The Plastidic 2-Cysteine Peroxiredoxin Is a Target for a Thioredoxin Involved in the Protection of the Photosynthetic Apparatus against Oxidative Damage

Abstract

The chloroplastic drought-induced stress protein of 32 kD (CDSP32) is composed of two thioredoxin modules and is induced by environmental and oxidative stress conditions. We investigated whether the plastidic protein BAS1, which is related to eubacterial 2-Cys peroxiredoxin, is a target for CDSP32. Using a CDSP32 active-site mutant, we showed that the BAS1 and CDSP32 proteins form a mixed disulfide complex in vitro. Moreover, affinity chromatography indicated that BAS1 is a major target for CDSP32 in chloroplasts. CDSP32 was able to reduce BAS1 in vitro, and BAS1 displayed CDSP32-dependent peroxidase activity. The function of CDSP32 was investigated in transgenic potato lines without detectable levels of the protein as a result of cosuppression. Under conditions of photooxidative stress induced by incubation with either methyl viologen or t -butyl hydroperoxide or by exposure to low temperature under high light, plants lacking CDSP32 exhibited decreased maximal photosystem II photochemical efficiencies compared with the wild type and transgenic controls. In addition, plants without CDSP32 retained much less chlorophyll than controls under stress, indicating increased damage to photosynthetic membranes. We conclude that CDSP32 is a thioredoxin with a critical role in plastid defense against oxidative damage and that this role is related to its function as a physiological electron donor to the BAS1 peroxiredoxin.

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

“…A similar specificity was found for CrPRX1, which is more efficiently reduced by Chlamydomonas TRX x (S. D. Lemaire, unpublished results) than by TRX m, f, or y (Lemaire et al 2003a). Besides TRX, chloroplastic 2-cys PRXs can also be reduced by CDSP32, a drought-induced TRX-like protein containing two TRX modules (Broin et al 2002) and by NTRC, a chloroplastic bipartite enzyme containing an NADPH-TRX reductase fused to its own TRX module (Moon et al 2006;Pérez-Ruiz et al 2006). Interestingly, genes encoding homologs of CDSP32 and NTRC are present in the Chlamydomonas genome and might therefore also act as reductants for PRX (Lemaire et al 2007).…”

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

“…Expression and Purification of Recombinant Proteins-Wildtype (WT) and mutated forms of MSRB1 and CDSP32 were purified as described previously (21,28). Recombinant C-terminal CDSP32 starts from Gly-181 and has a theoretical molecular mass of 14,956.1 Da.…”

“…These values are significantly less negative than those of the canonical plastidial Trxs f and m (approximately Ϫ360 mV at pH 7.9) (43,44) and lower than the redox midpoint potential of human Trx1 (approximately Ϫ290 mV) (45) but in the same range of those recorded for Escherichia coli Trx (46) or plastidial Trxs x and y (43,44). Interestingly, Trxs x and y are efficient reducers of the plastidial 2-Cys Prx (43) and Prx Q (44,47), respectively, two previously identified targets of CDSP32 (28,29). At pH 7.9, the redox midpoint potentials of 2-Cys Prx and Prx Q are around Ϫ370 mV (47,48), a value ϳ35 mV more negative than those of CDSP32, Trx x and Trx y, showing that efficient reduction of the disulfide bond formed in oxidized Prxs can occur only when the pool of Trxs is highly reduced.…”

“…CDSP32, induced under severe abiotic stress conditions (26,27), is composed of two Trx modules, with only one potential active redox disulfide center in the C-terminal domain (26). Affinity chromatography and co-immunoprecipitation assays identified plastidial 2-Cys Prxs and 1-Cys MSRB1 as potential targets of CDSP32 (28,29). Very interestingly, this Trx is able to regenerate the activity of plant and mammalian 1-Cys MSRBs without addition of GSH or any other thiol compound (30), raising the hypothesis that regeneration might proceed through an alternative mechanism, distinct from the glutathionylation/deglutathionylation process used by Grxs (21).…”

Reaction Mechanism of Plant 2-Cys Peroxiredoxin

“…Regeneration of oxidized plant 2-Cys Prx is coupled to the photosynthetic electron transport chain via the electron donors Trx m and Trx f (16,17). In addition to thioredoxin, reduction by a chloroplastic droughtinduced 32-kDa stress protein has been reported (18). The redox potential of Ϫ315 mV (16) places reductive regeneration of 2-Cys Prx between Calvin cycle activation and redox switching of the malate valve (16).…”