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The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m are also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, growth retardation being noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin–Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system.

The concerted regulation of chloroplast biosynthetic pathways and NADPH extrusion via malate valve depends on f and m thioredoxins (Trxs). The finding that decreased levels of the thiol-peroxidase 2-Cys peroxiredoxin (Prx) suppress the severe phenotype of Arabidopsis mutants lacking NADPH-dependent Trx reductase C (NTRC) and Trxs f uncovered the central function of the NTRC-2-Cys-Prx redox system in chloroplast performance. These results suggest that Trxs m is also regulated by this system; however, the functional relationship between NTRC, 2-Cys Prxs, and m-type Trxs is unknown. To address this issue, we generated Arabidopsis thaliana mutants combining deficiencies in NTRC, 2-Cys Prx B, Trxs m1, and m4. The single trxm1 and trxm4 mutants showed a wild-type phenotype, with growth retardation noticed only in the trxm1m4 double mutant. Moreover, the ntrc-trxm1m4 mutant displayed a more severe phenotype than the ntrc mutant, as shown by the impaired photosynthetic performance, altered chloroplast structure, and defective light-dependent reduction in the Calvin–Benson cycle and malate-valve enzymes. These effects were suppressed by the decreased contents of 2-Cys Prx, since the quadruple ntrc-trxm1m4-2cpb mutant displayed a wild-type-like phenotype. These results show that the activity of m-type Trxs in the light-dependent regulation of biosynthetic enzymes and malate valve is controlled by the NTRC-2-Cys-Prx system.

Redox regulation in heterotrophic organisms relies on NADPH, thioredoxins (TRXs), and an NADPH-dependent TRX reductase (NTR). In contrast, chloroplasts harbor two redox systems, one that uses photoreduced ferredoxin (Fd), an Fd-dependent TRX reductase (FTR), and TRXs, which links redox regulation to light, and NTRC, which allows the use of NADPH for redox regulation. It has been shown that NTRC-dependent regulation of 2-Cys peroxiredoxin (PRX) is critical for optimal function of the photosynthetic apparatus. Thus, the objective of the present study was the analysis of the interaction of NTRC and 2-Cys PRX in vivo and the identification of proteins interacting with them with the aim of identifying chloroplast processes regulated by this redox system. To assess this objective, we generated Arabidopsis thaliana plants expressing either an NTRC–tandem affinity purification (TAP)-Tag or a green fluorescent protein (GFP)–TAP-Tag, which served as a negative control. The presence of 2-Cys PRX and NTRC in complexes isolated from NTRC–TAP-Tag-expressing plants confirmed the interaction of these proteins in vivo. The identification of proteins co-purified in these complexes by MS revealed the relevance of the NTRC–2-Cys PRX system in the redox regulation of multiple chloroplast processes. The interaction of NTRC with selected targets was confirmed in vivo by bimolecular fluorescence complementation (BiFC) assays.

Thioredoxins (Trxs) are small, ubiquitous enzymes that catalyze disulphide–dithiol interchange in target enzymes. The large set of chloroplast Trxs, including f, m, x and y subtypes, use reducing equivalents fueled by photoreduced ferredoxin (Fdx) for fine-tuning photosynthetic performance and metabolism through the control of the activity of redox-sensitive proteins. Although biochemical analyses suggested functional diversity of chloroplast Trxs, genetic studies have established that deficiency in a particular Trx subtype has subtle phenotypic e ects, leading to the proposal that the Trx isoforms are functionally redundant. In addition, chloroplasts contain an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. Interestingly, Arabidopsis mutants combining the deficiencies of x- or f-type Trxs and NTRC display very severe growth inhibition phenotypes, which are partially rescued by decreased levels of 2-Cys peroxiredoxins (Prxs). These findings indicate that the reducing capacity of Trxs f and x is modulated by the redox balance of 2-Cys Prxs, which is controlled by NTRC. In this study, we explored whether NTRC acts as a master regulator of the pool of chloroplast Trxs by analyzing its functional relationship with Trxs y. While Trx y interacts with 2-Cys Prxs in vitro and in planta, the analysis of Arabidopsis mutants devoid of NTRC and Trxs y suggests that Trxs y have only a minor e ect, if any, on the redox state of 2-Cys Prxs.

Redox regulation in heterotrophic organisms relies on NADPH, thioredoxins (TRXs), and an NADPH-dependent TRX reductase (NTR). In contrast, chloroplasts harbor two redox systems, one that uses photoreduced ferredoxin (Fd), an Fd-dependent TRX reductase (FTR), and TRXs, which links redox regulation to light, and NTRC, which allows the use of NADPH for redox regulation. It has been shown that NTRC-dependent regulation of 2-Cys peroxiredoxin (PRX) is critical for optimal function of the photosynthetic apparatus. Thus, the objective of the present study was the analysis of the interaction of NTRC and 2-Cys PRX in vivo and the identification of proteins interacting with them with the aim of identifying chloroplast processes regulated by this redox system. To assess this objective, we generated Arabidopsis thaliana plants expressing either an NTRC–tandem affinity purification (TAP)-Tag or a green fluorescent protein (GFP)–TAP-Tag, which served as a negative control. The presence of 2-Cys PRX and NTRC in complexes isolated from NTRC–TAP-Tag-expressing plants confirmed the interaction of these proteins in vivo. The identification of proteins co-purified in these complexes by MS revealed the relevance of the NTRC–2-Cys PRX system in the redox regulation of multiple chloroplast processes. The interaction of NTRC with selected targets was confirmed in vivo by bimolecular fluorescence complementation (BiFC) assays.

Light is probably the most important environmental stimulus for plant development. As sessile organisms, plants have developed regulatory mechanisms that allow the rapid adaptation of their metabolism to changes in light availability. Redox regulation based on disulfide-dithiol exchange constitutes a rapid and reversible post-translational modification, which affects protein conformation and activity. This regulatory mechanism was initially discovered in chloroplasts when it was identified that enzymes of the Calvin-Benson cycle (CBC) are reduced and active during the day and become rapidly inactivated by oxidation in the dark. At present, the large number of redox-sensitive proteins identified in chloroplasts extend redox regulation far beyond the CBC. The classic pathway of redox regulation in chloroplasts establishes that ferredoxin (Fdx) reduced by the photosynthetic electron transport chain fuels reducing equivalents to the large set of thioredoxins (Trxs) of this organelle via the activity of a Fdx-dependent Trx reductase (FTR), hence linking redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. The presence in chloroplasts of this NADPH-dependent redox system raises the question of the functional relationship between NTRC and the Fdx-FTR-Trx pathways. Here, we update the current knowledge of these two redox systems focusing on recent evidence showing their functional interrelationship through the action of the thioldependent peroxidase, 2-Cys peroxiredoxin (2-Cys Prx). The relevant role of 2-Cys Prxs in chloroplast redox homeostasis suggests that hydrogen peroxide may exert a key function to control the redox state of stromal enzymes. Indeed, recent reports have shown the participation of 2-Cys Prxs in enzyme oxidation in the dark, thus providing an explanation for the long-lasting question of photosynthesis deactivation during the light-dark transition.