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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2005 Nov 1;102(45):16478–16483. doi: 10.1073/pnas.0507498102

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants

Laure Michelet *, Mirko Zaffagnini *,†, Christophe Marchand ‡, Valérie Collin *, Paulette Decottignies ‡, Pascale Tsan §, Jean-Marc Lancelin §, Paolo Trost †, Myroslawa Miginiac-Maslow *, Graham Noctor *, Stéphane D Lemaire *,
PMCID: PMC1283444  PMID: 16263928

Abstract

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions.

Keywords: Chlamydomonas, Arabidopsis, Calvin cycle, enzyme light-activation, thiol

Thioredoxins (TRXs) are small ubiquitous disulfide proteins with a conserved active site WC(G/P)PC that play key roles in redox signaling by oxidoreduction of disulfide bridges of various target proteins involved in a wide variety of functions (1). In plants, proteins of the TRX family are found in most subcellular compartments, but chloroplastic TRXs have been extensively studied because they are key regulators of photosynthesis. Under illumination, the photosynthetic electron-transfer chain reduces ferredoxin (Fd), which transfers electrons to acceptors, including TRXs through Fd-TRX reductase (FTR). Four types of TRXs (f, m, x, and y) are present in the chloroplast with, generally, several isoforms for each type (2). TRXs f and m are involved in the regulation of key carbon-fixation enzymes that are mostly inactive in the dark and activated by TRXs under illumination (3). Some of these enzymes, such as fructose-1,6-bisphosphatase, strictly depend on TRXf. TRXf also appears to be the most efficient activator of other carbon-metabolism enzymes, with the exception of glucose-6-phosphate dehydrogenase. TRXs x and y, discovered more recently, are, rather, implicated in responses to oxidative stress because they are particularly efficient for the reduction of peroxiredoxins (4, 5). Moreover, recent studies suggest the existence of numerous other TRX targets for which the specificity toward TRX types remains to be determined (6-8).

The thiol buffer glutathione (GSH) plays a key role in modulating plant stress responses (9-11), and work on animals has shown that GSH status can modulate protein activity through glutathionylation, a reversible posttranslational modification consisting of the formation of a mixed disulfide between the free thiol of a protein and GSH. This modification notably occurs in response to enhanced production of reactive oxygen species (ROS) and/or increases in oxidized glutathione (GSSG). Glutathionylation can protect proteins from irreversible oxidation and/or modulate their activity (12-17). Despite its theoretical appeal as a mechanism transmitting oxidative signals under stress, very little is known about glutathionylation in plants. Recently, two enzymes involved in sugar metabolism, namely aldolase and triose-phosphate isomerase, were shown to undergo glutathionylation, with a concomitant decrease of their activity (18).

Almost nothing is known about crosstalk between GSH and TRX systems in redox signaling in plants. Only a brief report has appeared on glutathionylation of extrachloroplastic TRXs in poplar (19). In humans, Casagrande et al. (13) have recently reported that human TRX (HsTRX) is able to undergo glutathionylation under oxidative stress. The modification specifically affects one of the extra cysteines the protein contains in addition to the two active-site cysteines and decreases TRX activity. These recent results have prompted us to investigate whether this posttranslational modification could constitute a mechanism of redox regulation of photosynthetic metabolism. For this purpose, we examined the glutathionylation of the nine chloroplastic TRXs from the higher plant Arabidopsis thaliana and the five TRXs from the green alga Chlamydomonas reinhardtii. We show that, among all chloroplastic TRXs, only f-type TRXs can undergo glutathionylation on a conserved extra cysteine, distinct from the two active-site cysteines. By reconstituting the thylakoid-dependent Fd-FTR-TRX system, we demonstrate that glutathionylation of TRXf strongly decreases its ability to activate target enzymes in the light and that this decrease is due to an impaired reduction of the glutathionylated form. Marked acceleration of TRXf glutathionylation by thiol oxidants suggests that this modification might represent an important chloroplastic mechanism that acts to modulate distribution of reductant in response to oxidative stress.

