Cytosolic NADP‐dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves

Cytosolic NADP‐dependent isocitrate dehydrogenase (cICDH) produces 2‐oxoglutarate (2‐OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T‐DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild‐type Col0. Metabolite profiling by gas chromatography–time of flight–mass spectrometry (GC–TOF–MS) and high‐performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H₂O₂ causes perturbed redox homeostasis and induction of stress‐related genes. Accumulation of oxidized glutathione and pathogen‐related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.     

NADP-isocitrate dehydrogenase polymorphism in the Atlantic salmon Salmo salar

Electrophoretic variation of Atlantic salmon NADP-isocitrate dehydrogenase in liver extracts of material from Ireland is interpreted in terms of two loci, IDH-A and IDH-B , coding for cytoplasmically-located isozymes. The IDH-A locus is polymorphic with qIDH-A ¹=0.18 and qIDH-A ²=0.82. This polymorphism is of potential value for investigating the population genetics of the two races of Atlantic salmon in Ireland. An additional, monomorphic band which appears in heart extracts may be a mitochondrial enzyme coded by a third locus.     

Studies on NADP-isocitrate dehydrogenase from maturing castor bean seeds

NADP-specific isocitrate dehydrogenase from the soluble fractionof maturing castor bean endosperm was partially purified (approximately180-fold) and some of its enzymatic properties were studied.Mg⁺⁺, Mn⁺⁺, Cd⁺⁺, Ba⁺⁺, Co⁺⁺, Zn⁺⁺, and Sr⁺⁺ were activatorsof the enzyme reaction at a concentration of 6.7x10 M. The optimumpH of this enzyme was about 8.5. The enzyme was stable in thenarrow range from pH 7.0 to pH 8.0. Km values for isocitrateand NADP at pH 8.5 were 3.5x10^–6 M and 3.6x10^–6M, respectively. Enzyme stability was not affected by NaCl concentrationand enzyme reaction was inhibited at 5x10^–6 M PCMB (80%inhibition). It is suggested that the condensation product ofglyoxylate and oxalacetate also inhibits the reaction. NADP-IDHin the crude extract from maturing castor bean endosperm washeat-stable but the dialyzed enzyme preparation and the partiallypurified enzyme were labile against heat treatment at 57°C.When Mg⁺⁺ was added to the partially purified enzyme in thepresence of isocitrate or NADP, the enzyme was stabilized againstheat treatment. Mn⁺⁺, Ca⁺⁺, Co⁺⁺, Sr⁺⁺ or Ba⁺⁺ could be substitutedfor Mg⁺⁺. Addition of only one of the factors, Mg⁺⁺, isocitrateor NADP, had no effect on the heat stability. Moreover, a combinationof isocitrate and NADP did not establish stabilization. A divalentcation plays a central role, while adenine nucleotide, especiallyATP, may have an important part in stabilization. (Received August 14, 1972; )     

Regulation of mitochondrial NADP-isocitrate dehydrogenase in rat heart during ischemia

The changes in the regulation of at mitochondrial NADP-isocitrate dehydrogenase (NADP-ICDH) in a rat heart during have been analysed. Increase of enzyme activity in the cytosol and mitochondria of the heart ischemia was detected. Catalytic properties of the mitochondrial NADP-ICDH at norm and pathology have been compared on homogeneous enzyme preparations. Enzyme from the normoxic and ischemic heart showed the same electrophoretical mobility and molecular mass. Enzyme isolated from the ischemic heart mitochondria demonstrated higher activation energy and lower thermal stability. NADP-isocitrate dehydrogenase at the normoxic and ischemic conditions exhibited different K_m for substrates and regulatory behaviour in relation to ATP, ADP, 2-oxoglutarate, citrate, malate, reduced and oxidised glutathione. The inhibitory effect of the Fe²⁺ and H₂O₂ mixture associated with the generation of hydroxyl radicals was lower in the ischemic enzyme. We hypothesise that the specific features of regulation behaviour of NADP-ICDH from the ischemic tissues permits the enzyme to supply NADPH to the glutathione reductase/glutathione peroxidase system.     

Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii

Chloroplastic NADP-isocitrate dehydrogenase isoenzyme (NADP-IDH₂; EC 1.1.1.42) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by a procedure which included affinity chromatography on Red-Sepharose as the key step. The 70-kDa isoenzyme was found to be a dimer formed by 40-kDa subunits. Antibodies raised against a recombinant tobacco cytosolic NADP-IDH cross-reacted strongly with the cytosolic NADP-IDH₁ and weakly with the NADP-IDH₂ isoenzyme from this alga. NADPH and GTP were found to inhibit both isoenzymes, whereas intermediates of the tricarboxylic acid cycle, glycolysis or reductive pentose phosphate cycle had no significant effect. The simultaneous presence of isocitrate and Mn²⁺ protected NADP-IDH₂ against thermal inactivation or inhibition by reagents specific for arginine or lysine.     

