NADP-dependent Malate Dehydrogenase Research Articles

Rice NADP-dependent malate dehydrogenase gene OsMDH8.2 is involved in heat tolerance

Convergent and divergent evolution lead to plants with stronger adaptability to higher temperatures, thus averting crop biomass yield reductions. NADP-dependent malate dehydrogenase (NADP-MDH) is a redox-regulated enzyme that catalyzes the reversible reduction of oxaloacetate to malate. The sole NADP-MDH gene in rice, OsMDH8.2, is expressed in mesophyll cells of photosynthetic organs and various sink tissues. However, it is unknown whether NADP-MDH functions in heat stress in rice. We characterized a transgenic OsMDH8.2 overexpression line under thermal stress treatment (40 °C). The transgenic line exhibited better adaptability to heat stress than the wild type; it better maintained biomass and a lower surface temperature through stomatal closure, and photosynthetic activity was less affected. OsMDH8.2 was found to affect peroxidase activity by reducing the hydrogen peroxide content in flag leaves after 3 and 5 days of thermal stress. Analysis results of OsMDH8.2 knockout lines confirmed that OsMDH8.2 contributes to heat tolerance. Transcriptome and metabolome analyses demonstrated that OsMDH8.2 plays a key role in energy homeostasis by reducing tricarboxylic acid cycle activity while inducing the glyoxylate cycle to produce more energy, and by regulating amino acid metabolism to rescue heat-stress damage. Collectively, these results suggest that OsMDH8.2 enhances glyoxylate cycle efficiency, resulting in lower carbon dioxide release into the environment through stomatal closure, providing an excellent strategy for improving plant heat tolerance through genetic engineering.

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.

Pathophysiological mechanisms of adaptation of muscle tissue of descendants of irradiated animals to altering influence of ionizing radiation

Biochemical processes occurring in a living organism take part in the development of radiation-induced structural disorders, realizing primary damage. As a result, morphological manifestations of radiation damage are preceded by chemical shifts determining them. In the descendants of animals irradiated in different doses, a decrease in physical performance is observed, which is due to a violation of the efficiency of the use of the unique biosubstrate of muscle tissue - creatinephosphate, a change in the ratio of the activity of aerobic and anaerobic metabolism, and processes of trans-deamination of amino acids. The purpose of the work is to investigate the the mechanisms of disruption of metabolic processes in the muscle tissue of the descendants of irradiated animals. It was concluded that, unlike skeletal muscle, the activity of the tricarboxylic acid cycle, in particular the NAD-dependent malate dehydrogenase, in the myocardium is quite significant both in the cytoplasm and in the mitochondria of the tissue, as evidenced by the higher level of tricarboxylic acid cycle metabolites acids - malic and oxaloacetic, as well as the activity of NADP-dependent malate dehydrogenase, which performs a connecting role between glycolysis and the cycle of tricarboxylic acids in providing them with metabolites and transferring protons from NADH+H+ to NADP. The author revealed that myocardium is characterized by a larger pool of adenyl nucleotides due to ATP. Tha data obrtained shiowed hyperglycemia which is observed in the blood of the descendants of irradiated animals. Gluconeogenesis is enhanced in the liver of the descendants of irradiated animals and this explains the hyperglycemia and accumulation of glycogen in the liver. At the same time, the penetration of glucose into muscle cells is weakened, which is associated with a decrease in their glycogen content, and this can be explained by the decrease in adaptation to physical exertion in the descendants of irradiated animals. The author made a cionclusion that the pathophysiological mechanisms of radiation-induced energy supply restructuring are aimed at short-term processes of strengthening the supply of energy to vital organs and systems for destroyed biochemical, physiological, functional and regulatory processes restoration and sanogenetic mechanisms activation.