Materials and Methods

Reagents and Protein Purification. Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich. All TRXs, sorghum NADP-malate dehydrogenase (NADP-MDH), A. thaliana GAPDH (B4), C. reinhardtii Fd, and Synechocystis FTR were produced and purified as described in refs. 4 and 20-23, respectively. GAPDH (B4) was oxidized before use. The active site of TRX is oxidized under the aerobic conditions used.

Chlamydomonas Culture. The C. reinhardtii cw-15 41A15 strand [cw-15 (cell-wall-less) (gift of J.-D. Rochaix, University of Geneva, Geneva)] was grown at 25°C under constant agitation and continuous light (100 μE·m-2·s-1) in Tris acetate phosphate medium (24).

Titration of Free Sulfhydryl (-SH) Groups. The number of free -SH groups was estimated spectrophotometrically with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) at 412 nm (εM = 13,600 M-1·cm-1) (25) or 14C-iodoacetamide (IAM, MP Biomedicals, Irvine, CA) by alkylation of free -SH groups followed by trichloroacetic acid precipitation and radioactivity measurements (26).

MALDI-TOF Mass Spectrometry. TRX and trypsin-treated TRX were analyzed by MALDI-TOF mass spectrometry, as described in ref. 27.

3D Structure Modeling. A. thaliana TRXs were modeled by using the program modeler 4.0 (28) with a representative structure of TRXm from C. reinhardtii (CrTRXm) (NMR structure 16 of the Protein Data Bank entry 1DBY) (29) as a template and a multiple sequence alignment with clustalw. Simple charge potentials associated to water-accessible surfaces and solvent accessibility were obtained by using the program molmol (30).

Extraction of Crude Thylakoids from C. reinhardtii. Cells were grown to 1-2 × 106 cells per ml and harvested by centrifugation of ≈400 ml of culture. The pellet was washed in 50 mM Hepes (pH 7.5)/1 mM MgCl2/0.3 M sorbitol (buffer 1) and resuspended in a 1/100 volume of buffer 1, and cells were broken with a French press at 6,000 psi. After centrifugation, the pellet was washed in 50 mM Hepes (pH 7.5) and 5 mM MgCl2 before resuspension in 50 mM Hepes (pH 7.5)/1 mM EDTA/5 mM NaCl (same volume). After centrifugation, membranes were resuspended in a 1/1,000 volume of buffer 1 and kept at 4°C. All centrifugations were performed at 3,000 × g for 5 min at 4°C. The final concentration of chlorophyll was ≈1 μg of chlorophyll per μl of thylakoids.

Measurement of NADP-MDH and GAPDH (B4) Activities. 2 μM NADP-MDH or 6 μM GAPDH were activated at 25°C by A. thaliana TRXf1 (AtTRXf1) (4 μM for NADP-MDH or 10 μM for GAPDH) in a reconstituted system composed of thylakoids (10 μg of chlorophyll equivalent), 5 μM Fd, and 1 μM FTR in buffer 1 under 150 μE·m-2·s-1. The NADP-MDH and GAPDH activity assays were performed as described in refs. 20 and 21, respectively.

Gel-Shift Assays. AtTRXf1 (4 μM) (glutathionylated or not) was incubated in the light in the reconstituted system described above. After the addition of 1 mM lucifer yellow, the samples were incubated for 10 additional minutes. After removal of the thylakoids by centrifugation, samples were loaded on a 12-18% urea gel (31).

Immunoblot. Proteins were blotted onto a nitrocellulose filter, incubated with anti-AtTRXf1 primary antibody at 4°C overnight, and reacted with peroxidase-linked anti-rabbit Ig for 1 h. Signals were visualized by enhanced chemiluminescence (32).

