Two opposing redox signals mediated by 2-cys peroxiredoxin shape the redox proteome during photosynthetic induction

Abstract

Photosynthetic induction, characterized by the lag in CO₂ assimilation rates during transition from darkness to light, has traditionally been attributed to Rubisco activase activity and stomatal opening. Yet, the faster induction of photosynthesis in the 2-Cys peroxiredoxins (Prxs) mutant (2cpab) suggested a role for oxidative signals in regulating photosynthetic rates, although the underlying molecular mechanism remains unclear. SPEAR, a redox proteomics approach, was used to systematically map redox changes occurring during photosynthesis induction and to unravel the role of 2-Cys Prxs in shaping these redox alterations. No significant difference was observed in protein expression levels between WT and 2cpab plants, suggesting that protein abundance does not account for the 2cpab phenotype. During the transition from dark to low light, 82 and 54 cysteine-containing peptides were reduced or oxidized, respectively, in WT plants. Most redox-regulated cysteines in photosynthetic proteins were found oxidized in the dark and became reduced in response to light. A reverse pattern was observed among redox-regulated cysteines in proteins involved in starch degradation and chloroplast glycolysis, which shifted from a reduced to an oxidized state in response to light. These findings demonstrate the initiation of two opposing redox responses, affecting distinct sets of metabolic proteins during the induction phase. Remarkably, a significantly lower number of cysteines were reduced or oxidized in 2cpab plants, highlighting the crucial role 2-Cys Prxs play in shaping both signals. Taken together, rotational shifts between metabolic pathways during the photosynthesis induction phase are regulated by two opposing redox signals mediated by 2-Cys Prx activity.

Keywords: 2-Cys peroxiredoxins; Photosynthesis induction; Redox proteomics; Redox signaling.

Graphical abstract

Fig. 1

Simultaneous quantification of Cys oxidation and protein expression in WT and 2cpab plants under dark and low light conditions. (a) Schematic representation of the workflow for this experiment. The upper graph in the Motivation part, was adapted from Lampl et al. (2022). (b) The number of identified d₀-NEM-labeled peptides, d₅-NEM-labeled peptides, peptides labeled with both d₀ and d₅-NEM and paired peptides detected in at least two replicates across all treatments. (c) The consensus motif of NEM-labeled peptides. The Arabidopsis thaliana proteome was used as a background population for probability calculation. A similar analysis performed for non-labeled Cys-containing peptides is presented in Supplemental Fig. 1(d, e) Volcano plot visualization of the expression profile of 9662 proteins in 2cpab compared to WT plants following treatment with low light (d) or dark (e). Colored dots highlight proteins with an average ≥ 1.5 log2-fold change in 2cpab mutant plants compared to WT plants and a p-value <0.05. (f) Subcellular distribution of the identified thiol proteins was determined using the SUBA tool. (g–j) Distribution plots displaying the oxidation degree of the peptides found in (g) WT dark, (h)2cpab dark, (i) WT low light and (j)2cpab low light plants.

Fig. 2

Photosynthetic induction is characterized by two opposing redox signals, both mediated by 2 Cys Prx.(a, b) Volcano plot visualization of the response of unique cysteine-containing peptides to the transition from dark to low light (LL) in the WT (a) and 2cpab(b) plants. Colored dots highlight Cys residues with a ≥5 % change in oxidation (ΔOxD) and a p-value <0.05 in LL-treated plants compared to plants in the dark. (c, d) Venn diagrams depicting the reduced (c) and oxidized (d) unique cysteine-containing peptides in the transition from dark to LL in WT and 2cpab plants. (e) Heat map visualization of K-means clustering of Cys oxidation state in response to LL in WT or 2cpab plants. (f, g) Violin plots of the two highlighted clusters taken from the K-means clustering displayed in (e). (h) Significantly enriched GO terms and KEGG pathways (hypergeometric test, p < 0.05) in the clusters displayed in (f, g). Bubble size indicates the number of proteins that are associated with the indicated biological function. The background population used for the enrichment analysis consisted of the proteins detected in the MS data. (i, j) Box plots showing changes in the redox state of reactive cysteines that underwent reduction (i) or oxidation (j), grouped by selected biochemical pathways, in WT and 2cpab plants.

Fig. 3

Redox-sensitive Cys sites in photosynthetic light reaction proteins. Schematic representation of the light-dependent reactions of photosynthesis. The oxidation degree of the specific identified Cys in dark and LL are presented as box plots for WT and 2cpab plants. Statistical differences are stated through asterisks on the bars. For proteins: FNR2, ATPC1 and psaC, the oxidation degree was calculated for two identified Cys in the same protein, and the structure showing these Cys is presented.

Fig. 4

Redox-sensitive Cys sites in proteins involved in the Calvin-Benson cycle and downstream reactions. Schematic presentation of the Calvin-Benson cycle (CBC) and proteins involved in downstream reactions, i.e., starch synthesis, starch degradation and plastidial glycolysis. Cysteine sites that underwent reduction or oxidation during the transition from dark to LL in WT plants are colored green and pink, respectively. The oxidation degree of the identified Cys sites is presented in box plots for WT and 2cpab plants under dark and LL conditions. Statistical differences are indicated by asterisks. For RCA, GAPB and PRK, the structures showing the participation of the identified Cys in forming disulfide bonds are presented.