The role of fatty acids in disturbance of energy processes in the early placenta with cytomegalovirus infection

Introduction. Cytomegalovirus (CMV) infection directly and indirectly can cause placental dysfunction. One of the reasons for its development may be a deficiency of energy supply due to changes in the level of fatty acids (FA) – the main sources of energy in the cell.Aim. Analysis of the fatty acids concentration and causes of its changes in the placenta during exacerbation of CMV infection in the first trimester of pregnancy.Materials and methods. Biosamples (venous blood, epithelium from the inner surface of the cheek, mucous membrane of the cervical canal, villous chorion) of 32 CMV-seropositive women with an exacerbation of CMV infection in the first trimester of pregnancy (main group) and 30 CMV-seronegative women (control group) were studied. Exacerbation of CMV infection was diagnosed by ELISA to detect IgM and IgG with avidity of 65% or more, PCR to detect CMV DNA. The profile and relative concentration of individual FA in villous chorion lipid extracts were studied by gas-liquid chromatography. The activity of pyruvate dehydrogenase, succinate dehydrogenase, NADP-dependent malate dehydrogenase, and glucose-6-phosphate dehydrogenase was determined by a histochemical method on sections of freshly frozen villous chorion tissues.Results. In the main group placentas, the concentration of medium-chain saturated fatty acids was statistically significantly lower: capric by 50%, lauric by 51%, unsaturated myristoleic by 44%; long-chain unsaturated acids: palmitoleic, oleic, linoleic and α-linolenic fatty acids by 52%, 55%, 57% and 64%, respectively; of polyunsaturated fatty acids with a very long chain: eicosapentaenoic and docosahexaenoic by 44% and 41%, respectively. The activity of enzymes: succinate dehydrogenase, pyruvate dehydrogenase, NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase decreased.Conclusion. Thus, we found a decrease in energy supply in the placenta during exacerbation of chronic CMV infection in the first trimester of pregnancy. Disorders of placental energy metabolism can cause placental insufficiency, which has adverse consequences for fetal development.

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.

The NADP-malate dehydrogenase (SmNADP-mdh), a C4 pathway gene from Suaeda monoica enhanced photosynthesis and biomass yield in C3 plants

Enhanced photosynthesis and biomass have been achieved in C 3 plants by over-expressing NADP dependent malate dehydrogenase ( SmNADP-mdh ), a C 4 pathway gene from Suaeda monoica . Over-expression of the SmNADP-mdh improved seed germination, seedling growth and accumulation of pigments, sugar, starch and C H N S elements in transgenic tobacco under in vitro conditions. Transgenic tobacco exhibited higher NADP-MDH activity, membrane stability index, photosynthesis efficiency, improved stomatal conductance, carboxylation efficiency, photosystem II (PSII) operating efficiency, electron transfer rate, photochemical quenching and better accumulation of sugar and starch at elevated (1200 ppm) CO 2 (eCO 2 ). Chlorophyll a fluorescence analysis showed improved pool size, electron transfer, water-splitting activity, photosynthetic performance indexes and efficient working of PSII at eCO 2 in transgenic tobacco over-expressing SmNADP-mdh . The higher expression of antioxidant defence genes indicated better scavenging and lesser accumulation of ROS at eCO 2 . Results suggest the potential of a single-cell C 4 halophytic NADP-mdh gene in the development of smart agriculture to improve the photosynthetic efficiency and biomass yield in C 3 plants. • The SmNADP-mdh transgenic tobacco exhibited higher NADP-mdh activity. • SmNADP-mdh gene improved seed germination and seedling growth. • Transgenic tobacco exhibited higher sugar contents under in vitro conditions. • Transgenic tobacco showed higher photosynthesis and carboxylation efficiency. • Transgenic tobacco showed higher contents of pigments and sugar at eCO 2 .

Change in expression levels of NAD kinase-encoding genes in Flaveria species

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3–C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.

Metabolomic analyses of highbush blueberry (Vaccinium corymbosum L.) cultivars revealed mechanisms of resistance to aluminum toxicity

