Regulation of plant cytosolic aldolase functions by redox-modifications.

From the five genes which code for cytosolic fructose 1,6-bisphosphate aldolases in Arabidopsis thaliana L., the cDNA clone of cAld2 (At2g36460), was heterologously expressed in E. coli and incubated under various oxidizing and reducing conditions. Covalent binding of a GSH moiety to the enzyme was shown by incorporation of biotinylated GSH (BioGEE) and by immunodetection with monoclonal anti-GSH serum. Nitrosylation after incubation with GSNO or SNP was demonstrated using the biotin-switch assay. Mass-spectrometry analysis showed glutathionylation and/or nitrosylation at two different cysteine residues: GSH was found to be attached to C68 and C173, while the nitroso-group was incorporated only into C173. Non-reducing SDS-PAGE conducted with purified wild-type and various Cys-mutant proteins revealed the presence of disulfide bridges in the oxidized enzyme, as described for rabbit muscle aldolase. Incubation of the purified enzyme with GSSG (up to 25 mM) led to partial and reversible inactivation of enzyme activity; NADPH, in the presence of the components of the cytosolic NADP-dependent thioredoxin system, could reactivate the aldolase as did DTT. Total and irreversible inactivation occurred with low concentrations (0.1 mM) of nitrosoglutathione (GSNO). Inactivation was prevented by co-incubation of cAld2 with fructose-1,6-bisphosphate (FBP). Nuclear localization of cAld2 and interaction with thioredoxins was shown by transient expression of fusion constructs with fluorescent proteins in isolated protoplasts.

Knockout of major leaf ferredoxin reveals new redox-regulatory adaptations in Arabidopsis thaliana.

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.

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

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.

Transcriptional regulation of NADP-dependent malate dehydrogenase: comparative genetics and identification of DNA-binding proteins.

The transcriptional regulation of NADP-malate dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution.

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.

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.

Strategies to maintain redox homeostasis during photosynthesis under changing conditions.

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.

Angiotensin II type 1-receptor mediated changes in heparan sulfate proteoglycans in human SV40 transformed podocytes.

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 microM 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.

Decreased content of leaf ferredoxin changes electron distribution and limits photosynthesis in transgenic potato plants.

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 CO2 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 (QA) 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.