Nardy Lamp et al.

PNAS, June 1, 2022; 119 (23) e2119719119

Significance

Identifying the intrinsic factors that regulate leaf photosynthetic rate may pave the way toward developing new strategies to enhance carbon assimilation. While the dependence of photosynthesis on the reductive activation of the Calvin–Benson cycle enzymes is well established, the role of oxidative signals in counterbalancing the reductive activity is just beginning to be explored. By developing 2-Cys peroxiredoxin-based genetically encoded biosensors, we demonstrated the induction of photosynthetically derived oxidative signals under habitual light conditions, a phenomenon typically masked by the dominance of the reductive power. Moreover, we unraveled the simultaneous activation of reductive and oxidative signals during photosynthesis induction phase and showed that 2-Cys peroxiredoxin activity attenuates carbon assimilation rates, demonstrating the restrictions imposed on photosynthetic performance by oxidative signals.

Abstract

Transmission of reductive and oxidative cues from the photosynthetic electron transport chain to redox regulatory protein networks plays a crucial role in coordinating photosynthetic activities. The tight balance between these two signals dictates the cellular response to changing light conditions. While the role of reductive signals in activating chloroplast metabolism is well established, the role of their counterbalanced oxidative signals is still unclear, mainly due to monitoring difficulties. Here, we introduced chl-roGFP2-PrxΔC_R, a 2-Cys peroxiredoxin-based biosensor, into Arabidopsis thaliana chloroplasts to monitor the dynamic changes in photosynthetically derived oxidative signaling. We showed that chl-roGFP2-PrxΔC_R oxidation states reflected oxidation patterns similar to those of endogenous 2-Cys peroxiredoxin under varying light conditions. By employing a set of genetically encoded biosensors, we showed the induction of 2-Cys peroxiredoxin-dependent oxidative signals, throughout the day, under varying light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals. Furthermore, we demonstrated the induction of 2-Cys peroxiredoxin-derived oxidative signals during a dark–to–low-light transition and uncovered a faster increase in carbon assimilation rates during the photosynthesis induction phase in plants deficient in 2-Cys peroxiredoxins compared with wild type, suggesting the involvement of oxidative signals in attenuating photosynthesis. The presented data highlight the role of oxidative signals under nonstress conditions and suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system.

See https://www.pnas.org/doi/10.1073/pnas.2119719119

Figure 1: Light responsiveness of the 2-Cys Prx redox state. (A–E) Plants were exposed for 5.5 h to the indicated light intensities at the onset of the light periods, following being kept in the dark for 8 h. Plants pretreated with DCMU were exposed to GL (120 µmol photons m⁻² s⁻¹) for the same light periods. Total protein was extracted using TCA, and free thiols were blocked with NEM. (A) An immunoblot, using anti-PRX, shows the oxidized and reduced states of 2-Cys Prx, as dimers and monomers, respectively. (B) Quantification of the relative abundance of the dimers out of the total of 2-Cys Prx using the Western blot analysis presented in A is visualized as box plots (n ≥ 3). Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Numbers above the vertical lines denote P values (Dunnett's test; control group, 120 µmol photons m⁻² s⁻¹). (C) Schematic illustration of the redox mobility gel assay. (D) Gel-shift assay with methoxypolyethylene glycol-maleimide5 (mPEG5) showing the in vivo redox state of 2-Cys Prx residues in plants exposed to light intensities as in A. Following NEM treatment, 1,4-dithiothreitol (DTT) was applied to reduce disulfides before mPEG labeling. The number of oxidized Cys residues (Ox1 and Ox2) was determined by the increased molecular mass of 2-Cys Prx after labeling with mPEG. (E) Quantification and statistical analysis of the relative abundance of the fully oxidized 2-Cys Prx (Ox2) as reflected in the gel-shift assay presented in D. Number above the vertical lines denotes P values (Student's t test). (F) Immunoblot assay showing the oxidized and reduced states of 2-Cys Prx (anti-PRX) as dimers and monomers, respectively, and 2-Cys Prx hyperoxidation (anti-SO3) throughout the day in plants exposed to the light intensities shown in G. (G) Light intensity over time and quantification of the relative abundance of the dimers out of the total of 2-Cys Prx using the data presented in F and SI Appendix, Fig. S4. This figure is shown in black and white in SI Appendix, Fig. S1.