Multiple redox and non-redox interactions define 2-Cys peroxiredoxin as a regulatory hub in the chloroplast.

In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.

Redox characterization of human cyclophilin D: identification of a new mammalian mitochondrial redox sensor?

Mitochondria are metabolically highly active cell organelles that are also implicated in reactive oxygen species production and in cell death regulation. Cyclophilin D, the only human mitochondrial isoform of cyclophilins, plays an essential role in the formation of the mitochondrial permeability transition pore leading to cell necrosis. Recently, it has been shown that redox environment modifies structural and functional properties of some plant cyclophilins. Here, it is shown that oxidation of human cyclophilin D influences the conformation of the enzyme but also its activity. Site-directed mutagenized variants of cyclophilin D allowed the identification of cysteine 203 as an important redox-sensitive residue. Moreover, the redox modulation of cyclophilin D was confirmed in human neuroblastoma SH-SY5Y cells exposed to oxidative stress. Altogether, our results suggest that cyclophilin D may play a role as a redox sensor in mitochondria of mammalian cells transmitting information on the redox environment to target proteins.

Redox regulation and overreduction control in the photosynthesizing cell: complexity in redox regulatory networks.

Regulation of the photosynthetic apparatus between efficient energy conversion at low light and avoidance of overreduction and damage development at excess light resembles dangerous navigating between Scylla and Charybdis. Photosynthesis is a high rate redox metabolic pathway that generates redox intermediates with extreme redox potentials and eventually reactive oxygen species and oxidative stress. Therefore it is not surprising that the states of defined redox reactions in the chloroplast provide the predominant information and thus directly or indirectly the decisive signals for the multilevel control of cell activities in the chloroplast, cytoplasm, mitochondrion and nucleus. This review elaborates on the diversity of photosynthesis-derived redox signals such as the plastoquinone and thiol redox state that regulate and coordinate light use efficiency, electron transport activity, metabolic reactions, gene transcription and translation not only in the chloroplast but through retrograde signaling also essentially in all other cell compartments. The synergistic and antagonistic interrelations between the redox-dependent signaling pathways and their interactions with other signals such as abscisic acid and tetrapyrol intermediates constitute a redundant and probably buffered regulatory network to optimize performance of photosynthesis on the cellular and whole leaf level.

Redox regulation and antioxidative defence in Arabidopsis leaves viewed from a systems biology perspective.

Redox regulation is a central control element in cell metabolism. It is employed to adjust photosynthesis and the antioxidant defence system of leaves to the prevailing environment. During recent years progress has been made in describing the redox-dependent alterations in metabolism, the thiol/disulfide proteome, the redox-dependent and cross-talking signalling pathways and the target genes of redox regulation. Some transcription factors have been identified as proteins that perform thiol/disulfide transitions linked to the redox-regulation of specific plant promoters. In addition first mathematical models have been designed to simulate antioxidant defence and predict its response. Taken together, a profound experimental data set has been generated which allows to approach a systems biology type of understanding of antioxidant defence in photosynthesising cells in the near future. Since oxidative stress is likely to limit plant growth under stress, such a systematic understanding of antioxidant defence will help to define novel targets for breeding stress-tolerant plants.

Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions.

Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

Regulation of gene expression by photosynthetic signals triggered through modified CO2 availability.

BACKGROUND: To coordinate metabolite fluxes and energy availability, plants adjust metabolism and gene expression to environmental changes through employment of interacting signalling pathways. RESULTS: Comparing the response of Arabidopsis wild-type plants with that of the mutants adg1, pgr1 and vtc1 upon altered CO2-availability, the regulatory role of the cellular energy status, photosynthetic electron transport, the redox state and concentration of ascorbate and glutathione and the assimilatory force was analyzed in relation to the transcript abundance of stress-responsive nuclear encoded genes and psaA and psbA encoding the reaction centre proteins of photosystem I and II, respectively. Transcript abundance of Bap1, Stp1, psaA and psaB was coupled with seven metabolic parameters. Especially for psaA and psaB, the complex analysis demonstrated that the assumed PQ-dependent redox control is subordinate to signals linked to the relative availability of 3-PGA and DHAP, which define the assimilatory force. For the transcripts of sAPx and Csd2 high correlations with the calculated redox state of NADPH were observed in pgr1, but not in wild-type, suggesting that in wild-type plants signals depending on thylakoid acidification overlay a predominant redox-signal. Strongest correlation with the redox state of ascorbate was observed for 2CPA, whose transcript abundance regulation however was almost insensitive to the ascorbate content demonstrating dominance of redox regulation over metabolite sensing. CONCLUSION: In the mutants, signalling pathways are partially uncoupled, demonstrating dominance of metabolic control of photoreaction centre expression over sensing the redox state of the PQ-pool. The balance between the cellular redox poise and the energy signature regulates sAPx and Csd2 transcript abundance, while 2CPA expression is primarily redox-controlled.

