Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses

Abstract

SummaryThe chloroplastic drought-induced stress protein of 32 kDa (CDSP32) is a thioredoxin induced by environmental stress conditions. To gain insight into the function of CDSP32, we applied two strategies to analyze its targets. First, using affinity chromatography with an immobilized CDSP32 active site mutant, we identified six plastidic targets of CDSP32. Three of them are involved in photosynthetic processes: ATP-ase c-subunit, Rubisco and aldolase. The three others participate in the protection against oxidative damage: two peroxiredoxins, PrxQ and the BAS1 2-Cys peroxiredoxin, and a B-type methionine sulfoxide reductase. Then, we developed a novel strategy to trap targets directly in leaf extracts. The method, based on coimmunoprecipitation using extracts from plants overexpressing Wt CDSP32 or CDSP32 active site mutant, confirmed the interaction in vivo between CDSP32 and the PrxQ and BAS1 peroxiredoxins. We showed that CDSP32 is able to form heterodimeric complexes with PrxQ and that the peroxiredoxin displays CDSP32-dependent peroxidase activity. Under photooxidative stress induced by methyl viologen, plants overexpressing CDSP32 active site mutant exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared with Wt plants and with plants overexpressing Wt CDSP32. We propose that the increased sensitivity results from trapping in planta of the targets involved in the protection against oxidative damage. We conclude that CDSP32, compared with other plant thioredoxins, is a thioredoxin more specifically involved in plastidic responses against oxidative stress.

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

“…It is possible that ACHT1 might have a transient reductive role in this time frame, during which the Calvin cycle is activated and the oxidative pentose phosphate pathway is inactivated. In case the reduction of additional Trxs, such as other ACHT family members (Dangoor et al, 2009) or the stress-induced chloroplast CDSP32 (Rey et al, 2005), is as limiting as the reduction of ACHT1, they may form a similar oxidative signaling pathway with 2-Cys Prx, which may be used to regulate other plant responses.…”

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

“…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.…”

“…matography experiments (29). Moreover, activity assays showed that CDSP32 provides MSRB1 with electrons (16,30).…”

“…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).…”

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

“…To answer these questions, much recent work has been devoted to large-scale searches for their putative targets. Various improved in vitro proteomic approaches [such as affinity chromatography (16)(17)(18)(19)(20)(21), labeled gel electrophoresis in which target proteins are revealed after reduction of protein extract by a TRX͞NADPH thioredoxin reductase (NTR) system (22)(23)(24), or coimmunoprecipitation (25)] suggested Ͼ100 putative TRX targets, but for which further investigations will be required for target validation regarding TRX specificity. Other methods devoted to the large-scale isolation of TRX͞target complexes established in vivo have also been developed, including affinity chromatography of TRX͞target complexes established in vivo during cell culture or the yeast͞mammalian two-hybrid methods (26)(27)(28)(29)(30)(31).…”