Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts

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.

Keywords: 2-Cys peroxiredoxin; TrxL2; chloroplast; oxidation; redox regulation.

Fig. 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).

Fig. 2.

Biochemical characterization of target-reducing activity of Trx and TrxL2. (A) Redox shift visualization of FBPase, SBPase, and RCA. (B) Redox shift visualization of 2CPA and PrxQ. (A and B) Each target protein (oxidized form; 2 μM) was incubated with Trx-f1, Trx-m1, TrxL2.1, or TrxL2.2 (1 μM) in the presence of DTT (0.5 mM for FBPase, SBPase, RCA, and 2CPA; 0.05 mM for PrxQ) for 30 min. Free thiols were labeled with AMS (for FBPase, SBPase, RCA, and PrxQ) or N-ethylmaleimide (for 2CPA), and proteins were subjected to nonreducing SDS/PAGE. (C–F) Comparison of Prx-reducing activity of Trx-f1 and TrxL2. Reduction level of 2CPA (C and D) or PrxQ (E and F) was calculated as the ratio of the reduced form to the total, and plotted against the DTT concentration (C and E) or the reaction time (D and F). Raw images of the SDS/PAGE and immunoblotting are shown in SI Appendix, Fig. S6 A–D. Each value represents the mean ± SD (n = 3). Ox, oxidized form; Red, reduced form.

Fig. 3.

Biochemical characterization of target-oxidizing activity of TrxL2. (A) An example of redox shift visualization. RCA (after reduction treatment; 2 μM) was incubated with TrxL2 (oxidized form; 2 μM) for the indicated time. Free thiols were labeled with AMS, and proteins were subjected to nonreducing SDS/PAGE. (B–D) Comparison of target-oxidizing activity of Trx-f1 and TrxL2. Reduction level of RCA (B), FBPase (C), or SBPase (D) was calculated as the ratio of the reduced form to the total, and plotted against the reaction time. Data on the reduction level of Trx-f1 or TrxL2 are also shown. Raw images of the SDS/PAGE are shown in SI Appendix, Fig. S8. Each value represents the mean ± SD (n = 3). Ox, oxidized form; Red, reduced form.

Fig. 4.

Reconstitution of the TrxL2/2CP redox cascade in vitro for oxidizing target protein and draining reducing power to H₂O₂. Reduction level of RCA (A), FBPase (B), or SBPase (C) was calculated as the ratio of the reduced form to the total and plotted against the reaction time. Data on the reduction level of TrxL2 are also shown. Raw images of the SDS/PAGE and immunoblotting are shown in SI Appendix, Fig. S11. Each value represents the mean ± SD (n = 3).

Fig. 5.

Redox dynamics in vivo. Arabidopsis wild-type (WT) and mutant (2cpa, 2cpb, and 2cpa 2cpb) plants were dark adapted for 12 h, irradiated by HL (700 μmol photons m⁻²⋅s⁻¹) for 30 min, and transferred back to the dark. During this period, the redox state of ATP synthase CF₁-γ subunit (A), RCA (B), FBPase (C), and SBPase (D) was sequentially determined. Reduction level was calculated as the ratio of the reduced form to the total. Each value represents the mean ± SD from three biological replicates. Ox, oxidized form; Red, reduced form; R.I., redox-insensitive form of RCA.

Fig. 6.

Redox dynamics in vivo. Arabidopsis wild-type (WT) and mutant (2cpa, 2cpb, and 2cpa 2cpb) plants were dark adapted for 12 h, irradiated by HL (700 μmol photons m⁻²⋅s⁻¹) for 30 min, and transferred back to the dark. During this period, the redox state of Trx-f1 (A), Trx-m2 (B), Trx-x (C), Trx-y2 (D), TrxL2.1 (E), TrxL2.2 (F), and 2CP (G) was sequentially determined. (A and D) Oxidation index was calculated as the ratio of the Ox1 form to the total. Reduction index was calculated as the ratio of the Red2 form to the total. (B, C, E, and G) Reduction level was calculated as the ratio of the reduced form to the total. (E) Asterisk indicates a possible nonspecific band. (G) As a loading control, Rubisco large subunit was stained with CBB. Each value represents the mean ± SD from three biological replicates. Ox, oxidized form; Red, reduced form.

Fig. 7.

Simplified working model of chloroplast redox regulation. (A) Classically recognized “light-side” reaction. In response to light-dependent excitation of electron transport chain (ETC), redox-regulated proteins are reduced via the FTR/Trx pathway. (B) Newly emerging “dark-side” reaction. Redox-regulated proteins are oxidized via the TrxL2/2CP pathway. Several other factors may also be involved in this process directly or indirectly. See the main text for details. Ox, oxidized form; Red, reduced form.