Results

AtTRXf1 Is Specifically Glutathionylated on Cys-60. Sequence analysis of chloroplastic TRXs identified numerous TRXs with one or several extra cysteines in addition to the two active-site cysteines. However, the presence and/or position of these extra cysteines did not appear to be conserved, with the exception of f-type TRXs, which share a strictly conserved extra cysteine not required for TRX activity (33). TRXf thus appeared to be the best candidate to initiate this study. Hence, we tested whether AtTRXf1 could undergo glutathionylation. The protein was first reduced with DTT to make its three cysteines accessible to glutathione. After DTT removal, the TRX was incubated with 5 mM GSSG for various times. A progressive disappearance of the number of free thiols, determined by DTNB derivatization, was observed (data not shown). Samples with 0, 1, 2, or 3 thiols accessible to DTNB were analyzed by MALDI-TOF. The spectrum of the untreated protein presents two peaks because of partial excision of the initial methionine (Fig. 1). The spectra of the treated samples revealed the progressive disappearance of these peaks, concomitant with the appearance of two new peaks with molecular masses increased by ≈305 Da (Fig. 1). This shift is consistent with the presence of one glutathione adduct per TRX molecule. To determine the position of the glutathionylated residue on AtTRXf1, the protein was digested with trypsin before MALDI-TOF analysis. The peptide profile of the glutathionylated TRX reveals two additional peaks, compared with the profile obtained for the untreated protein (Fig. 2). These peaks correspond to the peptides [L58-K69] and [A45-K69] containing the additional Cys-60, shifted by 305 Da. If the observed mass increase is due to the formation of a mixed disulfide between GSH and the thiol of Cys-60, this modification should be reversed by the addition of a disulfide reductant. Indeed, after DTT treatment, the additional peaks could no longer be detected (data not shown). It should also be noted that peptide [L23-K37], containing the two active-site cysteines, was never identified in a shifted peak. Therefore, during the GSSG treatment, the two active-site cysteines were progressively reoxidized into a disulfide bridge while Cys-60 was undergoing glutathionylation, thus explaining why the decrease of free thiols exceeds the number of glutathionylated cysteines.

Fig. 1.

Fig. 1.

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AtTRXf1 is glutathionylated. MALDI-TOF analysis on whole protein for untreated AtTRXf1 (A) and AtTRXf1 treated with 5 mM GSSG for 5 h (B). The spectrum of the protein presents two peaks because of partial excision of the initial methionine.

Fig. 2.

Fig. 2.

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Glutathionylation of AtTRXf1 is specific to Cys-60. MALDI-TOF analysis after tryptic digestion of untreated AtTRXf1 (A) and AtTRXf1 treated with 5 mM GSSG for 5 h (B). The peptides containing the two active-site cysteines (WCGPC) or Cys-60 are indicated.

f-Type TRXs Are the Only Glutathionylated TRXs in the Chloroplast. To determine whether other chloroplastic TRXs could undergo glutathionylation, we tested all chloroplastic TRXs from A. thaliana and C. reinhardtii. This analysis was restricted to TRXs containing at least one additional cysteine because the active-site cysteines are not stably glutathionylated in HsTRX or AtTRXf1. We also tested another TRXf to determine whether the glutathionylation of the conserved extra cysteine is a general feature of f-type TRXs. Because TRXf from Chlamydomonas is difficult to produce and purify, we used spinach TRXf1 (SoTRXf1) for these assays. Because AtTRXf2 shares 89% identity with At-TRXf1, glutathionylation of AtTRXf2 has not been tested. The number of free thiols accessible on the TRXs was determined by IAM derivatization, followed by MALDI-TOF analysis. The active site of each TRX was in the oxidized form. SoTRXf1 and AtTRXy1 have one free accessible thiol, and CrTRXm has two, although, in the latter case, one of the cysteines was only slightly derivatized. Thus, all of the extra cysteines on these TRXs are accessible. The additional cysteine of AtTRXm3 is not accessible (data not shown). The extent of glutathionylation after 1 or 5 h of treatment with 5 mM GSSG was determined by 14C IAM labeling, and the results were confirmed by MALDI-TOF experiments. After 1 h, only f-type TRXs (AtTRXf1 and SoTRXf1) show significant glutathionylation (≈35%). After 5 h, glutathionylation of f-type TRX reached 80%, whereas a weak glutathionylation was observed on one of the additional cysteines of CrTRXm. No glutathionylation was observed for the other TRXs tested. In conclusion, not all accessible cysteines on a TRX molecule are able to undergo glutathionylation, and TRXf glutathionylation is likely to be a general feature of f-type TRXs.