Peroxiredoxin Q of Arabidopsis thaliana is attached to the thylakoids and functions in context of photosynthesis.

Peroxiredoxin Q (Prx Q) is one out of 10 peroxiredoxins encoded in the genome of Arabidopsis thaliana, and one out of four that are targeted to plastids. Peroxiredoxin Q functions as a monomeric protein and represents about 0.3% of chloroplast proteins. It attaches to the thylakoid membrane and is detected in preparations enriched in photosystem II complexes. Peroxiredoxin Q decomposes peroxides using thioredoxin as an electron donor with a substrate preference of H(2)O(2) > cumene hydroperoxide >> butyl hydroperoxide >> linoleoyl hydroperoxide and insignificant affinity towards complex phospholipid hydroperoxide. Plants with decreased levels of Prx Q did not have an apparently different phenotype from wildtype at the plant level. However, similar to antisense 2-cysteine (2-Cys) Prx plants [Baier, M. et al. (2000)Plant Physiol., 124, 823-832], Prx Q-deficient plants had a decreased sensitivity to oxidants in a leaf slice test as indicated by chlorophyll a fluorescence measurements. Increased fluorescence ratios of photosystem II to I at 77 K and modified transcript levels of plastid- and nuclear-encoded proteins show that regulatory mechanisms are at work to compensate for the lack of Prx Q. Apparently Prx Q attaches to photosystem II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absence causes metabolic changes that are sensed and trigger appropriate compensatory responses.

Influence of the photoperiod on redox regulation and stress responses in Arabidopsis thaliana L. (Heynh.) plants under long- and short-day conditions.

Arabidopsis thaliana L. (Heynh.) plants were grown in low light (150 micromol photons m(-2) s(-1) and 20 degrees C) either in short days (7.5 h photoperiod) or long days (16 h photoperiod), and then transferred into high light and low temperature (350-800 micromol photons m(-2) s(-1) at 12 degrees C). Plants grown in short days responded with a rapid increase in NADP-malate dehydrogenase (EC 1.1.1.82) activation state. However, persisting overreduction revealed a new level of regulation of the malate valve. Activity measurements and Northern-blot analyses indicated that NADP-malate dehydrogenase transcript and protein levels increased within a few hours. Using macroarrays, additional changes in gene expression were identified. Transcript levels for several enzymes of glutathione metabolism and of some photosynthetic genes increased. The cellular glutathione level increased, but its redox state remained unchanged. A different situation was observed in plants grown in long-day conditions. Neither NADP-malate dehydrogenase nor glutathione content changed, but the expression of several antioxidative enzymes increased strongly. We conclude that the endogenous systems that measure day length interact with redox regulation, and override the interpretation of the signals, i.e. they redirect redox-mediated acclimation signals to allow for more efficient light usage and redox poising in short days to systems for the prevention of oxidative damages when grown under long-day conditions.

pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.

Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Deltarpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

The mitochondrial type II peroxiredoxin F is essential for redox homeostasis and root growth of Arabidopsis thaliana under stress.

Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation, and disease, as they function both in antioxidant defense by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism. At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion. Therefore, it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localizations. This paper presents a characterization of PrxII F at the level of subcellular distribution, activity, and reductive regeneration by mitochondrial thioredoxin and glutaredoxin. By employing tDNA insertion mutants of A. thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase. However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared with wild type is observed under stress conditions induced by CdCl2 as well as after administration of salicylhydroxamic acid, an inhibitor of cyanide-insensitive respiration. Simultaneously, major changes in the abundance of both nuclear and mitochondria-encoded transcripts were observed. These results assign a principal role to PrxII F in antioxidant defense and possibly redox signaling in plants cells.

