Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

Plants perform photosynthesis and assimilatory processes in a continuously changing environment. Energy production in the various cell compartments and energy consumption in endergonic processes have to be well adjusted to the varying conditions. In addition, dissipatory pathways are required to avoid any detrimental effects caused by over-reduction. A large number of short-term and long-term mechanisms interact with each other in a flexible way, depending on intensity and the type of impact. Therefore, all levels of regulation are involved, starting from energy absorption and electron flow events through to post-transcriptional control. The simultaneous presence of strong oxidants and strong reductants during oxygenic photosynthesis is the basis for regulation. However, redox-dependent control also interacts with other signal transduction pathways in order to adapt metabolic processes and redox-control to the developmental state. Examples are given here for short-term and long-term control following changes of light intensity and photoperiod, focusing on the dynamic nature of the plant regulatory systems. An integrating network of all these mechanisms exists at all levels of control. Cellular homeostasis will be maintained as long as the mechanisms for acclimation are present in sufficiently high capacities. If an impact is too rapid, and acclimation on the level of gene expression cannot occur, cellular damage and cell death are initiated.

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.

Ferredoxins are the major distributors for electrons to the various acceptor systems in plastids. In green tissues, ferredoxins are reduced by photosynthetic electron flow in the light, while in heterotrophic tissues, nicotinamide adenine dinucleotide (reduced) (NADPH) generated in the oxidative pentose-phosphate pathway (OPP) is the reductant. We have used a Ds-T-DNA insertion line of Arabidopsis thaliana for the gene encoding the major leaf ferredoxin (Fd2, At1g60950) to create a situation of high electron pressure in the thylakoids. Although these plants (Fd2-KO) possess only the minor fraction of leaf Fd1 (At1g10960), they grow photoautotrophically on soil, but with a lower growth rate and less chlorophyll. The more oxidized conditions in the stroma due to the formation of reactive oxygen species are causing a re-adjustment of the redox state in these plants that helps them to survive even under high light. Redox homeostasis is achieved by regulation at both, the post-translational and the transcriptional level. Over-reduction of the electron transport chain leads to increased transcription of the malate-valve enzyme NADP-malate dehydrogenase (MDH), and the oxidized stroma leads to an increased transcription of the OPP enzyme glucose-6-P dehydrogenase. In isolated spinach chloroplasts, oxidized conditions give rise to a decreased activation state of NADP-MDH and an activation of glucose-6-P dehydrogenase even in the light. In Fd2-KO plants, NADPH-requiring antioxidant systems are upregulated. These adjustments must be caused by plastid signals, and they prevent oxidative damage under rather severe conditions.

A complete ferredoxin (Fd) cDNA clone was isolated from potato (Solanum tuberosum L. cv Desiree) leaves. By molecular and immunoblot analysis, the gene was identified as the leaf-specific Fd isoform I. Transgenic potato plants were constructed by introducing the homologous potato fed 1 cDNA clone as an antisense construct under the control of the constitutive cauliflower mosaic virus 35S promoter. Stable antisense lines with Fd contents between 40% and 80% of the wild-type level were selected by northern-and western-blot analysis. In short-term experiments, the distribution of electrons toward their stromal acceptors was altered in the mutant plants. Cyclic electron transport, as determined by the quantum yields of photosystems I and II, was enhanced. The CO 2 assimilation rate was decreased, but depending on the remaining Fd content, some lines showed photoinhibition. The leaf protein content remained largely constant, but the antisense plants had a lower total chlorophyll content per unit leaf area and an increased chlorophyll a/b ratio. In the antisense plants, the redox state of the quinone acceptor A in photosystem II (Q A ) was more reduced than that of the wild-type plants under all experimental conditions. Because the plants with lower Fd amounts reacted as if they were grown under a higher light intensity, the possibility that the altered chloroplast redox state affects light acclimation is discussed.Ferredoxins (Fds) are small, iron-and sulfurcontaining proteins that act as low-potential oneelectron carriers. In chloroplasts, Fd distributes the electrons from PSI onto the various electronconsuming reactions in the chloroplast stroma (Arnon, 1988). During nitrogen (N) and sulfur (S) assimilation, reduced Fd is directly used by nitrite reductase, Gln synthase, sulfite reductase (Knaff and Hirasawa, 1991), and by enzymes of secondary metabolism, such as choline monooxygenase (Brouquisse et al., 1989). Moreover, Fd supplies electrons via Ferredoxin-NADP ϩ -Reductase for NADP reduction (Knaff and Hirasawa, 1991). The generated NADPH serves as a soluble reductant in the chloroplast stroma, mainly for the reduction of 1,3bisphos-phoglycerate by NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase during CO 2 assimilation (Leegood, 1996). Fd is involved in the regulation of redox-modulated chloroplast enzymes via electron flow toward ferrodoxin-thioredoxin-reductase (FTR) and thioredoxins (Knaff and Hirasawa, 1991). Thioredoxins reduce their target enzymes and, thus, in interplay with specific metabolic effectors, adjust the enzyme activation states to the actual demand (Scheibe, 1991). Moreover, the redox state of thioredoxins also acts on chloroplast gene expression by regulating transcription and translation of several chloroplast proteins (Kim and Mayfield, 1997;Link, 2001).Fd is furthermore involved in nonassimilatory electron fluxes that act to adjust the stromal ATP/2e Ϫ ratio. Electrons are often generated in excess to the amount required for CO 2 fixation or photorespiration (Backhausen et al., 1...