Aluminum (Al) is an important factor that limits plant growth under acidic soil conditions. However, several plant species developed distinct mechanisms that limit the damage caused by high Al concentrations. In highbush blueberry (Vaccinium corymbosum), the Al resistance mechanisms are not fully understood. This study was designed to evaluate the effect of Al toxicity on roots and leaves of highbush blueberry genotypes with contrasting Al resistance [Star (Al-sensitive) and Camellia and Cargo (Al-resistant)] and identify the main molecular and physiological strategies underpinning adaptive Al stress responses in nutrient solution. After 48 h of Al treatment, the reduced form of ascorbate (ASC) was higher in roots, but unchanged in leaves of Cargo and Camellia genotypes compared to the control. We also observed decreased root exudation of oxalate in the Al-treated sensitive cultivar Star throughout the treatment period. However, in the resistant cultivar (Camellia), the exudation of oxalate increased 2.4- and 2.8-fold at 24 and 48 h, respectively. Al treatment differentially affected the enzyme activity and gene expression of the tricarboxylic acid (TCA) cycle enzymes. NAD-dependent malate dehydrogenase (NAD-MDH) expression in roots of cultivar Cargo was reduced at 24 h and increased at 48 h, whereas in leaves the expression was higher at 24 h and decreased at 48 h compared to the control. Citrate synthase (CS) activity in Al-resistant Cargo roots diminished at 24 h, increasing afterwards, without variation in the CS gene expression, compared with the initial time point (t = 0). In Al-resistant Camellia roots, the gene expression and the activity of CS decreased during Al exposure. NADP-dependent malate dehydrogenase (NADP-MDH) activity showed increased activity and gene expression at 24 h, in the leaves of cultivar Cargo, whereas in roots the gene expression decreased, but the activation state of NADP-MDH increased. The expression of genes encoding TCA cycle enzymes did not differ significantly in the Al-sensitive cultivar Star during Al exposure. In conclusion, the exudation of organic acid anions, particularly oxalate, plays an important role in Al resistance of highbush blueberry genotypes whilst elevated levels of ASC in roots, also contribute to the Al-resistance mechanisms exhibited by genotypes Camellia and Cargo.

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.

Genomic dissection and expression analysis of stress-responsive genes in C4 panicoid models, Setaria italica and Setaria viridis

The study reports the identification and expression profiling of five major classes of C4 pathway-specific genes, namely, carbonic anhydrase (CaH), phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-dependent malate dehydrogenase (MDH) and NADP-dependent malic enzyme (NADP-ME), in the model species, Setaria italica and Setaria viridis. A total of 42 and 41 genes were identified in S. italica and S. viridis, respectively. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. RNA-Seq derived expression profiles of the gene family members showed their differential expression pattern in tissues and dehydration stress. Comparative genome mapping and Ks dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiαCaH1, SiβCaH5, SiPEPC2, SiPPDK2, SiMDH8, and SiNADP-ME5 in the tolerant cultivar. Overexpression of SiNADP-ME5 in heterologous yeast system enabled the transgenic cells to survive and grow in dehydration stress conditions, which highlights the putative role of SiNADP-ME5 in conferring tolerance to dehydration stress. Altogether, the study highlights key genes that could be potential candidates for elucidating their functional roles in abiotic stress response.

Induced Mutagenesis Enhances Lodging Resistance and Photosynthetic Efficiency of Kodomillet (Paspalum Scrobiculatum)

The present research was focused in the development of photosynthetically efficient (PhE) and non-lodging mutants by utilizing ethyl methane sulphonate (EMS) and gamma radiation in the kodomillet variety CO 3, prone to lodging. Striking variations in a number of anatomical characteristics of leaf anatomy for PhE and culm thickness for lodging resistance was recorded in M2 (second mutant) generation. The identified mutants were subjected to transcriptomic studies to understand their molecular basis. Expression profiling was undertaken for pyruvate phosphate dikinase (PPDK), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen—(NADPH) and NADP-dependent malate dehydrogenase (NADP-MDH) in the mutants CO 3-100-7-12 (photosynthetically efficient) and in CO 3-200-13-4 (less efficient). For lodging trait, two mutants CO 3-100-18-22 (lodged) and CO 3-300-7-4 (non-lodged) were selected for expression profiling using genes GA2ox6 and Rht-B. The studies confirmed the expression of PPDK increased 30-fold, NADP-ME2 ~1-fold and NADP-MDH10 was also highly expressed in the mutant CO 3-100-7-12. These expression profiles suggest that kodomillet uses an NADP-malic enzyme subtype C4 photosynthetic system. The expression of Rht-B was significantly up regulated in CO 3-300-7-4. The study highlights the differential expression patterns of the same gene in different lines at different time points of stress as well as non-stress conditions. This infers that the mutation has some effect on their expression; otherwise the expression levels will be unaltered. Enhancement in grain yield could be best achieved by developing a phenotype with high PhE and culm with thick sclerenchyma cells.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK. Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis.

