The contribution of glutathione peroxidases to chloroplast redox homeostasis in Arabidopsis.

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy.

Redox-control of chlorophyll biosynthesis mainly depends on thioredoxins.

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the pale green phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes.

The NADPH-Dependent Thioredoxin Reductase C-2-Cys Peroxiredoxin Redox System Modulates the Activity of Thioredoxin x in Arabidopsis Chloroplasts.

The chloroplast redox network is composed of a complex set of thioredoxins (Trxs), reduced by ferredoxin (Fdx) via a Fdx-dependent Trx reductase (FTR), and an NADPH-dependent Trx reductase with a joint Trx domain, NTRC, which efficiently reduces 2-Cys peroxiredoxins (2-Cys Prxs). Recently, it was proposed that the redox balance of 2-Cys Prxs maintains the redox state of f-type Trxs, thus allowing the proper redox regulation of Calvin-Benson cycle enzymes such as fructose 1,6-bisphosphatase (FBPase). Here, we have addressed whether the action of 2-Cys Prxs is also exerted on Trx x. To that end, an Arabidopsis thaliana quadruple mutant, ntrc-trxx-Δ2cp, which is knocked out for NTRC and Trx x, and contains severely decreased levels of 2-Cys Prxs, was generated. In contrast to ntrc-trxx, which showed a severe growth inhibition phenotype and poor photosynthetic performance, the ntrc-trxx-Δ2cp mutant showed a significant recovery of growth rate and photosynthetic efficiency, indicating that the content of 2-Cys Prxs is critical for the performance of plants lacking both NTRC and Trx x. Light-dependent reduction of FBPase was severely impaired in mutant plants lacking NTRC or NTRC plus Trx x, despite the fact that neither NTRC nor Trx x is an effective reductant of this enzyme. However, FBPase reduction was recovered in the ntrc-trxx-Δ2cp mutant. Our results show that the redox balance of 2-Cys Prxs, which is mostly dependent on NTRC, modulates the activity of Trx x in a similar way as f-type Trxs, thus suggesting that the activity of these Trxs is highly interconnected.

Photosynthetic activity of cotyledons is critical during post-germinative growth and seedling establishment.

Thioredoxins (Trxs) play a relevant role in thiol-dependent redox regulation, which allows the rapid adaptation of chloroplast metabolism to unpredictable environmental conditions. In chloroplasts, Trxs use reducing equivalents provided by photoreduced ferredoxin (Fdx) via the action of a ferredoxin-thioredoxin reductase (FTR), thus linking redox regulation to light. In addition, these organelles contain an NADPH-thioredoxin reductase, NTRC, with a Trx domain at the C-terminus. NTRC efficiently reduces 2-Cys peroxiredoxins (Prxs), hence having antioxidant function. However, NTRC also participates in the redox regulation of processes, such as starch and chlorophyll biosynthesis, which are known to be regulated by Trxs. Thus, the question arising is whether there is a cross-talk between the 2 redox systems. Arabidopsis mutants simultaneously devoid of NTRC and Trx x or Trxs f show a dramatic growth inhibition phenotype, indicating that NTRC is required for the function of these unrelated Trxs. Remarkably, both the ntrc-trxx double mutant and, to a higher extent, the ntrc-trxf1f2 triple mutant show high mortality at the seedling stage, which is rescued by sucrose. These findings show the relevant role of redox regulation for chloroplast performance and uncover the key function of cotyledons chloroplasts at the transition to autotrophic metabolism during seedling establishment.

The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.

Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide.

Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs), thiol-based peroxidases able to reduce hydrogen and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide.

Sigma factor-mediated plastid retrograde signals control nuclear gene expression.

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.

Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants.

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.

NTRC new ways of using NADPH in the chloroplast.

Despite being the primary source of energy in the biosphere, photosynthesis is a process that inevitably produces reactive oxygen species. Chloroplasts are a major source of hydrogen peroxide production in plant cells; therefore, different systems for peroxide reduction, such as ascorbate peroxidase and peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power required for hydrogen peroxide reduction by these systems is provided by Fd reduced by the photosynthetic electron transport chain; hence, the function of these systems is highly dependent on light. Recently, it was described a novel plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR) domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus. NTRC is able to conjugate both NTR and Trx activities to efficiently reduce 2-Cys Prx using NADPH as a source of reducing power. Based on these results, it was proposed that NTRC is a new pathway to transfer reducing power to the chloroplast detoxification system, allowing the use of NADPH, besides reduced Fd, for such function. In this article, the most important features of NTRC are summarized and the implications of this novel activity in the context of chloroplast protection against oxidative damage are discussed.

Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.