Glutathionylation of AtTRXf1 Affects the Activation of NADP-MDH and GAPDH. The effect of glutathionylation on the activation of TRXf targets, namely NADP-MDH and GAPDH, was analyzed. For this purpose, AtTRXf1 was completely glutathionylated (>95% estimated by MALDI-TOF analysis). Target enzyme-activation assays were performed in a complete reconstituted thylakoid-dependent system under light. In this system, no DTT is needed, thus preventing the removal of glutathione from AtTRXf1-SG (glutathionylated AtRXf1). The kinetics obtained showed a significant decrease in activity when AtTRXf1-SG was used, compared with AtTRXf1-SH (Fig. 3). The remaining 5% nonglutathionylated AtTRXf1 present cannot account for the activation kinetics observed for AtTRXf1-SG (data not shown). The activation of both enzymes was similarly affected, with activation rates decreased 5.5 ± 1.9-fold (n = 13) for NADP-MDH and 4.1 ± 1.2-fold (n = 10) for GAPDH (data not shown).

Fig. 3.

Fig. 3.

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Glutathionylation of AtTRXf1 affects the activation of NADP-MDH and GAPDH. Activations of both proteins have been performed in the reconstituted system composed of thylakoids, Fd, and FTR under light. After various times of illumination, the activities of the proteins were measured (see Materials and Methods). (A) NADP-MDH (2 μM) was activated with 4 μM untreated AtTRXf1 (filled circles) or glutathionylated AtTRXf1 (open circles). (B) GAPDH (6 μM) was activated with 10 μM untreated AtTRXf1 (filled squares) or glutathionylated AtTRXf1 (open squares).

Reduction of AtTRXf1 Is Less Efficient When the Protein Is Glutathionylated. The decrease in the activation of NADP-MDH and GAPDH could be because of an effect on the interaction of AtTRXf1-SG with its targets or with FTR. To distinguish between these two possibilities, the extent of TRX reduction was estimated. Samples containing AtTRXf1-SH or AtTRXf1-SG in the reconstituted system were exposed to dark or to light and subsequently treated with lucifer yellow, which adds 500 Da per derivatized thiol. The active site of AtTRXf1-SH appeared mainly oxidized in the dark and became completely reduced after exposure to light (Fig. 4). For AtTRXf1-SG, the active site is oxidized in the dark but only slightly reduced in the light, indicating that the glutathionylated AtTRXf1 is affected in its reduction and, thus, in its interaction with FTR. Even if a problem of interaction of AtTRXf1-SG with its targets cannot be excluded, the decreased reduction of AtTRXf1-SG could account for the lower activation of NADP-MDH and GAPDH (Fig. 3). Moreover, this result suggests that the effect of glutathionylation of TRXf would not be restricted to the two targets tested but might affect the activation of all TRXf targets.

Fig. 4.

Fig. 4.

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Reduction of AtTRXf1 is less efficient when the protein is glutathionylated. Reduction of 4 μM AtTRXf1 was performed in the reconstituted system composed of thylakoids, Fd, and FTR under dark or light for 10 min before derivatization with 1 mM lucifer yellow. The various forms of AtTRXf1 are visualized by Western blot on a 12-18% urea gel. LY, lucifer yellow-derivatized cysteine; S-S, disulfide bridge; SH, underivatized cysteine.

Thiol Oxidants Greatly Enhance TRXf Glutathionylation. All experiments described above were performed in the presence of 5 mM GSSG, the classical conditions generally used to test glutathionylation. However, this concentration of GSSG is higher than the estimated concentration in chloroplasts. Indeed, the standard concentration of the glutathione pool has been estimated at between 1 and 4.5 mM (34), and GSSG represents only ≈10% of this pool. Because protein glutathionylation in vivo may be favored by oxidative stress, we tested the ability of different oxidants (1 mM diamide, 1 mM H2O2, or 1 mM CuCl2) to promote AtTRXf glutathionylation in the presence of glutathione at a concentration and an initial GSH/GSSG ratio close to physiological conditions found in the chloroplasts (2 mM GSH/0.2 mM GSSG). In these conditions, MALDI-TOF analysis did not reveal the presence of TRXf dimers. All these oxidants enhanced the glutathionylation efficiency of TRXf, none of them promoting glutathionylation of other TRXs (data not shown). These conditions seemed even more efficient for glutathionylation than 5 mM GSSG, especially when diamide was used as an oxidant. Indeed, in the presence of 1 mM diamide and 2 mM GSH/0.2 mM GSSG, 80% of the TRX could be glutathionylated in 1 h, whereas this level was reached only after 5-6 h in the presence of 5 mM GSSG. A comparable loss of free SH groups on TRXf was observed when 5 mM cysteine was used instead of 5 mM GSSG or when, in the presence of 1 mM diamide, 2 mM cysteine was used instead of 2 mM GSH (data not shown). This finding indicates that f-type TRXs are more generally S-thiolatable. However, because glutathione represents the most important free thiol in the cell, conditions leading to S-thiolation will result mainly in glutathionylation of target protein cysteines. Moreover, we could trigger glutathionylation by addition of oxidants in the presence of glutathione at a concentration and an initial GSH/GSSG ratio close to physiological conditions. Thus, although all experiments presented here were performed in vitro, these results suggest that glutathionylation of TRX f could occur in vivo under oxidative stress.

