NADH-dependent Glutamate Dehydrogenase Research Articles

Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin.

The access to the enantiopure noncanonical amino acid l-phosphinothricin (l-PPT) by applying biocatalysts is highly appealing in organic chemistry. In this study, a NADH-dependent glutamate dehydrogenase from Lachnospiraceae bacterium (LbGluDH) was chosen for the asymmetric synthesis of l-PPT. Three flexible loops undergoing big conformational shifts during the catalysis were identified and rationally engineered following the initial mutagenesis. The enzyme's specific activity toward the key precursor of l-PPT, 2-oxo-4-[(hydroxy) (methyl) phosphinyl] butyric acid (PPO), was improved from negligible to 9 U/mg, and the Km value was reduced to 17 mM. The computational analysis showed that the modified loops broadened the enzyme's narrow tunnels, allowing the substrate to access the binding pocket and get closer to the crucial residue D165, thereby enhancing the catalytic process. Utilizing the variant as the catalyst, the preparation of l-PPT achieved a 100% conversion rate within 60 min, coupled with a stereoselectivity exceeding 99.9%, demonstrating its practical capacity for industrial application. Similar enhancement in catalytic activity was obtained applying the same strategy to a typical NADH-dependent GluDH from Pyrobaculum islandicum (PisGluDH), indicating the effectiveness of our strategy for the protein engineering of GluDHs targeted to the biosynthesis of unnatural compounds.

Concentration-dependent dual effects of exogenous sucrose on nitrogen metabolism in Andrographis paniculata

The effects of exogenous sucrose (Suc) concentrations (0, 0.5, 1, 5, 10 mmol L−1) on carbon (C) and nitrogen (N) metabolisms were investigated in a medicinal plant Andrographis paniculata (Chuanxinlian). Suc application with the concentration of 0.5–5 mmol L−1 significantly promoted plant growth. In contrast, 10 mmol L−1 Suc retarded plant growth and increased contents of anthocyanin and MDA and activity of SOD in comparison to 0.5–5 mmol L−1 Suc. Suc application increased contents of leaf soluble sugar, reducing sugar and trerhalose, as well as isocitrate dehydrogenase (ICDH) activity, increasing supply of C-skeleton for N assimilation. However, total leaf N was peaked at 1 mmol L−1 Suc, which was consistent with root activity, suggesting that exogenous Suc enhanced root N uptake. At 10 mmol L−1 Suc, total leaf N and activities of glutamine synthase (GS), glutamate synthase (GOGAT), NADH-dependent glutamate dehydrogenase (NADH-GDH) and glutamic–pyruvic transaminase (GPT) were strongly reduced but NH4+ concentration was significantly increased. The results revealed that exogenous Suc is an effective stimulant for A. paniculata plant growth. Low Suc concentration (e.g. 1 mmol L−1) increased supply of C-skeleton and promoted N uptake and assimilation in A. paniculata plant, whereas high Suc concentration (e.g. 10 mmol L−1) uncoupled C and N metabolisms, reduced N metabolism and induced plant senescence.

Organic nitrogen sources promote andrographolide biosynthesis by reducing nitrogen metabolism and increasing carbon accumulation in Andrographis paniculata

Nitrogen (N) form affects secondary metabolites of medicinal plants, but the physiological and molecular mechanisms remain largely unknown. To fully understand the response of andrographolide biosynthesis to different N forms in Andrographis paniculata, the plants were fed with nutritional solution containing sole N source of nitrate (NO3−), ammonium (NH4+), urea or glycine (Gly), and the growth, carbon (C) and N metabolisms and andrographolide biosynthesis were analyzed. We found that plants grown in urea and Gly performed greater photosynthetic rate and photosynthetic N use efficiency (PNUE) than those grown in NO3− and NH4+. Organic N sources reduced the activities of enzymes involving in C and N metabolisms such as glutamine synthase (GS), glutamate synthase (GOGAT) and NADH-dependent glutamate dehydrogenase (NADH-GDH), invertase (INV), isocitrate dehydrogenase (ICDH) and glycolate oxidase (GO), resulting in reduced depletion of carbohydrates and increased starch accumulation. However, they enhanced andrographolide content by up-regulating the key genes in its biosynthetic pathway including HMGR, DXS, GGPS and ApCPS. Besides, NH4+ decreased leaf SPAD value, contents of soluble protein and amino acids and GO activity, but increased photosynthetic rate and contents of soluble sugar and starch in comparison to NO3−. Andrographolide biosynthesis was also up-regulated. The results revealed that increasing accumulation of carbohydrates, especially starch, was beneficial to the biosynthesis of andrographolide; organic N sources decreased carbohydrate depletion by reducing N metabolism, and promoted plant growth and andrographolide biosynthesis synergistically.

Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli.

l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.

Impact of exogenously applied ABA on proline metabolism conferring drought and salinity stress tolerance in wheat genotypes

Drought and salinity are the most catastrophic abiotic stresses that affect plant growth and development. Plant initiates alterations at physiological, biochemical and molecular levels to combat adverse effects of drought in which abscisic acid (ABA) plays a pivotal role. The present investigation aims to study the modulation of enzymatic changes conferring stress tolerance in the 7-day-old shoots of five wheat genotypes influenced by exogenously applied ABA during stress. The results revealed that application of ABA (10 μM) positively influenced the shoot length and biomass of Gladius and Drysdale under water deficit and water withholding condition, while Kharchia was the only genotype showing positive response under salt stress condition and nearly negligible effect being observed in PBW 175 and PBW 660. The content of thiobarbituric acid reactive substances was lowered while proline content along with 2,2 diphenyl-picrylhydrazyl activity increased drastically with exogenous ABA especially in Gladius, Drysdale and Kharchia. The genotypes, i.e., Gladius, Drysdale and Kharchia, also possessed increased activity of proline metabolizing enzymes, i.e., NADH-dependent glutamate dehydrogenase, pyrroline-5-carboxylate synthetase (1.5–2-fold) in response to ABA while proline dehydrogenase remained unresponsiveness to ABA indicating the ABA-dependent and ABA-independent regulation of enzymes, respectively, and depicting the role of ABA and proline in conferring drought and salinity tolerance.

CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.

Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for L-ornithine overproduction. In fed-batch fermentation, L-ornithine of 88.26g/L with productivity of 1.23g/L/h (over 72h) and yield of 0.414g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of L-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote L-ornithine production by C. glutamicum strains.

Benzothiadiazole and nitrogen source modify the nitrogen metabolism in cucumber infected with Pseudomonas syringae pv. lachrymans

The influence of nitrogen source and benzothiadiazole (BTH) pretreatment on nitrogen metabolism-related enzyme activities was studied in cucumber plants infected with Pseudomonas syringae pv. lachrymans (Psl). The plants were grown hydroponically in media containing nitrate and urea at different ratios: 100% NO3− (100N), 75% NO3−/25% urea (75N) and 50% NO3−/50% urea (50N) and pretreated with BTH prior to Psl inoculation.In the presence of urea in the nutritional medium the activities of most enzymes in the control plants decreased. Disease development was influenced by nitrogen nutrition and the weakest symptoms occurred in the 75N plants. The BTH-induced disease restriction was dependent on the type of nitrogen nutrition and was most visible in the 50N plants (about 2-fold reduction of infected leaf area compared to the plants not pretreated with BTH). The nitrogen metabolism was modified by BTH and this effect was stronger than that of urea. Irrespective of the type of nitrogen nutrition, in the BTH-treated plants, the activities of nitrate reductase, ferredoxin- and NADH-dependent glutamate synthase decreased whereas those of NADH-dependent glutamate dehydrogenase (NADH-GDH), aminotransferases and NADP-dependent isocitrate dehydrogenase were enhanced compared to the controls. After infection, in all nutritional variants, the strongest effect of BTH pretreatment was observed for NADH-GDH on the 2 day, when its activity increased by 85–256% compared to the infected non-pretreated plants. Our results concerning the glutamate-metabolizing enzymes suggest that BTH treatment combined with low dose of urea may have a positive influence on the nitrogen management leading to the inhibition of disease symptom progress.

Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings

Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and β-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

Reduced levels of NADH-dependent glutamate dehydrogenase decrease the glutamate content of ripe tomato fruit but have no effect on green fruit or leaves.

Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development.