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP+ ) and pool size (sum of NADPH and NADP+ ), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP+ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found invitro to inefficiently reduce m-type thioredoxin, which activates Calvin cycle enzymes, and NADP-dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP-malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m-type thioredoxin exhibited similar light-dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m-type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP+ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis.

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.

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.

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster

ABSTRACTObjectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na2MoO4).Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food.Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration.Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.

Enzyme Changes in the Offspring of Female Rats due to Long-Term Administration of Cyclic AMP and Insulin before Pregnancy.

We studied the effects of insulin and cAMP on the offspring of female rats after daily treatment with these substances over 4weeks. In adult offspring from cAMP-treated females, activities of pyruvate kinase and glucose-6-phosphate dehydrogenase decreased in the liver and brain and activities of NADP-dependent malate dehydrogenase and 6-phosphogluconate dehydrogenase decreased in the liver. In the offspring of insulin-treated females, we observed only activation of glucose-6-phosphate dehydrogenase and malate dehydrogenase in the liver and only in females. Enzyme activity probably correlates with their content, as no changes in their kinetic properties were observed under these conditions. Long-term hormone treatment before pregnancy can affect the expression of genes for some enzymes in the offspring due to transmission of epigenetic signals by the ovum. However, further studies are required to confirm this mechanism.

Proteomics dissection of cold responsive proteins based on PEG fractionation in Arabidopsis

Proteome profiling was performed on Arabidopsis plant exposed to cold stress at 4 °C for 24 h in an attempt to explore the mechanisms of plant climate adaptation. The polyethylene glycol(PEG) fractionation protocol developed in this lab was used to identify as many differentially expressed low-abundance proteins as possible. In comparison with those of the biological controls, 67 protein spots with at least two-fold difference in expression were identified for the plant exposed to cold temperatures; and from these spots, 50 proteins were successfully identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS). Bioinformatics studies on these proteins show that 57.8% of these proteins were localized in the chloroplast. Of these proteins, 8 ones have functions in photosynthesis, including glycine hydroxymethyltransferase, Rubisco large subunit, Rubisco activase, PSBO2, fructose-1,6-bisphosphate aldolase, NADP-dependent malate dehydrogenase, sedoheptulose bisphosphatase and photosystem II reaction center PsbP family protein, suggesting that photosynthesis is greatly affected by cold stress. The identified proteins were validated by quantitative real-time polymerase chain reaction(qPCR). Taken together, our results suggest that the chloroplast might also act as a cold stress sensor and that photosynthesis-related proteins may play important roles in cold acclimation for Arabidopsis.

Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis

In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis.

Effect of inland salt-alkaline stress on C<sub>4</sub> enzymes, pigments, antioxidant enzymes, and photosynthesis in leaf, bark, and branch chlorenchyma of poplars

The effects of soil salt-alkaline (SA) stress on leaf physiological processes are well studied in the laboratory, but less is known about their effect on leaf, bark and branch chlorenchyma and no reports exist on their effect on C4 enzymes in field conditions. Our results demonstrated that activities of C4 enzymes, such as phospholenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), and NADP-dependent malate dehydrogenase (NADP-MDH), could also be regulated by soil salinity/alkalinity in poplar (Populus alba × P. berolinensis) trees, similarly as the already documented changes in activities of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), pigment composition, photosynthesis, and respiration. However, compared with 50-90% changes in a leaf and young branch chlorenchyma, much smaller changes in malondialdehyde (MDA), antioxidative enzymes, and C4 enzymatic activities were observed in bark chlorenchyma, showing that the effect of soil salinity/alkalinity on enzymatic activities was organ-dependent. This suggests that C4 enzymatic ratios between nonleaf chlorenchyma and leaf (the commonly used parameter to discern the operation of the C4 photosynthetic pathway in nonleaf chlorenchyma), were dependent on SA stress. Moreover, much smaller enhancement of these ratios was seen in an improved soil contrary to SA soil, when the fresh mass (FM) was used as the unit compared with a calculation on a chlorophyll (Chl) unit. An identification of the C4 photosynthesis pathway via C4 enzyme difference between chlorenchyma and leaf should take this environmental regulation and unit-based difference into account.