Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.)

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.     

Enzyme Activities in Urtica dioica: Effects of Daylength and Leaf Age on Glutamate Dehydrogenase, Aspartate Aminotransferase and Alanine Aminotransferase

Enzymes, important to protein synthesis, were investigated in young and old leaves of Urtica dioica. The plants, divided into two groups, were exposed to either 18-hour or 12-hour photo-periods. One group of plants from each photoperiodic regime was subjected to an irradiance of 28 W × m^-2, and the other group of plants to 42 W × m^-2. The enzymes investigated were glutamate dehydrogenase (GDH), aspartate aminotransferase (glutamate-oxaloacetate transaminase, GOT), and alanine aminotransferase (glutamate-pyruvate transaminase, GPT), GDH and GOT were determined by means of electrophoretic separation on polyacrylamide and spectrophotometric measurements. GPT was determined only by the latter method. Plants exposed to 18-hour photoperiods showed much higher GDH activity than did those exposed to 12-hour photoperiods. The activity of GDH also increased with leaf age. Besides one uniform NAD⁺-dependent GDH, two other NAD⁺-independent enzymes, showing GDH activity, were identified on polyacryl-amide gel electrophoresis. The distribution of NADH and NAD⁺-dependent GDH activity between young and old leaves was similar under different growth conditions. The activity of GOT was insensitive to environmental changes. The results regarding GPT indicate that this enzyme responded to different photoperiods in the same way as GDH. A correlation coefficient of 0.928 was obtained for the relationship between GDH and GPT activity.     

Tentoxin. An uncompetitive inhibitor of lettuce chloroplast coupling factor 1.

The interaction of tentoxin [cyclo-(-L-leucyl-N-methyl-(Z)-dehydrophenylalanyl-glycyl-N-methyl-L-alanyl-)] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 . 10(8) M-1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.     

Effects of ADP on different inhibitory properties of brain glutamate dehydrogenase isoproteins by perphenazine

Incubation of glutamate dehydrogenase isoproteins (GDH I and GDH II) from bovine brains with perphenazine resulted in a time-dependent loss of enzyme activity. 2-Oxoglutarate and NADH, separately or together, gave partial but not complete protection against the inhibition. Although there were no detectable differences between GDH I and GDH II in inhibition by perphenazine in the absence of ADP, the sensitivities to the inhibition by the drug were significantly distinct for the two isoproteins in the presence of ADP. Low concentrations of ADP (0.05-0.20 mM) did not interfere with the inhibition of GDH I and GDH II by perphenazine. However, in the presence of high concentrations of ADP (0.5-1.0 mM), inhibitory effects of perphenazine on GDH isoproteins were significantly diminished as determined by enzyme kinetics and quantitative affinity chromatography on perphenazine-Sepharose. GDH I was more sensitively reacted with ADP than GDH II on the inhibition by perphenazine. Since physiological ADP levels can vary from 0.05 to > 1.0 mM depending on the rate of oxidative phosphorylation, our results suggest a possibility that two types of GDHs are differently regulated by the antipsychotic actions of perphenazine depending on the physiological concentrations of ADP. GTP and L-leucine, other well-known allosteric regulators, did not affect the inhibitory actions of perphenazine on bovine brain GDH isoproteins.     

Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.     

核桃凋落叶分解对莴笋抗氧化系统及光合特性的影响

为探讨核桃对农作物的化感作用,该试验采用盆栽法,设置4个凋落叶施用量水平(0、30、60、90g/盆),研究了核桃凋落叶在土壤中自然分解过程中对莴笋(播种后80、100、120和140d)抗氧化系统、光合生理特征及其生长的影响。结果显示:(1)核桃凋落叶处理的莴笋叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性在播种80d时得到促进,在100d时受到抑制,而在120d之后基本恢复至正常水平,并以SOD表现最为敏感。(2)核桃凋落叶处理100和120d时,莴笋叶片可溶性蛋白(SP)含量显著降低,而可溶性糖(SS)含量显著增加。(3)核桃凋落叶处理100、120d时,莴笋叶片净光合速率(Pn)受到显著抑制,各处理气孔导度(Gs)和蒸腾速率(Tr)显著低于对照。(4)核桃凋落叶处理的莴笋株高、地上部分生物量及地上部分占总生物量比重在处理120d时均显著低于对照,在140d时基本恢复正常。研究表明,核桃凋落叶在土壤中分解对莴笋产生的化感作用强度随分解时间延长呈现出逐渐增强后再减弱的变化趋势;莴笋可以通过调控自身的保护酶活性和渗透调节物质含量在一定程度上缓解化感物质伤害,对核桃凋落叶的化感作用有较强的耐受能力,生产中可以在核桃林下进行间作或者套作莴笋。     

