Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.

Summary: Retrograde signalling from plastids to the nucleus is necessary to regulate the organelle's proteome during the establishment of photoautotrophy and fluctuating environmental conditions. Studies that used inhibitors of chloroplast biogenesis have revealed that hundreds of nuclear genes are regulated by retrograde signals emitted from plastids. Plastid gene expression is the source of at least one of these signals, but the number of signals and their mechanisms used to regulate nuclear gene expression are unknown. To further examine the effects of plastid gene expression on nuclear gene expression, we analyzed Arabidopsis mutants that were defective in each of the six sigma factor (SIG) genes that encode proteins utilized by plastid‐encoded RNA polymerase to transcribe specific sets of plastid genes. We showed that SIG2 and SIG6 have partially redundant roles in plastid transcription and retrograde signalling to control nuclear gene expression. The loss of GUN1 (a plastid‐localized pentatricopeptide repeat protein) is able to restore nuclear (but not plastid) gene expression in both sig2 and sig6, whereas an increase in heme synthesis is able to restore nuclear gene expression in sig2 mutants only. These results demonstrate that sigma factor function is the source of at least two retrograde signals to the nucleus; one likely to involve the transcription of tRNA^Glu. A microarray analysis showed that these two signals accounted for at least one subset of the nuclear genes that are regulated by the plastid biogenesis inhibitors norflurazon and lincomycin. Together these data suggest that such inhibitors can induce retrograde signalling by affecting transcription in the plastid.

Thiol-dependent redox regulation allows the rapid adaptation of chloroplast function to unpredictable changes in light intensity. Traditionally, it has been considered that chloroplast redox regulation relies on photosynthetically reduced ferredoxin (Fd), thioredoxins (Trxs), and an Fd-dependent Trx reductase (FTR), the Fd-FTR-Trxs system, which links redox regulation to light. More recently, a plastid-localized NADPH-dependent Trx reductase (NTR) with a joint Trx domain, termed NTRC, was identified. NTRC efficiently reduces 2-Cys peroxiredoxins (Prxs), thus having antioxidant function, but also participates in redox regulation of metabolic pathways previously established to be regulated by Trxs. Thus, the NTRC, 2-Cys Prxs, and Fd-FTR-Trxs redox systems may act concertedly, but the nature of the relationship between them is unknown. Here we show that decreased levels of 2-Cys Prxs suppress the phenotype of the Arabidopsis thaliana ntrc KO mutant. The excess of oxidized 2-Cys Prxs in NTRC-deficient plants drains reducing power from chloroplast Trxs, which results in low efficiency of light energy utilization and impaired redox regulation of Calvin–Benson cycle enzymes. Moreover, the dramatic phenotype of the ntrc-trxf1f2 triple mutant, lacking NTRC and f-type Trxs, was also suppressed by decreased 2-Cys Prxs contents, as the ntrc-trxf1f2-Δ2cp mutant partially recovered the efficiency of light energy utilization and exhibited WT rate of CO₂ fixation and growth phenotype. The suppressor phenotype was not caused by compensatory effects of additional chloroplast antioxidant systems. It is proposed that the Fd-FTR-Trx and NTRC redox systems are linked by the redox balance of 2-Cys Prxs, which is crucial for chloroplast function. Significance Chloroplasts harbor a complex redox network formed by two systems, the FTR- thioredoxins (Trxs), which relies on photoreduced ferredoxin (Fd), and the NADPH-dependent Trx reductase C NTRC. Thus, an important issue in chloroplast biology is to establish the relationship between these redox pathways. Here we propose that the Fd-FTR-Trxs and NTRC redox systems are integrated via the redox balance of 2-Cys peroxiredoxins (Prxs), which therefore has a key role in chloroplast function. NTRC controls the redox balance of 2-Cys Prxs, which maintains the reducing capacity of the pool of chloroplast Trxs and, consequently, proper regulation of photosynthetic carbon assimilation enzymes. Therefore, redox regulation of chloroplast enzymes and hydrogen peroxide reduction are linked by the action of the NTRC-2-Cys Prxs system.

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.