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.

No abstract

Abstract. In patients with diabetic nephropathy, glomerular staining for heparan sulfate proteoglycans (HSPG) side chains and for agrin is decreased. In the present study, the influence of angiotensin II (AngII) on the production of HSPG in SV40 transformed podocytes was investigated. SV40 transformed human podocytes were cultivated with or without 1 M AngII, and HSPG production was measured by sequential DEAE-anion exchange chromatography and HPLC-DEAE separation. Expression of agrin was studied by indirect immunofluorescence and Western blot analysis using specific mono-and polyclonal antibodies. DEAE separation of total glycosaminoglycans (GAG) revealed a significant increase of GAG in the culture supernatant and decrease in the cell and matrix layer when podocytes were cultured for 72 h in the presence of AngII. This was particularly found for HS-GAG.Qualitative analysis of HSPG, using gel filtration of HNO 2 -treated fractions, showed that AngII treatment decreased N-sulfation of HS-GAG side chains. Indirect immunofluorescence staining with anti-agrin polyclonal antibody was strongly decreased after AngII stimulation. A reduction in agrin expression in cell extracts could also be detected in Western blot analysis using an mAb. No changes in agrin mRNA were found after AngII stimulation. It is concluded from this study that AngII decreases the amount of HSPG on the cell surface and in the extracellular matrix of podocytes. Because HSPG play a fundamental role in the permselectivity of the glomerular basement membrane, these results thus may explain at least partially the antiproteinuric effects of angiotensin-converting enzyme inhibition in patients with diabetic nephropathy.Diabetic nephropathy (DN) is characterized by mesangial matrix expansion, thickening of the glomerular basement membrane (GBM), and a concomitant loss of heparan sulfate proteoglycans (HSPG) (1-4). HSPG consist of a core protein to which one or more HS glycosaminoglycan (GAG) side chains are attached. To date, three HSPG core proteins-perlecan, agrin, and collagen XVIII (5-7)-have been identified in the GBM.The permselectivity of the GBM is partly due to the high negative charge of HSPG and their interaction with other matrix components (8 -10). Most studies on the decrease of glomerular HSPG in patients with DN have suggested that a selective dysregulation in sulfation of HSPG is underlying the observed reduction in HSPG expression (11-13). However, we recently provided in vivo and in vitro evidence that under hyperglycemic conditions, the expression of the core protein of agrin in the GBM of these patients and in cultured podocytes is also affected (14).In patients with DN, an initial increase in the GFR is followed by a linear decrease in GFR over time (15). It is believed that in these patients, autoregulation of renal blood flow is impaired and, consequently, systemic pressure is transferred to the glomerular capillary loops, resulting in hyperfiltration (15,16). Several clinical studies have demonstrated that antihypertensive drugs...