Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

Plant chloroplasts have evolved multiple strategies with which to adapt photosynthesis to fluctuating light environments. One such strategy involves the redox regulation of various enzymes that function in photosynthesis reactions. Multiple photosynthesis-related proteins, such as the four Calvin–Benson cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase FBPase], sedoheptulose-1,7-bisphosphatase SBPase], and phosphoribulokinase PRK]), possess redox-active Cys residues (1, 2). In addition, the γ-subunit of ATP synthase (CF₁-γ) and two regulatory proteins associated with Calvin–Benson cycle enzymes, CP12 and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA), are also redox-regulated (2–4). In the 1970s thioredoxin (Trx) was identified as a reducing power mediator for FBPase and SBPase in chloroplasts (5, 6). In a light-containing environment, reducing power is transferred from the photosynthetic electron transport system to Trx via ferredoxin and ferredoxin-Trx reductase (6). Trx then achieves light-dependent activation of its target enzymes by reducing the disulfide bond on these enzymes.In chloroplasts, NADPH-Trx reductase C (NTRC) works in parallel with the Trx-dependent system as another redox pathway. NTRC is also a redox-responsive protein containing both NADPH-dependent Trx reductase and Trx domains; these enable NTRC to reduce its target proteins using the reducing power of NADPH (7). NTRC can reduce 2-Cys peroxiredoxin (2-Cys Prx) in addition to several Trx-targeted proteins (8–12). 2-Cys Prx utilizes reducing power to reduce reactive oxygen species such as H₂O₂ (13). In chloroplasts, NTRC is thought to be a major electron donor for 2-Cys Prx (14) because the reducing power transfer efficiency from NTRC is extremely high compared with that from typical chloroplast Trx proteins (12). Plants deficient in NTRC show severe phenotypes, such as stunted growth, low chlorophyll content, and very high nonphotochemical quenching (NPQ) (7, 11, 12, 14–17). Thus, it is clear that NTRC plays important physiological roles in chloroplasts.Redox-regulated proteins in the stroma are reduced in the light and then reoxidized in the dark (18, 19). Reoxidation is an important process in plants; for example, we recently showed that the reoxidation of chloroplast NADP-malate dehydrogenase is important for maintaining NADPH homeostasis in chloroplasts, particularly in an environment with fluctuating light (20). Despite the importance of the oxidation process, the proteins involved in target oxidation have yet to be clarified. Recently, Trx-like proteins, such as Trx-like 2 (TrxL2) and atypical Cys His-rich Trx (ACHT), have been suggested as oxidation factors (21–27). These reports were mainly based on the results of in vitro experiments, suggesting that Trx-like proteins transfer the reducing power of Trx-targeted proteins to H₂O₂ via 2-Cys Prx. However, the functions of these proteins in vivo are not known very well. The so-called common Trxs belonging to f-, m-, x-, y- (or z-?) types were also thought to be the candidate of the oxidation factor. Because it is known that, particularly, Trx-f can oxidize its target proteins under certain conditions in vitro (25, 28), we focused this work on Trx-f.Target oxidation by ACHT1 and ACHT2, among five ACHT isoforms in Arabidopsis thaliana (29), has been demonstrated in vitro (25). ACHT1 and ACHT2 are broadly conserved in photosynthetic organisms, including green algae, moss, and seed plants (30). Their amino acid sequences and biochemical properties are similar (SI Appendix, Fig. S1A) (25, 29). In addition, ACHT1 and ACHT2 (designated also as Lilium5 and Lilium2, respectively) are predicted to originate from the same ancestral gene (31). Comparison of the expression patterns of ACHT1 and ACHT2 in the database shows that ACHT2 is expressed more than ACHT1, especially in leaves (SI Appendix, Fig. S1B) (32), suggesting that ACHT2 may play a dominant role in A. thaliana leaves.Oxidation of target proteins by the TrxL2 isoforms from A. thaliana, namely TrxL2.1 and TrxL2.2, has been demonstrated in vitro (22). TrxL2 genes are also conserved in photosynthetic organisms, such as seed plants, moss, and some green algae, but not in Chlamydomonas reinhardtii (30). Although the amino acid sequences and biochemical properties of TrxL2.1 and TrxL2.2 are similar (SI Appendix, Fig. S2A) (22), their expression patterns are different, and TrxL2.1 is reported to be more expressed than TrxL2.2, particularly in leaves (SI Appendix, Fig. S2B) (32). In addition, TrxL2.1 expression seems to be regulated by the circadian rhythm and the rhythm of temperature change (SI Appendix, Fig. S2C). The expression of TrxL2.1 is more strongly induced before and during light-to-dark transitions than TrxL2.2, suggesting that TrxL2.1 plays a predominant role during these periods.In the present study, we generated A. thaliana mutant plant lines deficient in Trx-f1 and Trx-f2, TrxL2.1, or ACHT1 and ACHT2, whose target oxidation activities are well studied in vitro, and used these plants to investigate redox state changes in chloroplasts. We found that Trx-f, TrxL2.1, ACHT1, and ACHT2 are involved in the oxidation of FBPase, CF₁-γ, and RCA. ACHT2 also seemed to be involved in the regulation of NPQ. Furthermore, the knockout of Trx-like proteins suppressed the impact of NTRC deficiency in plants, suggesting that a connection existed between the NTRC system and Trx-like protein-involving system.