Relationship between chemiluminescent and enzymatic activity of blood neutrophils interactions in patients with community6 acquired pneumonia

The aim of the study was to investigate enzymatic and chemiluminescent activity in blood neutrophils of patients with moderate and severe communityacquired pneumonia (CAP). Methods. The study involved 48 patients with moderate CAP and 40 patients with severe CAP; they were examined on admission. Synthesis of primary and secondary reactive oxygen species (ROS) was evaluated using the chemiluminescence assay. Neutrophil NAD(P)dependent dehydrogenase activity was assessed using the bioluminescent method. Results. The «respiratory burst» was found in neutrophils of CAP patients. Terminal reactions of the anaerobic glycolysis and the tricarbonic acid cycle were intensified in patients with severe CAP only. Increased lipid anabolism activity and substrate outflow from the tricarbonic acid cycle via NADHdependent glutamate dehydrogenase were shown regardless of CAP severity. The canonical analysis showed abnormalities of metabolic mechanisms of the «respiratory burst» in blood neutrophils in CAP. Conclusion. The metabolic disorders and «respiratory burst» intensity in CAP arize the need for pathogenic correction. High NADHdepend ent dehydrogenase activity and lower chemiluminescent activity in blood neutrophils could be used as additional markers of severe course of CAP.

Хемилюминесцентная и энзиматическая активность нейтрофильных гранулоцитов у больных распространенным гнойным перитонитом в зависимости от исхода заболевания

Aim was to study the chemiluminescent activity and the activity of NAD- and NADP-dependent dehydrogenases of neutrophils depending on the outcome of the widespread purulent peritonitis (WPP). 51 patients with a mean age of RSE 54.2 ± 19.2 years were observed. As a control 75 healthy people of similar age range were examined. Lucigenin- and luminol-dependent chemiluminescence activity and activity of NAD(P)-dependent dehydrogenases peripheral blood neutrophils were studied. In patients with a favorable outcome of WPP the maximum intensity increased and the magnitude of the activation index of lucigenine-dependent spontaneous chemiluminescence reduced. Regardless to the outcome of WPP in patients with increased activation index and maxima of the luminol-dependent spontaneous and zymosan-induced chemiluminescence. At the unfavorable outcome of the disease in neutrophils NAD- dependent isositrate dehydrogenase and anaerobic lactate dehydrogenase reaction activity increased. Regardless to the outcome WPP in patients neutrophils aerobic reaction of lactate dehydrogenase, glucose-6-phosphate dehydrogenase and NADP-dependent glutamate dehydrogenase activity reduced but levels of NADH-dependent reaction of malate dehydrogenase, NADH-dependent glutamate dehydrogenase and NADP-dependent isocitrate dehydrogenase activity increased. With the reduction of the intensity ofplastic processes and imbalances enzymatic activity in nitrogen metabolism in patients with WPP at unfavorable outcome of the disease increases the activity of enzymes that characterize the level of anaerobic and aerobic respiration. In the absence of marked changes in the activity of enzymes that characterize the level of energy processes in cells of patients with favorable outcome of the WPP, increases the intensity of spontaneous lucigenin-dependent chemiluminescence and reduced neutrophil activation index.

Beneficial Role of Exogenous Spermidine on Nitrogen Metabolism in Tomato Seedlings Exposed to Saline-alkaline Stress

We studied the effects of exogenous spermidine (Spd) on plant growth and nitrogen metabolism in two cultivars of tomato (Solanum lycopersicum) that have differential sensitivity to mixed salinity-alkalinity stress: ‘Jinpeng Chaoguan’ (salt-tolerant) and ‘Zhongza No. 9’ (salt-sensitive). Seedling growth of both tomato cultivars was inhibited by salinity-alkalinity stress, but Spd treatment alleviated the growth reduction to some extent, especially in ‘Zhongza No. 9’. Exogenous Spd may help reduce stress-induced increases in free amino acids, ammonium (NH4+) contents, and NADH-dependent glutamate dehydrogenase (NADH-GDH) activities; depress stress-induced decreases in soluble protein and nitrate content; and depress nitrate reductase, nitrite reductase, glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) activities, especially for ‘Zhongza No. 9’. Based on our results, we suggest that exogenous Spd promotes the assimilation of excess toxic NH4+ by coordinating and strengthening the synergistic action of NADH-GDH, GS/NADH-GOGAT, and transamination pathways, all during saline-alkaline stress. Subsequently, NH4+ and its related enzymes (GDH, GS, GOGAT, GOT, and GPT), in vivo, are maintained in a proper and balanced state to enable mitigation of stress-resulted damages. These results suggest that exogenous Spd treatment can relieve nitrogen metabolic disturbances caused by salinity-alkalinity stress and eventually promote plant growth.

