Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxindependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed.O ur knowledge on redox regulation of various physiological processes through thiol-disulfide interchange with proteins of the thioredoxin (TRX) superfamily is rapidly progressing as genome-wide approaches and functional genomics studies are expanding. In the plant biology domain, thioredoxin-dependent regulation has been first discovered for chloroplastic enzymes involved in carbon assimilation (1). The completion of the sequencing of the Arabidopsis genome revealed that TRXs constituted a multigene family and led to the assumption that numerous TRX targets were still to be discovered (2-4). Thus, proteomic approaches aimed at identifying the largest possible number of TRX targets have been developed. The first attempts took advantage of the reaction mechanism of TRXs with their targets, in which a transient heterodisulfide forms between the reduced TRX and the oxidized protein, followed by the release of the reduced target due to the attack of the mixed disulfide by the second cysteine of TRX. Model studies showed that when this second cysteine was mutated, the heterodisulfide was stabilized (5, 6). This property was applied in vivo by transforming a TRX-null mutant yeast with a monocysteinic TRX and allowed isolating a peroxiredoxin (PRX) (7). Subsequently, monocysteinic TRX mutants were used in affinity chromatography to trap interacting proteins (8-11). Native TRX affinity columns also allowed retaining some targets by electrostatic interaction (12). Another technique was also developed that consists of a reduction of a crude soluble protein extract with reduced TRX followed by a separation of the proteins on a 2D gel after derivatization of the newly appeared thiols either with a fluorescent (13) or with a radioactive (14) reagent. In one case, both techniques have been applied in parallel (15), yielding similar results.Most of the disulfide proteomics have been led with higher plants: spinach (8, 10, 12), Arabidopsis (11, 14), or cereal grains (12). A single study was led with cyanobacteria and revealed targets mostly different from those of higher plants (16). The unicellular eukaryotic green alga Chlamydomonas reinhardtii possesses a TRX syste...

Proteomics was used to search for putative thioredoxin (TRX) targets in leaves of the model plant, Arabidopsis thaliana. About forty different proteins have been found to be reduced by TRX, after TRX itself has been specifically reduced by its NADPH-dependent reductase. Twenty-one of the identified proteins were already known or recently proposed to be TRX-dependent and nineteen of the proteins were new potential targets. The identified proteins are involved in a wide variety of processes, including the Calvin cycle, metabolism, photosynthesis, folding, defense against oxidative stress and amino acid synthesis. Two proteins from the glycine cleavage complex were also identified as putative TRX targets, and a new role can be postulated in leaves for TRX in defense against herbivores and/or pathogens.

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