant tetrapyrrole biosynthesis pathway have been identified to be redox regulated by thioredoxins (TRXs) and NADPH-dependent thioredoxin reductase C (NTRC). Among these proteins, Mg protoporphyrin IX methyltransferase (encoded by CHLM) was identified to be activated and stabilized by interaction with NTRC. CHLM catalyzes a methyl group transfer by using S-adenosylmethionine (SAM). Here we demonstrate that three conserved cysteine residues of Arabidopsis CHLM are essential for catalytic function and redox-dependent activation of the enzyme. In vitro and in planta biochemical assays of recombinant CHLM and the Arabidopsis chlm knockout mutant overexpressing wild-type and cysteine substitution mutants of CHLM revealed modified methyltransferase activity, when the conserved cysteine residues of CHLM are replaced by serine. While C177 is responsible for redox-dependent enzyme activation, exchange of the two cysteine residues, C111 and C115, has a strong impact on enzyme activity. The modified CHLM activity of single and double mutants with cysteine substitution is presented, and the role of each cysteine residue is discussed based on a modeled structure of CHLM. These studies contribute to enhanced understanding of the physiological and enzymatic significance of redox-regulated CHLM.

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98–99%). In addition of being part of a redox directed activity “light switch”, reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.

Deletion of chloroplast NADPH-dependent thioredoxin reductase results in inability to regulate starch synthesis and causes stunted growth under short-day photoperiods.

Plastid-localized NADPH-dependent thioredoxin reductase C (NTRC) is a unique NTR enzyme containing both reductase and thioredoxin domains in a single polypeptide. Arabidopsis thaliana NTRC knockout lines (ntrc) show retarded growth, especially under short-day (SD) photoperiods. This study identified chloroplast processes that accounted for growth reduction in SD-acclimated ntrc. The strongest reduction in ntrc growth occurred under photoperiods with nights longer than 14h, whereas knockout of the NTRC gene did not alter the circadian-clock-controlled growth of Arabidopsis. Lack of NTRC modulated chloroplast reactive oxygen species (ROS) metabolism, but oxidative stress was not the primary cause of retarded growth of SD-acclimated ntrc. Scarcity of starch accumulation made ntrc leaves particularly vulnerable to photoperiods with long nights. Direct interaction of NTRC and ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was confirmed by yeast two-hybrid analysis. The ntrc line was not able to maximize starch synthesis during the light period, which was particularly detrimental under SD conditions. Acclimation of Arabidopsis to SD conditions also involved an inductive rise of ROS production in illuminated chloroplasts that was not counterbalanced by the activation of plastidial anti-oxidative systems. It is proposed that knockout of NTRC challenges redox regulation of starch synthesis, resulting in stunted growth of the mutant lines acclimated to the SD photoperiod.

Posttranslational Influence of NADPH-Dependent Thioredoxin Reductase C on Enzymes in Tetrapyrrole Synthesis

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steady-state levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.