NADP-dependent Malate Dehydrogenase Activity Research Articles

Genome-wide characterization of soybean malate dehydrogenase genes reveals a positive role of GmMDH2 in salt stress response

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and the stress response. However, information on MDH genes in the soybean genome is limited. Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the presence of conserved protein motifs was analyzed. The genes were divided into five clusters according to their phylogenetic relationships. The intracellular localizations of six GmMDHs were determined by confocal microscopy on Arabidopsis mesophyll protoplasts. Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR). GmMDHs displayed unique expression patterns in diverse soybean tissues. It is noteworthy that under salt stress, the expression levels of a chloroplast isoform (GmMDH2) were unusually high, presumably indicating a critical role in soybean responses to salinity. Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme had NADP-dependent MDH activity. The redox states of the nicotinamide adenine dinucleotide phosphate (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance. Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance. A polymorphism possibly associated with salt tolerance was discovered in the promoter region of GmMDH2. These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but it also identified a potential candidate gene for the future enchancement of salt tolerance in the soybean.

A Semi-throughput Procedure for Assaying Plant NADP-malate Dehydrogenase Activity Using a Plate Reader.

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a redox regulated enzyme playing an important role in plant redox homeostasis. Leaf NADP-MDH activation level is considered a proxy for the chloroplast redox status. NADP-MDH enzyme activity is commonly assayed spectrophotometrically by following oxaloacetate-dependent NADPH oxidation at 340 nm. We have developed a plate-adapted protocol to monitor NADP-MDH activity allowing faster data production and lower reagent consumption compared to the classic cuvette format of a spectrophotometer. We provide a detailed procedure to assay NADP-MDH activity and measure the enzyme activation state in purified protein preparations or in leaf extracts. This protocol is provided together with a semi-automatized data analysis procedure using an R script.

Antagonistic Regulation by CPN60A and CLPC1 of TRXL1 that Regulates MDH Activity Leading to Plant Disease Resistance and Thermotolerance

Global warming and emerging plant diseases challenge agricultural food/feed production. We identify mechanism(s) regulating both plant thermotolerance and disease resistance. Using virus-induced gene silencing (VIGS)-based genetic screening, we identify a thioredoxin-like 1 (TRXL1) gene involved in plant nonhost disease resistance and thermotolerance. TRXL1 is reduced, partly degraded via proteases and proteasome, and alters its chloroplast localization during heat stress. TRXL1 interacts with more than 400 proteins, including chaperonin CPN60A, caseinolytic protease (CLPC1), and NADP-dependent malate dehydrogenase (NADP-MDH). Chaperonin 60A (CPN60A) guards TRXL1 from degradation, whereas CLPC1 degrades TRXL1 during heat stress. TRXL1 regulates NADP-MDH activity, leading to an increase in malate level and inhibition of superoxide radical formation. We show that CPN60A and NADP-MDH positively regulate nonhost resistance, and CPN60A positively and CLPC1 negatively regulate thermotolerance. This study shows an antagonistic post-translational regulation of TRXL1 by CPN60A and CLPC1 and regulation of MDH by TRXL1, leading to plant disease resistance and thermotolerance.

Fruit Spray of 24-Epibrassinolide and Fruit Shade Alter Pericarp Photosynthesis Activity and Seed Lipid Accumulation in Styrax tonkinensis

As demand for biofuel feedstock production increases, the substitutable woody oilseed benzoin (Styrax tonkinensis (Pierre) Craib ex Hartwich) has received increased attention among Chinese researchers. However, the mechanism of seed oil regulation has not been revealed in this species. Maternal control of seed lipid content has been observed in many plant species. In this study, in planta treatments were applied to create different pericarp photosynthesis activation levels in benzoin. Compared with fruit shading treatment, superior photosynthetic physiological characteristics were found in the brassinosteroid hormone 24-epibrassinolide-treated pericarps during maturation (56–112 days after flowering); specifically, these superior characteristics were reflected by higher average and peak values of the Chl a/b ratio, NADPH/NADP+ ratio, NADP-dependent malate dehydrogenase activity, and ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Next, we found that the pericarp-encapsulated seed yield and oil content were closely connected to the maternal-specific photosynthetic events. The enhanced pericarp photosynthesis activity (treated with 10 μM brassinosteroid) was accompanied by higher fatty acid synthase activity and a faster lipid deposition rate, which was verified by transmission electron microscopy. These changes led to a final increase in the total fatty acid (FA) content of mature seeds of 19%, which could be largely accounted for by the elevated saturated FA (mostly long-chain FAs) percentage in benzoin oil. When benzoin fruits grew in low light (bagged in three layers of black non-woven fabrics), lipid synthesis capability was markedly impaired. The dynamics of cellular storage reserves were further analyzed, suggesting that the pericarp-seed carbohydrate translocation efficiency in fruits and the carbon partitioning among seed reserves were directly responsible for the seed oil diversification under the treatments. Taken together, our results highlighted the maternal control, via pericarp photosynthesis, over the regulation of the seed oil content in benzoin.

