Keisuke Yoshida, Ayaka Hara, Kazunori Sugiura, Yuki Fukaya, and Toru Hisabori

PNAS August 28, 2018 115 (35) E8296-E8304

Significance

To ensure efficient photosynthetic carbon gain, plant chloroplasts have to adjust their own physiology toward changes in light environments. Specific chloroplast proteins are reversibly activated–inactivated during light–dark cycles by switching the reduction–oxidation states of their Cys residues, which is termed redox regulation. A long-standing issue in plant biology is the manner in which redox-regulated proteins are reoxidized upon the interruption of light exposure. In this study, we identified the thioredoxin-like2 (TrxL2)/2-Cys peroxiredoxin (2CP) redox cascade as a molecular basis for oxidative thiol modulation in chloroplasts. This finding dissects the “dark side” of chloroplast redox regulation, providing an insight into how plants rest their photosynthetic activity at night.

Abstract

Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H₂O₂). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light–dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.

See: http://www.pnas.org/content/115/35/E8296

Figure 1: An overview of midpoint redox potentials (E_m) of FTR-C, 10 Trx isoforms, NTRC (including NTR domain and Trx domain), and two TrxL2 isoforms from Arabidopsis. Each E_m was determined at pH 7.5. The data for TrxL2 are shown in SI Appendix, Fig. S4. The data for other proteins can be found in our earlier studies (11, 16, 17).