Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses.
The 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 gamma-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 co-immunoprecipitation 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.
Rey
Pascal
P
CEA/Cadarache, DSV, DEVM, Laboratoire d'Ecophysiologie de la Photosynthèse, 13108 Saint-Paul-lez-Durance Cedex, France. pascal.rey@cea.fr
Cuiné
Stéphan
S
Eymery
Françoise
F
Garin
Jérome
J
Court
Magali
M
Jacquot
Jean-Pierre
JP
Rouhier
Nicolas
N
Broin
Mélanie
M
D002846:Chromatography, Affinity; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D018384:Oxidative Stress; D009195:Peroxidase / Q000378:metabolism; D010788:Photosynthesis; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis*; D018087:Plastids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D011198:Solanum tuberosum / Q000378:metabolism; D013879:Thioredoxins / Q000032:analysis*
D010936:Plant Extracts; D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D009195:Peroxidase