CP12-mediated protection of Calvin-Benson cycle enzymes from oxidative stress

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin-Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin-Benson cycle from oxidative stress.

Keywords: 1,3-bisphosphoglycerate; 5,5-dithiobis(2-nitrobenzoic acid); Arabidopsis thaliana; At; BPGA; Bio-GSSG; Calvin–Benson cycle; Chlamydomonas reinhardtii; Cr; DTNB; DTT; GAPDH; GSH; GSNO; GSSG; Glutathionylation; Grxs; IAM; MALDI-ToF; N-ethylmaleimide; NEM; Oxidative stress; PRK; Protein–protein interaction; TCEP; Thiol-based redox regulation; Tris(2-carboxyethyl)phosphine; Trxs; biotinylated oxidized glutathione; dithiothreitol; glutaredoxins; glyceraldehyde-3-phosphate dehydrogenase; iodoacetamide; matrix-assisted laser-desorption ionization-time-of-flight; nitrosoglutathione; oxidized glutathione; phosphoribulokinase; reduced glutathione; thioredoxins.