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.

Transmission of reductive and oxidative cues from the photosynthetic electron transport chain to redox regulatory protein networks plays a crucial role in coordinating chloroplast metabolism and photosynthetic activities. The tight balance between these two signals dictates the cellular response to changing light conditions. The highly abundant stromal peroxidase, 2-Cys peroxiredoxin (2-Cys Prx), derives light-dependent oxidative signals by transferring oxidizing equivalents of photosynthetically produced H2O2 to redox-regulated proteins. However, a comprehensive physiological understanding of the role of oxidative signals under changing light intensities is still lacking, mainly due to the difficulties in accurately and nondestructively monitoring them. Here, we introduced chl-roGFP2-PrxΔCR, a 2-Cys Prx-based biosensor, into Arabidopsis thaliana chloroplasts to monitor the dynamic changes in photosynthetically-derived oxidative signaling. We showed that chl-roGFP2-PrxΔCR oxidation states reflected the similar oxidation patterns of endogenous 2-Cys Prx under varying light conditions, as determined by both redox western blots and fluorescence measurements. Furthermore, systematic monitoring of probe oxidation throughout the day led to the identification of the dynamic light intensity range of 2-Cys Prx-mediate redox signaling and demonstrated the induction of quantifiable redox signals under low-light conditions. The presented data highlights the role of oxidative signals under nonstressed conditions and suggests 2-Cys Prx-based biosensor as a powerful tool to explore their dynamic in higher plants.

Nitrate reduction and subsequent ammonium assimilation require reducing equivalents directly produced by the photosynthetic electron transport chain. Therefore, it has been suggested that nitrate assimilation provides a valuable sink for excess electrons under high-light (HL) conditions, which protects the photosynthetic apparatus from excessive harmful reactive oxygen species. This work experimentally tested this hypothesis by monitoring photosynthetic efficiency and the chloroplastic glutathione redox state (chl-EGSH) of plant lines with mutated glutamine synthetase 2 (GS2) and ferredoxin-dependent glutamate synthase 1 (GOGAT1), two key enzymes of the nitrogen assimilation pathway. Unlike wild-type (WT) plants, mutant lines incorporated significantly less isotopically-labeled nitrate into amino acids, demonstrating impaired nitrogen assimilation. When nitrate assimilation was compromised, photosystem II (PSII) proved more vulnerable to photodamage, as shown by the low PSII quantum yields recorded in the mutant lines. High temporal resolution monitoring of the redox state of chloroplast-targeted reduction-oxidation sensitive green fluorescent protein 2 (chl- roGFP2), expressed in the background of the mutant lines, enabled assessment of the effect of the nitrate assimilation pathway on the chl-EGSH. Remarkably, while oxidation followed by reduction of chl-roGFP2 was detected in WT plants in response to HL, oxidation values were stable in the mutant lines, suggesting that the relaxation of chl-EGSH after HL-induced oxidation is achieved by diverting excess electrons to the nitrogen assimilation pathway. Together, these findings indicate that the nitrogen assimilation pathway serves as a sustainable energy dissipation route, ensuring efficient photosynthetic activity and fine-tuning redox metabolism under light-saturated conditions.