Inactivation of thioredoxin f1 leads to decreased light activation of ADP-glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants.

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose-dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light-activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch-to-sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography-mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.

New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin.

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of approximately 30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 +/- 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A(4)-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A(4)-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX.

Specificity of thioredoxins and glutaredoxins as electron donors to two distinct classes of Arabidopsis plastidial methionine sulfoxide reductases B.

Methionine sulfoxide reductases (MSRs) A and B reduce methionine sulfoxide (MetSO) S- and R-diastereomers, respectively, back to Met using electrons generally supplied by thioredoxin. The physiological reductants for MSRBs remain unknown in plants, which display a remarkable variety of thioredoxins (Trxs) and glutaredoxins (Grxs). Using recombinant proteins, we show that Arabidopsis plastidial MSRB1 and MSRB2, which differ regarding the number of presumed redox-active cysteines, possess specific reductants. Most simple-module Trxs, especially Trx m1 and Trx y2, are preferential and efficient electron donors towards MSRB2, while the double-module CDSP32 Trx and Grxs can reduce only MSRB1. This study identifies novel types of reductants, related to Grxs and peculiar Trxs, for MSRB proteins displaying only one redox-active cysteine.

Thioredoxins in chloroplasts.

Thioredoxins (TRXs) are small disulfide oxidoreductases of ca. 12 kDa found in all free living organisms. In plants, two chloroplastic TRXs, named TRX f and TRX m, were originally identified as light dependent regulators of several carbon metabolism enzymes including Calvin cycle enzymes. The availability of genome sequences revealed an unsuspected multiplicity of TRXs in photosynthetic eukaryotes, including new chloroplastic TRX types. Moreover, proteomic approaches and focused studies allowed identification of 90 potential chloroplastic TRX targets. Lately, recent studies suggest the existence of a complex interplay between TRXs and other redox regulators such as glutaredoxins (GRXs) or glutathione. The latter is involved in a post-translational modification, named glutathionylation that could be controlled by GRXs. Glutathionylation appears to specifically affect the activity of TRX f and other chloroplastic enzymes and could thereby constitute a previously undescribed regulatory mechanism of photosynthetic metabolism under oxidative stress. After summarizing the initial studies on TRX f and TRX m, this review will focus on the most recent developments with special emphasis on the contributions of genomics and proteomics to the field of TRXs. Finally, new emerging interactions with other redox signaling pathways and perspectives for future studies will also be discussed.

Thioredoxins, glutaredoxins, and glutathionylation: new crosstalks to explore.

Oxidants are widely considered as toxic molecules that cells have to scavenge and detoxify efficiently and continuously. However, emerging evidence suggests that these oxidants can play an important role in redox signaling, mainly through a set of reversible post-translational modifications of thiol residues on proteins. The most studied redox system in photosynthetic organisms is the thioredoxin (TRX) system, involved in the regulation of a growing number of target proteins via thiol/disulfide exchanges. In addition, recent studies suggest that glutaredoxins (GRX) could also play an important role in redox signaling especially by regulating protein glutathionylation, a post-translational modification whose importance begins to be recognized in mammals while much less is known in photosynthetic organisms. This review focuses on oxidants and redox signaling with particular emphasis on recent developments in the study of functions, regulation mechanisms and targets of TRX, GRX and glutathionylation. This review will also present the complex emerging interplay between these three components of redox-signaling networks.