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.

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 thioredoxinlike 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.

NADP-thioredoxin reductase ͉ sugar sensing ͉ redox regulation ͉ ADP-glucose pyrophosphorylase

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx x is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx x knock-out mutant has been identified and a double mutant (denoted Δ2cp) with <5% of 2-Cys Prx content has been generated. The phenotypes of the three mutants, ntrc, trxx, and Δ2cp, were compared under standard growth conditions and in response to continuous light or prolonged darkness and oxidative stress. Though all mutants showed altered redox homeostasis, no difference was observed in response to oxidative stress treatment. Moreover, the redox status of the 2-Cys Prx was imbalanced in the ntrc mutant but not in the trxx mutant. These results show that NTRC is the most relevant pathway for chloroplast 2-Cys Prx reduction in vivo, but the antioxidant function of this system is not essential. The deficiency of NTRC caused a more severe phenotype than the deficiency of Trx x or 2-Cys Prxs as determined by growth, pigment content, CO2 fixation, and Fv/Fm, indicating additional functions of NTRC.

SignificanceChloroplasts 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.

Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP + and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light-and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. Reversible disulfide bond formation between two Cys residues regulates structure and function of many proteins in diverse organisms (Cook and Hogg, 2013). Thiol disulfide exchange is controlled by thioredoxins (Trxs), which are small proteins containing a redoxactive disulfide group in their active site (Holmgren, 1985;Baumann and Juttner, 2002). The latter can be reduced to a dithiol by Trx reductases using NADPH or ferredoxin (Fdx) as electron donors. Due to their low redox midpoint potential, reduced Trxs are able to reductively cleave disulfide bonds in many target proteins and, thus, modulate their functions.Plants contain the most versatile Trx system found in all organisms with respect to the multiplicity of different isoforms and reduction systems (Buchanan and Balmer, 2005;Nikkanen and Rintamäki, 2014;Geigenberger and Fernie, 2014). The Arabidopsis (Arabidopsis thaliana) genome contains a complex family of Trxs, including up to 20 different isoforms grouped into seven subfamilies (Schürmann and Buchanan, 2008;Dietz and Pfannschmidt, 2011). Trxs f1-2, m1-4, x, y1-2, and z are located exclusively in the chloroplast, and Trx o is located exclusively in the mitochondria, while the eight Trx h representatives are distributed between the cytosol, nucleus, endoplasmic reticulum, and mitochondria (Meyer et al., 2012). The different Trxs can be reduced by two different redox systems, dependent on Fdx and Fdx-Trx reductase in the chlorop...

Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5Ј-CCTTTT-3Ј. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point ␣-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.

High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.