3D Structure Modeling. Three-dimensional structures were available for HsTRX (35, 36), SoTRXf1 (37), and CrTRXm (29). Structures of A. thaliana TRXs were modeled by using a representative structure of CrTRXm. The cysteines that can be easily glutathionylated in HsTRX, AtTRXf1, and SoTRXf1 are located in the loop connecting helix α3 and strand β4, close to the active site (Fig. 5). In contrast, nonglutathionylated extra cysteines are located in secondary structures and are remote from the active site. Solvent accessibility surfaces show that the cysteine sulfur is largely exposed in Cys-73 of HsTRX, Cys-85 of AtTRXy1, and Cys-86 of CrTRXm. Surface electrostatic potentials are also shown (Fig. 5).

Fig. 5.

Fig. 5.

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Modeling of chloroplast thioredoxins displaying additional cysteines. (Left) Ribbon representation of TRXs, displaying the cysteines of the active site in green and the other cysteines in pink. PDB coordinates were used for HsTRX [1ERU (35)], SoTRXf1 [1F9M(A) (37)], and CrTRXm (structure 16 of 1DBY). Models generated with the program modeler were used for AtTRXf1, AtTRXm3, and AtTRXy1. (Center) Position of the cysteines (sulfur atoms in yellow) at the surface of the various TRXs. (Right) Electrostatic potentials associated to water-accessible surfaces are represented from negative to positive by an orange to blue continuous color range. Nt, N-terminal; Ct, C-terminal.

Discussion

The aims of this study were (i) to establish whether specific chloroplastic TRXs undergo glutathionylation, (ii) to examine the effect this glutathionylation has on the activity of these TRXs toward target enzymes, (iii) to investigate the structural determinants of TRX glutathionylation, and (iv) to explore whether TRX glutathionylation is promoted by conditions that typically occur in plants under stress conditions. The results clearly show that, among all chloroplastic TRXs, only f-type TRXs are glutathionylated. These TRXs are specifically required to activate several Calvin-cycle enzymes and are the most efficient in the activation of all other TRX targets involved in carbon metabolism, such as NADP-MDH (3, 4). The glutathionylatable Cys of TRXf is invariant among all species but is not required for TRX activity (33). Here, we have shown that the status of this third Cys likely plays a key role in modulating TRX efficiency: the activation of two distinct TRXf target enzymes, NADP-MDH and GAPDH, is strongly decreased when TRXf is glutathionylated. Whereas this effect may occur through changes in both the reductase-TRX and TRX-target interaction, gel-shift assays revealed a clear effect of glutathionylation on the reduction of AtTRXf1 by light. This result strongly suggests that the physiological significance of TRX modulation is target-independent, i.e., glutathionylation will affect the activation of all proteins subject to regulation by TRXf.