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

Root or secondary leaf segments from maize ( Zea mays L. cv. Ganga safed-2) seedlings were incubated with 9-amino acids and two amides separately, each at 5 m M for 24 h, to study their effects on glutamate dehydrogenase (GDH) activity. Most of the compounds tested inhibited the specific activity of NADH-GDH and increased that of NAD⁺-GDH in the roots in the presence as well as in the absence of ammonium. In the leaves, such effects were recorded only with a few amino acids. Total soluble protein in the root and leaf tissues increased with the supply of most of the amino compounds. The effect of glutamate on enzyme activity and protein was concentration dependent in both tissues. When the enzyme extracts from root or leaf tissues were incubated with some of the amino acids, NADH-GDH declined while NAD⁺-GDH increased in most cases. The inhibition of NADH-GDH increased with increasing concentration of cysteine from 1 to 5 m M . The experiments demonstrate that most of the amino acids regulated GDH activity, possibly through some physicochemical modulation of the enzyme molecule.     

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants

Plants accumulate high levels of Gamma amino butyric acid (GABA) in response to different environmental stresses and GABA metabolism has different functions such as osmotic and pH regulation, bypass of tricarboxylic acid cycle, and C:N balance. The cytoplasmic male sterile (CMS) II mutant of Nicotiana sylvestris has a deletion in the mitochondrial gene nad7 which encodes the NAD7 subunit of complex I which causes increased leaf respiration, impaired photosynthesis, slower growth and increased amino acid levels. In this study we aimed to elucidate the role of GABA and GABA metabolism in different genotypes of the same plant system under salt stress (100mM NaCl) in short (24h) and long (7, 14 and 21 days) terms. We have investigated the differences in leaf fresh and dry weights, relative water content, photosynthetic efficiency (F(v)/F(m)), glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15) enzyme activities, GABA content and GAD gene expression profiles. GDH activity showed variations in CMSII and wild type (WT) plants in the first 24h. GAD gene expression profiles were in good agreement with the GAD enzyme activity levels in CMSII and WT plants after 24h. In long-term salinity, GAD activities increased in WT but, decreased in CMSII. GABA accumulation in WT and CMSII plants in short and long term was induced by salt stress. Variations in GDH and GAD activities in relation to GABA levels were discussed and GABA metabolism has been proposed to be involved in better performance of CMSII plants under long term salinity.     

Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L

Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Western blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues.     

不同生育期花生叶片蛋白质含量及氮代谢相关酶活性分析

以5个珍珠豆型花生(Arachis hypogaea Linn.)品种(系)‘汕E’(‘Shan E’)、‘汕G’(‘Shan G’)、‘TH’、‘TJ’和‘泉花7号’(‘Quanhua No.7’)为研究对象,分析了花针期、结荚期和饱果期花生叶片中可溶性蛋白质含量及硝酸还原酶(NR)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)活性的变化趋势,并比较了5个品种(系)荚果和秆产量的差异。结果表明:在3个生育期内,5个花生品种(系)叶片可溶性蛋白质含量和GDH活性的变化趋势基本一致,而NR和GS活性的变化趋势则有差异。其中,可溶性蛋白质含量均呈"低—高—低"的变化趋势,在结荚期最高;GDH活性均逐渐升高,至饱果期达最高;‘泉花7号’叶片NR活性呈"高—低—高"的变化趋势,而其他4个品种(系)叶片NR活性均逐渐降低;‘汕E’、‘TJ’和‘泉花7号’叶片GS活性呈逐渐降低趋势,而‘汕G’和‘TH’叶片GS活性呈"低—高—低"的变化趋势。总体上看,5个品种(系)中,‘汕G’和‘泉花7号’叶片的可溶性蛋白质含量及NR和GDH活性、‘汕E’叶片的NR和GS活性以及‘TH’叶片的GDH活性均较高。5个品种(系)的2个产量指标(单株荚果鲜质量和单株秆鲜质量)均有明显差异,总体上看,‘汕G’、‘泉花7号’和‘TH’的2个产量指标均较高,而‘汕E’和‘TJ’的2个产量指标均较低。综合分析结果显示:‘汕G’和‘泉花7号’叶片可溶性蛋白质含量及NR和GDH活性均相对较高,其荚果和秆产量也均较高,表明花生荚果和秆产量与不同生育期叶片氮代谢水平有一定关系。     

Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism

The subcellular localisation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in grapevine (Vitis vinifera L.) leaves and flowers was investigated using immunogold-labelling experiments. In mature leaf tissue or fully developed flowers, GS was visualised both in the cytosol and in the chloroplasts, a high proportion of the protein being present in the phloem companion cells. GDH was preferentially located in the mitochondria of the phloem companion cells in both leaves and flowers. This observation suggests that, in conjunction with GS, GDH plays a major role in controlling the translocation of organic carbon and nitrogen metabolites in both vegetative and reproductive organs. Significant amounts of GDH protein were also visualised in multivesicular bodies within the flower receptacle. Although the function of such organelles is still unknown, its is possible that the presence of GDH in such cellular structures is important for the recycling of carbon and nitrogen molecules in senescing tissues in which the enzyme is generally induced.     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.     

From pancreatic islets to central nervous system, the importance of glutamate dehydrogenase for the control of energy homeostasis

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme linking the Krebs cycle to the multifunctional amino acid glutamate. Thereby, GDH plays a pivotal role between carbohydrate and protein metabolisms, controlling production and consumption of the messenger molecule glutamate in neuroendocrine cells. GDH activity is under the control of several regulators, conferring to this enzyme energy-sensor property. Indeed, GDH directly depends on the provision of the co-factor NADH/NAD⁺, rendering the enzyme sensitive to the redox status of the cell. Moreover, GDH is allosterically regulated by GTP and ADP. GDH is also regulated by ADP-ribosylation, mediated by a member of the energy-sensor family sirtuins, namely SIRT4. In the brain, GDH ensures the cycling of the neurotransmitter glutamate between neurons and astrocytes. GDH also controls ammonia metabolism and detoxification, mainly in the liver and kidney. In pancreatic β-cells, the importance of GDH as a key enzyme in the regulation of insulin secretion is now well established. Inhibition of GDH activity decreases insulin release, while activating mutations are associated with a hyperinsulinism syndrome. Although GDH enzyme catalyzes the same reaction in every tissue, its function regarding metabolic homeostasis varies greatly according to specific organs. In this review, we will discuss specificities of GDH regulation in neuroendocrine cells, in particular pancreatic islets and central nervous system.     

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase,and its spatial and temporal changes during leaf development

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.     

Physiology of maize II: Identification of physiological markers representative of the nitrogen status of maize (Zea mays) leaves during grain filling

To illustrate the development of the source-to-sink transition in maize leaves during the grain-filling period, an integrated physiological-agronomic approach is presented in this study. The evolution of physiological markers such as total leaf nitrogen (N), chlorophyll, soluble protein, amino acid and ammonium contents was monitored from silking to a period close to maturity in different leaf stages of three maize genotypes grown at high and low levels of N fertilization. In addition, the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH), two enzymes known to play a direct or an indirect role during leaf N remobilization, were measured. In the three genotypes examined, we found that a general decrease of most metabolic and enzyme markers occurred during leaf ageing and that this decrease was enhanced when plants were N starved. In contrast, such variations were not observed between different sections of a single leaf even at an advanced stage of leaf senescence. We found that there is a strong correlation between total N, chlorophyll, soluble protein and GS activity, which is not dependent upon the N fertilization level, which indicates the N status of the plant, either in a single leaf or during ageing. In contrast, ammonium, amino acids and GDH activity were not subject to such variations, thus suggesting that they are indicators of the metabolic activity of the whole plant in response to the level of N fertilization. The use of these markers to predict the N status of maize as a function of both plant development and N availability is discussed.     

Induction of a specific isoenzyme of glutamate dehydrogenase during isolation and the first 48 h of culture of Brassica napus leaf protoplasts

The specific activity of NADH‐glutamate dehydrogenase (GDH, EC 1.4.1.2) in leaf protoplasts ( Brassica napus L. cv. Bronowski) was initially low and progressively increased during culture in Murashige and Skoog (MS) medium and MS (−NH₄) (ammonium nitrate‐free MS) medium in the dark. Native polyacrylamide gel electrophoresis (PAGE) and tetrazolium staining revealed that the high specific activity of NAD‐GDH (deamination) in leaves correlated with the cathodal isoenzymes, and the high specific activity of NADH‐GDH (amination) in leaf protoplasts to the anodal ones. Changes in isoenzyme pattern were correlated with an increase in the specific activity of NADH‐GDH but not with the NADH‐GDH/NAD‐GDH ratio. The increase in NADH‐GDH (amination) activity of leaf protoplasts was correlated with the occurrence of the isoenzyme GDH7, which was not detected in leaves.