Universally occurring phenylpropanoid and species-specific indolic metabolites in infected and uninfected Arabidopsis thaliana roots and leaves

A total of eleven alkali-released, aromatic compounds were identified by HPLC, MS and NMR analyses in cell wall extracts from Arabidopsis thaliana roots. Nine of them together constituted the three complete series of 4-hydroxy-, 4-hydroxy-3-methoxy, and 4-hydroxy-3,5-dimethoxy-substituted benzaldehydes, benzoic acids and cinnamic acids. The other two were indolic metabolites: indole-3-carboxylic acid and indole-3-carbaldehyde. Qualitatively similar, but quantitatively distinct profiles were obtained using cell-wall extracts from A. thaliana leaves. Several of these compounds, particularly indole-3-carboxylic acid, 4-hydroxybenzoic acid and all four aldehydes, increased considerably in concentration upon infection of roots with Pythium sylvaticum, as did at least some of them upon infection of leaves with Pseudomonas syringae pv tomato. Comparison of these results with analogous data on a variety of different plant species suggests a remarkable structural uniformity among the majority of constitutive as well as infection-induced, aromatic cell wall-bound compounds throughout the entire plant kingdom-in sharp contrast to the highly species-specific, chemically highly divers bouquets of soluble aromatic metabolites.     

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli

Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.     

Changes in NADP-isocitrate dehydrogenase isoenzyme levels in Acinetobacter calcoaceticus in response to acetate

Abstract During adaptation of Acinetobacter calcoaceticus to growth on acetate the specific activity of NADP-isocitrate dehydrogenase increased. This response is unique, as in other bacteria grown under the same conditions the activity of the enzyme decreases as a result of covalent phosphorylation. Moreover, A. calcoaceticus is also unusual in containing two distinct isoenzymes of NADP-isocitrate dehydrogenase. It has here been shown that the adaptation of A. calcoaceticus to acetate is accompanied by an increase in the relative proportion of the larger, allosteric, isoenzyme with a concomitant decrease in the level of the smaller, non-allosteric, isoenzyme.     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.     

Aluminum: a pH-dependent inhibitor of NADP-isocitrate dehydrogenase from porcine heart

Aluminum showed a pH-dependent inhibitory effect on NADP-isocitrate dehydrogenase from porcine heart. Aluminum ions (Al³⁺) acted as a partial competitive inhibitor of the enzyme with respect to the substratethreo-Ds-isocitrate and inhibited the enzyme non-competitively with respect to NADP at pH 6.85. Fractional velocity plot analysis showed theK _i of the enzyme for aluminum ions to be 0.88m. When pH was elevated to 8.0, aluminum ions, which occur as a form of the Al(OH)₄ ^– anion, acted as partial uncompetitive and non-competitive inhibitors of the enzyme with respect to the substrates isocitrate and NADP, respectively. TheK _i of the enzyme was determined to be 5.64 m at pH 8.0 by fractional velocity plot analysis. The inhibition of NADP-isocitrate dehydrogenase by two forms of aluminum ions may explain aluminum toxicity in various tissues and organs.     

Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase

Kinetic and regulatory properties of NADP-isocitrate dehydrogenase (NADP-IDH) and aspartate aminotransferase (AsAT) responsible for 2-oxoglutarate metabolism in the cytoplasm and mitochondria of rat liver were studied. Based on the subcellular location of these enzymes and their kinetic parameters (Km, Ksi) obtained with highly purified enzyme preparations, it is suggested that synthesis of 2-oxoglutarate should be mainly determined by cytoplasmic NADP-IDH (86% of the total activity in the cell), whereas its utilization should depend on cytoplasmic AsAT (78% of the total activity). AsAT from the rat liver was specified by substrate inhibition and also by changes in the enzyme affinity for the substrates under the influence of some intermediates of the tricarboxylic acid cycle: isocitrate, succinate, fumarate, and citrate. Key intermediates of nitrogen metabolism (glutamate, glutamine, and aspartate) are involved in the regulation of NADP-IDH and AsAT. These enzymes are regulated oppositely, and the catalytic activity of one enzyme can be stimulated concurrently with a decrease in the activity of the other. Obviously, carbon and nitrogen metabolism in the rat liver can be controlled through redistribution of 2-oxoglutarate between different metabolic processes via regulatory mechanisms influencing differently located forms of NADP-IDH and AsAT.     

Aluminum decreases the glutathione regeneration by the inhibition of NADP-isocitrate dehydrogenase in mitochondria

Effect of aluminum on the NADPH supply and glutathione regeneration in mitochondria was analyzed. Reduced glutathione acted as a principal scavenger of reactive oxygen species in mitochondria. Aluminum inhibited the regeneration of glutathione from the oxidized form, and the effect was due to the inhibition of NADP-isocitrate dehydrogenase the only enzyme supplying NADPH in mitochondria. In cytosol, aluminum inhibited the glutathione regeneration dependent on NADPH supply by malic enzyme and NADP-isocitrate dehydrogenase, but did not affect the glucose 6-phosphate dehydrogenase dependent glutathione formation. Aluminum can cause oxidative damage on cellular biological processes by inhibiting glutathione regeneration through the inhibition of NADPH supply in mitochondria, but only a little inhibitory effect on the glutathione generation in cytosol.     

Thioredoxin targets in Arabidopsis roots

The importance of redox‐regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol‐disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y‐type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy‐two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid‐dependent biosynthesis of isoprenoids as a new redox‐mediated process. The redox‐regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia‐lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT‐reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.