Comparative analysis of the accessibility of the cysteines and of the electrostatic charge surfaces around them provide a number of elements that may explain the specificity of TRX glutathionylation (Fig. 5). Studies of a model system showed that introducing positively charged residues around cysteines increases glutathionylation rates by favoring electrostatic interaction with the overall negative charge of glutathione and by lowering the pKa of the cysteine (38). The high accessibility to solvent of Cys-73 in HsTRX and its electrostatic environment are consistent with its high susceptibility to glutathionylation (13). Like Cys-73 in HsTRX, glutathionylated cysteines in AtTRXf1 and SoTRXf1 are located in the flexible α3-β4 loop (39). These cysteines are accessible to IAM derivatization, and the solvent-accessible surfaces around them are charged mainly positively, favoring the interaction with glutathione. The absence of reactivity of AtTRXm3 may be because of the fact that its cysteine is buried and located in a rigid β-sheet. One of the CrTRXm cysteines is slightly glutathionylated. From the structure, Cys-86 is a good candidate because it is exposed to solvent within a large positively charged patch, whereas Cys-54 is completely buried. The case of AtTRXy1 is more difficult to explain: Cys-85 is not glutathionylated, even though the sulfur is exposed, and the presence of several positively charged residues would be expected to stabilize the interaction. The structural model obtained by sequence homology may not reflect the exact conformation of AtTRXy1. NMR or crystallographic data would be needed for a more precise interpretation.

Protein S-glutathionylation in vitro can be triggered by protein thiol oxidation by ROS followed by reaction with GSH or by thiol-disulfide exchange with GSSG (40). Both ROS and GSSG can accumulate in plants under stress (11), and it has not yet been determined which of these two mechanisms is predominant in determining glutathionylation in vivo. However, our results indicate that the most efficient way to glutathionylate TRX f in vitro is the use of an oxidant in the presence of GSH, as already observed for other proteins (17). Indeed, exposure of cells to ROS appears to be the more effective mechanism in vivo (15, 16). Our data suggest that glutathionylation could be a way of decreasing TRXf activity under conditions of enhanced ROS production. As a consequence, the activity of TRXf targets, including Calvin-cycle enzymes, will also decrease, thus slowing down the Calvin cycle. The glutathionylation of some enzymes of this cycle for which TRX-dependence has not been shown (18) might also contribute to this slowing down. Because this cycle is the main consumer of reducing power (NADPH) in chloroplasts, the effect of down-regulating the Calvin cycle would be to enhance electron availability at the reducing side of photosystem I. Two opposing scenarios could result from this redistribution of reducing power. In the first, more reductant would be available for ROS detoxification by ascorbate peroxidase, glutathione peroxidase, and monodehydroascorbate reductase (34), as well as peroxiredoxins that use other forms of TRXs (4, 5) that do not undergo glutathionylation and, so, remain active. Once ROS have been detoxified, there presumably exists a system to deglutathionylate f-type TRXs and the other glutathionylated proteins. Glutaredoxins (GRXs), members of the TRX super-family, seem to be specifically required for deglutathionylation of proteins (14, 41). Chloroplasts do not apparently contain classical CPYC GRXs but, rather, CGFS GRXs (42). However, the major, presumably, chloroplastic GRX is not reduced by glutathione in standard conditions and, thus, is unable to reverse the glutathionylation of TRXf (L.M. and S.D.L., unpublished results). A specific GRX reductase will have to be identified to study the physiological mechanism controlling TRXf deglutathionylation. Alternatively, overreduction of photosystem I acceptors could lead to enhanced ROS generation, representing a feedback mechanism that reinforces the initial oxidative signal. Further work is required to elucidate which of these scenarios is favored by the specific glutathionylation of TRXf and to establish how the process is reversed.

Acknowledgments

We thank Pr. Peter Schürmann (University of Neuchâtel, Neuchâtel, Switzerland) for the kind gift of SoTRXf and the Synechocystis FTR production vector.

Author contributions: M.M.-M. and S.D.L. designed research; L.M., M.Z., C.M., P.D., P. Tsan, and S.D.L. performed research; V.C. contributed new reagents/analytic tools; C.M., P.D., J.-M.L., P. Trost, G.N., and S.D.L. analyzed data; and L.M., P. Tsan, M.M.-M., G.N., and S.D.L. wrote the paper.

Conflict of interest statement: No conflicts declared.

Abbreviations: TRX, thioredoxin; AtTRX, A. thaliana TRX; AtTRXf1-SG, glutathionylated AtTRXf1; CrTRX, C. reinhardtii TRX; Fd, ferredoxin; FTR, Fd TRX reductase; GSH/GSSG, reduced/oxidized glutathione; Hs TRX, human TRX; NADP-MDH, NADP-malate dehydrogenase; ROS, reactive oxygen species; SoTRX, spinach TRX.

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