The antioxidant status of photosynthesizing leaves under nutrient deficiency: redox regulation, gene expression and antioxidant activity in Arabidopsis thaliana.

Redox signals provide important information on plant metabolism during development and in dependence on environmental parameters and trigger compensatory responses and antioxidant defence. The aim of the study was to characterize the redox and antioxidant status of photosynthesizing leaves under N, P and S deficiency on a comparative basis. Therefore, redox signals, indicators of the cellular redox environment and parameters of antioxidant defence were determined and related to general growth parameters, namely (1) transcript levels of all chloroplast encoded genes; (2) ascorbate and glutathione; (3) activities of catalase (CAT) and ascorbate peroxidase (APX); and (4) transcript amounts of eight peroxiredoxins, three catalases and three ascorbate peroxidases. The results reveal distinct patterns of redox responses dependent on the type of nutrient deficiency. (1) Nitrogen deprivation caused up-regulation of psbA, psbC, petA, petG and clpP transcripts, down-regulation of psbG, psbK and ndhA, a five-fold increase in ascorbic acid, a severe drop in CAT and APX activities, although cat1 mRNA levels were increased in young and old leaves. (2) With the exception of psbA and psaJ transcripts, P-starvation induced a general trend to decreased mRNA abundance of plastome genes; ascorbate and glutathione levels were increased, as was the activity of APX and CAT. In accordance with that result, transcripts of all cat genes and stromal apx, as well as prxIIC, prxIID, were elevated under P deprivation. (3) Sulphur depletion increased transcripts of petA, petB, petD, petG, ndhJ and rpo-genes. mRNAs of psbG, psbK, atpA, atpB, atpE and atpF were decreased. Glutathione levels dropped to less than 25% of control, in parallel activities of APX were stimulated in young leaves. Transcripts of many antioxidant enzymes were unaltered or decreased, only cat2 mRNA was increased. It is concluded that N-, P- and S-nutrient deprivation trigger distinct redox changes and induce oxidative stress with a rather defined pattern in the context of nutrient-specific alterations in metabolism.

cDNA cloning of 12 subunits of the V-type ATPase from Mesembryanthemum crystallinum and their expression under stress.

The vacuolar-type ATPase (V-ATPase) and the vacuolar H(+)-pyrophosphatase are electrogenic proton pumps at plant endomembranes that create the proton motive force required for secondary activated transport and metabolite accumulation during development and adaptation to a variety of adverse growth conditions. Twelve distinct vacuolar H(+)-ATPase (VHA) subunits are suggested to constitute the functional V-ATPase complex. Starting from the available expressed sequence tag (EST) sequences and by homology screening, the complete set of 12 VHA subunits was cloned as cDNAs from the halophyte Mesembryanthemum crystallinum, vha-A-H, -a,-c, -d and -e. Transcript levels of all 12 VHA subunits as well as of tonoplast pyrophosphatase and P-ATPase were analysed in root and leaf tissue under conditions of osmotic (700 mM mannitol), heat and cold stress, and salinity. Distinct coordinated changes of stress-induced expression were observed for most subunits in roots and leaves, with mostly paralleled changes in transcript levels of all subunits. In some cases, contrasting responses were seen for vha-B and -c transcript amounts.

Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E(m)') of Prx II B, Prx II C, and Prx II E were around -290 mV and, thus, less negative than E(m)' of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (-307 to -322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.

Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction.

The mechanism of the post-translational modulation of nitrate reductase activity (NR, EC 1.6.6.1) is briefly summarized, and it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co-immunoprecipitation experiments, 14-3-3 binding to phospho-NR and the function of Mg(2+) in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14-3-3 binding in controlling NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation of NR of Ricinus communis is described as an interesting special case that differs from the 'normal' mechanism in several important aspects.

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source.

Phosphoenolpyruvate (PEP) carboxylation was measured as dark 14CO2 fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO2 fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO2-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO2 fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4-5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.