METABOLIC PLATELET STATUS IN PATIENTS WITH STABLE ANGINA

Aim. To study the activity of NAD- and NADP-dependent platelet dehydrogenases in patients with various functional classes of angina. Material and methods. The study included 91 men with Functional Class II–IV angina, aged 45–72 years. In all participants, the bioluminescent method was used to measure the activity of the following enzymes: glucose-6-phosphate dehydrogenase (G6PD), glycerol-3-phosphate dehydrogenase (GPDH), NADPmalate dehydrogenase (NADP-MDH), NAD- and NADH-dependent lactate dehydrogenase reaction (LDH and NADH-LDH, respectively), NAD- and NADHdependent malate dehydrogenase reaction (MDH and NADH-MDH, respectively), NADP- and NADPH-dependent glutamate dehydrogenase (NADP-GDH and NADPH-GDH, respectively), NAD- and NADH-dependent glutamate dehydrogenase (NAD-GDH and NADH-GDH, respectively), NAD- and NADP-dependent isocitrate dehydrogenase (NAD-IDH and NADP-IDH, respectively), and glutathione reductase (GR). Results. The metabolic platelet status was associated with the functional class of angina. In patients with Functional Class II angina, minimal platelet changes, linked to the inhibition of plastic processes, were observed. Patients with Functional Class III angina demonstrated an inhibition of lipid metabolism and an increase in glycolytic activity. In Functional Class IV angina, the metabolic disturbances were most pronounced and manifested in inhibited plastic processes and lipid catabolism, together with activated anaerobic reactions. Conclusion. In stable angina patients, the changes in intracellular metabolic activity of platelets mirror the increase in the functional class of angina. Modification of platelet metabolism could be one of the methods of decreasing the receptor and, therefore, aggregation platelet activity.

Characterization of a NADH-Dependent Glutamate Dehydrogenase Mutant of Arabidopsis Demonstrates the Key Role of this Enzyme in Root Carbon and Nitrogen Metabolism

The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

Physiological responses of Catharanthus roseus to different nitrogen forms

The responses of carbon and nitrogen metabolisms in the medical plant Catharanthus roseus to the nitrogen solutions (N1, N2 and N3) containing different ratio of nitrate to ammonium (1:0, N1; 1:1, N2; 1:3, N3) were investigated here. The plants in N3 nitrogen solution were strongly inhibited in photosynthetic gas exchange and carbohydrate accumulation, reflecting the toxicity symptom of excess ammonium continuously accumulated in plants. The treatment with N2 nitrogen solution, however, displayed an obviously synergistic effect on plant growth and metabolisms in contrast to nitrate as the sole source. The short-term (7 days) exposure of plants to N2 nitrogen solution resulted in an increased shoot/root ratio, leaf mass ratio, and Pn, as well as the elevated levels of sucrose, glutamate, aspartate, proline and threonine. The plants in N2 nitrogen solution accumulated twofold catharanthine and vinblastine than did the plants in N1 or N3 nitrogen solution after the long-term incubation. Internal nitrate had an increased accumulation in the plants in N2 nitrogen solution compared to the counterparts. The supply of N2 or N3 nitrogen solution to plants for 7 days induced an over tenfold increase of ammonium in leaves as compared to the case using N1 nitrogen solution. The increased ammonium ion promoted the activities of NADH-dependent glutamate dehydrogenase (NADH-GDH) both in the leaf and root of plants. Under the ammonium-containing solution (N2 and N3 nitrogen solutions), there was a significantly increased activity for glutamine synthase (GS) in the root during experiment and for nitrate reductase (NR) in the leaf and root only after 21 days of treatment. The performed correlation analysis revealed a negative relation between soluble sugars and internal ammonium, whereas a positive correlation of alkaloid production with glutamate and aspartate.

Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase encoded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain.