BmNPV alters NADP-dependent malate dehydrogenase activity and associated macromolecules and retards growth and development of the mulberry silkworm,Bombyx moriL., during the final instar

Changes in tissue-specific NADP-dependent malate dehydrogenase (MDH) activity and protein and cholesterol contents and retardation of growth and development in the mulberry silkworm, Bombyx mori L., induced by infection with a baculovirus, the B. mori nucleopolyhedrovirus (BmNPV), were investigated. The study revealed that the relative growth rate and development of the fifth-instar larva was significantly inhibited during the progression of nuclear polyhedrosis disease at various times post inoculation (p.i.). Percent pupation and reproductive output were also adversely affected. NADP-dependent MDH activity in the hemolymph peaked sharply at 6 h p.i. and then gradually decreased, reaching a minimum at 264 h p.i., with exceptions at 30 and 72 h p.i. These levels of MDH activity were different from those in non-infected controls. In fat-body tissues, NADP-dependent MDH activity was significantly higher in infected insects than in non-infected controls of the same age. These results indicate that baculovirus infection causes significant changes in intermediary metabolic pathways, causing a significant fall and rise in protein and cholesterol contents in tissues during development of fifth-instar larvae.

Photosynthetic electron transport and carbon-reduction-cycle enzyme activities under long-term drought stress in Casuarina equisetifolia Forst. & Forst.

The inhibition of photosynthetic electron transport and the activity of photosynthetic carbon reduction cycle (PCR) enzymes under long-term water stress after slow dehydration was studied in non-nodulated Casuarina equisetifolia Forst. & Forst. plants. Initially, drought increased the fraction of closed Photosystem II (PS II) reaction centres (lowered qP) and decreased the quantum yield of PS II electron transport (ΦPSII) with no enhancement of non-radiative dissipation of light energy (qN) because it increased the efficiency of electron capture by open PS II centres (F′v/F′m). As drought progressed, F′v/F′m fell and the decrease in ΦPSII was associated with an increased qN. The kinetics of dark relaxation of fluorescence quenching pointed to an increase in a slowly-relaxing component under drought, in association with increased contents of zeaxanthin and antheraxanthin. Total NADP-dependent malate dehydrogenase activity increased and total stromal fructose-1,6-bisphosphatase activity decreased under drought, while the activation state of these enzymes remained unchanged. Water stress did not alter the activity and the activation state of ribulose bisphosphate carboxylase oxygenase.

Identification of Residues of Spinach Thioredoxin f That Influence Interactions with Target Enzymes

The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77. Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared. All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax. In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins. The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f. Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.

Essential histidine at the active site of sorghum leaf NADP-dependent malate dehydrogenase.

Chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent malate dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.

NADP regulates the light activation of NADP-dependent malate dehydrogenase

The chloroplastic NADP-dependent malate-dehydrogenase (EC 1.1.1.82) activity is modulated by light and dark. The enzyme is activated upon illumination of intact or broken chloroplasts or by incubation with dithiothreitol, whereas dark has the opposite effect. The present communication shows an additional regulation of the light modulation: in isolated intact pea chloroplasts, light activation was inhibited in the presence of electron acceptors such as sodium bicarbonate, 3-phosphoglycerate or oxaloacetate, which consume NADPH2 and produce NADP. With broken chloroplasts, addition of NADP resulted in a pronounced lag phase of NADP-dependent malate dehydrogenase light activation, while NADPH2 was without any effect. The extent of the lag phase was correlated to the amount of NADP added. When light was replaced by dithiotreitol, the inhibition effect was even more pronounced. It was assumed that NADP inhibits the modulation reaction directly: reduced thioredoxin, a potent mediator of activation by light, or dithiotreitol appear to counteract NADP in a competitive manner. The results indicate a physiological role of NADP in the regulation of chloroplastic NADP-dependent malate dehydrogenase which is capable of removing electrons from the chloroplast, via oxaloacetate reduction and malate export. Thus an NADP concentration sufficient for continuous photosynthetic electron flow may be achieved.

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C3 Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Mesembryanthemum crystallinum, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C(3)-photosynthetic net CO(2) fixation, whereas plants exposed to the high salinity level exhibited net CO(2) dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.Experiments established the extraorganellar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts. NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent of pretreatment with dithiothreitol. Protoplast extracts of M. crystallinum performing CAM exhibited higher activities (expressed per mg chlorophyll per min) of phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malic enzyme, NAD-malic enzyme, NADP-malate dehydrogenase, enolase, phosphoglyceromutase, NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphohexose isomerase than protoplast extracts from M. crystallinum not exhibiting CAM. The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm.

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O(2) evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.

Histochemical Observations of Steroid Dehydrogenases and Related Enzymes in the Livers of Laying and Moulting Hens

The livers of laying White Leghorn hens showed moderate 17β-hydroxysteroid dehydrogenase activity, whereas those of moulting hens had none or were weak. This activity had an intimate relationship with ovarian hormone synthesis. No Δ5-3β-hydroxysteroid dehydrogenase activity was found in the livers of laying or moulting hens which indicated an absence of steroid hormone synthesis in chicken livers. Moderate NADP-dependent malate dehydrogenase activity appeared in the livers of laying hens but there was little in those of moulting ones. No glucose-6-phosphate dehydrogenase activity was found. From these results, it was assumed that the NADPH2 utilized in steroid metabolism must be supplied from NADP-dependent malate dehydrogenase. Although NADP-dependent isocitrate dehydrogenase was strong in chicken livers, it is doubtful that it participates in steroid synthesis because this enzyme is located only in mitochondria. The lactate and succinate dehydrogenase activities were strong in the livers of laying hens but weak in those that were moulting. NAD-dependent malate dehydrogenase activity was strong, and that of NAD-dependent isocitrate dehydrogenase weak, with no differences between laying and moulting hens.