Cellular response of light/dark-grown green alga Chlorella vulgaris Beijerinck (Chlorophyceae) to exogenous adenine- and phenylurea-type cytokinins

Treatment exposed to light Chlorella vulgaris Beijerinck (Chlorophyceae) with adenine- (BA, Kin, Z) and phenylurea-type (DPU) cytokinins effects positively on alga viability by 1.5- to twofold increase in cell number, chlorophylls, carotenoids, monosaccharides and glycolate content as well as NADH-dependent hydroxypyruvate reducing enzyme activity (NADH-HPR) extensively involved in carbon metabolism. Cytokinins enhance nitrogen assimilation by stimulation of NADH-dependent glutamate dehydrogenase (NADH-GDH) aminating activity finally leading to higher protein level and its secretion as well as polypeptide accumulation in the range of molecular weight 12–195 kDa. In the dark, cytokinins mimic the regulatory effect of light upon algal cell division, metabolite content and stimulate carbon recycling for Calvin cycle reactions by enhancing of light-dependent NADH-HPR activity. The delaying of protein degradation and stimulation of their secretion to environment, triggering polypeptide accumulation and twofold higher NADH-GDH activity catalysing amino acids biosynthesis are observed in the dark-grown microalgae in response to cytokinins. Chlorella vulgaris exhibits sensitivity on cytokinins in the following order of their stimulating properties: DPU > Z > Kin > BA in both light and dark conditions. Understanding of cytokinin role in lower plants under different light conditions could be a step toward the elucidation of the evolution of hormone regulation and their action at molecular level.

Physiological and biochemical effects of cadmium toxicity in enzymes involved in nitrogen and amino—acid metabolism in tomato plants

The activities of several enzymes related to amino—acid metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. 63/5F1) in control and treated leaves and roots. Glutamine synthetase and glutamate synthase ferredoxine dependent activities rapidly declined in stress condition. In presence of cadmium, the decline of glutamine synthetase activity was retarded, while the activity of glutamate synthase significantly and more rapid declined. The activity of NADH—dependent glutamate dehydrogenase was markedly increased in presence of cadmium. Western blots analysis from cadmium treated plants, showed a decrease in chloroplastic GS (GS2) protein and an increase in cytosolic GS (GS1) and glutamate dehydrogenase (GDH) proteins. The activities of phosphoenol pyruvate carboxylase and NADP—dependent isocitrate dehydrogenase also increased in both organs of plantes treated. The activity of NAD and NADP—dependent malate dehydrogenase were decreased especially in leaves.

Differential capacity of nitrogen assimilation between apical and basal leaf portions of a tank epiphytic bromeliad

The leaf is the main organ of the vegetative body of tank epiphytic bromeliads, which may be subdivided into apical and basal parts. Little is known about the existence of morphological, anatomical or physiological differences between these two leaf portions. The objective of this study was to verify the existence of a differential capacity to assimilate nitrogen from urea between basal and apical leaf portions of Vriesea gigantea, a tank epiphytic bromeliad. Plants were cultivated in vitro in the presence of 5 mM urea for 15 days. During this period, the activities of the enzymes glutamine synthetase (GS, EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) were quantified, as well as the densities of trichomes and stomata found at the leaf surface of both regions. The highest activities of GS and NADH-GDH were always detected at the top portion, and on the 3rd day of in vitro cultivation an increase in these activities was detected. An inverse correlation between number of trichomes and stomata in these two leaf portions was verified: the top region presented 30% less trichomes and twice as many stomata as the basal region. This suggests that the upper region of the leaf may be preferentially involved with the assimilation of nitrogen from urea via GS and NADH-GDH, whereas the basal region may be involved with its absorption. This is the first study to report that ammonium assimilation may take place preferentially in a specific portion of tank-forming bromeliad leaves.

Transgenic Tomato Plants that Overexpress a Gene for NADH-dependent Glutamate Dehydrogenase (legdh1)

Tomato plants were transformed with a plasmid that included a legdh1 gene for NADH-dependent glutamate dehydrogenase (NADH-GDH) from Lycopersicon esculentum Mill. coupled, in the sense orientation, with the constitutively active 35S promoter from cauliflower mosaic virus. Three independent transformants were obtained. In these transgenic lines, high-level expression of legdh1 mRNA was detected in the leaves, and the NADH-GDH activity in the leaves of the transgenic plants was approximately twice that in the leaves of the non-transgenic plant. In the transgenic tomato fruits examined for six successive weeks after flowering, the levels of total free amino acids were higher (2.1- to 2.3-fold) than those in the controls. In particular, the level of glutamate was about twice that in the control fruits.