Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions.

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.

An integrated "omics" approach to the characterization of maize (Zea mays L.) mutants deficient in the expression of two genes encoding cytosolic glutamine synthetase.

BACKGROUND: To identify the key elements controlling grain production in maize, it is essential to have an integrated view of the responses to alterations in the main steps of nitrogen assimilation by modification of gene expression. Two maize mutant lines (gln1.3 and gln1.4), deficient in two genes encoding cytosolic glutamine synthetase, a key enzyme involved in nitrogen assimilation, were previously characterized by a reduction of kernel size in the gln1.4 mutant and by a reduction of kernel number in the gln1.3 mutant. In this work, the differences in leaf gene transcripts, proteins and metabolite accumulation in gln1.3 and gln1.4 mutants were studied at two key stages of plant development, in order to identify putative candidate genes, proteins and metabolic pathways contributing on one hand to the control of plant development and on the other to grain production. RESULTS: The most interesting finding in this study is that a number of key plant processes were altered in the gln1.3 and gln1.4 mutants, including a number of major biological processes such as carbon metabolism and transport, cell wall metabolism, and several metabolic pathways and stress responsive and regulatory elements. We also found that the two mutants share common or specific characteristics across at least two or even three of the "omics" considered at the vegetative stage of plant development, or during the grain filling period. CONCLUSIONS: This is the first comprehensive molecular and physiological characterization of two cytosolic glutamine synthetase maize mutants using a combined transcriptomic, proteomic and metabolomic approach. We find that the integration of the three "omics" procedures is not straight forward, since developmental and mutant-specific levels of regulation seem to occur from gene expression to metabolite accumulation. However, their potential use is discussed with a view to improving our understanding of nitrogen assimilation and partitioning and its impact on grain production.

Resolving the role of plant glutamate dehydrogenase: II. Physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously.

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and ß-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.

The Key Role of Glutamate Dehydrogenase 2 (GDH2) in the Control of Kernel Production in Maize (Zea mays L.).

The agronomic potential of glutamate dehydrogenase 2 (GDH2) in maize kernel production was investigated by examining the impact of a mutation on the corresponding gene. Mu-insertion homozygous and heterozygous mutant lines lacking GDH2 activity were isolated and characterized at the biochemical, physiological and agronomic levels. In comparison to the wild type and to the homozygous ghd2 mutants, the heterozygous gdh2 mutant plants were characterized by a decrease in the root amino acid content, whereas in the leaves an increase of a number of phenolic compounds was observed. On average, a 30 to 40% increase in kernel yield was obtained only in the heterozygous gdh2 mutant lines when plants were grown in the field over two years. The importance of GDH2 in the control of plant productivity is discussed in relation to the physiological impact of the mutation on amino acid content, with primary carbon metabolism mostly occurring in the roots and secondary metabolism occurring in the leaves.

Metabolic profiling of maize mutants deficient for two glutamine synthetase isoenzymes using 1H-NMR-based metabolomics.

INTRODUCTION: Maize mutants deficient for the expression of two genes encoding cytosolic glutamine synthetase (GS) isoenzymes GS1.3 and GS1.4 displayed reduced kernel number and kernel size, respectively, the effect of the mutation being cumulative in the double mutant. However, at maturity, shoot biomass production was not modified in all the mutants, indicating that the reaction catalysed by the enzyme is specifically involved in the control of grain yield. OBJECTIVE: To examine the physiological impact of the GS mutations on the leaf metabolic profile during the kernel filling period, during which nitrogen is remobilized from the shoots to be further exported to the kernels. METHODOLOGY: An (1)H-NMR spectroscopy metabolomic was applied to the investigation of metabolic change of the gln1.3, gln1.4 and gln1.3/1.4 double mutant. RESULTS: In the three GS mutants, an increase in the amount of several N-containing metabolites such as asparagine, alanine, threonine and phophatidylcholine was observed whatever the level of nitrogen fertilisation. In addition, we found an accumulation of phenylalanine and tyrosine, two metabolites involved the primary steps of the phenylpropanoid pathway. CONCLUSION: Changes in the metabolic profile of the GS mutants suggest that, when cytosolic GS activity is strongly reduced, either alternative metabolic pathways participate in the reassimilation of ammonium released during leaf protein remobilization or that premature leaf senescence is induced when kernel set and kernel filling are affected. The accumulation of phenylalanine and tyrosine in the mutant plants indicates that lignin biosynthesis is altered, thus possibly affecting ear development.

Resolving the role of plant glutamate dehydrogenase. I. In vivo real time nuclear magnetic resonance spectroscopy experiments.

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

New insights towards the function of glutamate dehydrogenase revealed during source-sink transition of tobacco (Nicotiana tabacum) plants grown under different nitrogen regimes.

The metabolic, biochemical and molecular events occurring in the different leaf stages along the main axis of tobacco (Nicotiana tabacum) plants grown either on a nitrogen-rich medium, on a medium containing ammonium as sole nitrogen source or on a nitrogen-depleted medium, are presented. This study shows that the highest induction of cytosolic glutamine synthetase (GS1) protein and transcript occurs when nitrogen remobilization is maximal as the result of nitrogen starvation, whereas both glutamate dehydrogenase (GDH) transcript and activity remain at a very low level. In contrast, GDH is highly induced when plants are grown on ammonium as sole nitrogen source, a physiological situation during which leaf protein nitrogen remobilization is limited. It is therefore concluded that GDH does not play a direct role during the process of nitrogen remobilization but is rather induced following a built up of ammonium provided externally or released as the result of protein hydrolysis during natural leaf senescence.

Further insights into the isoenzyme composition and activity of glutamate dehydrogenase in Arabidopsis thaliana.

Following the discovery that in Arabidopsis, a third isoenzyme of NADH-dependent glutamate dehydrogenase (GDH) is expressed in the mitochondria of the root companion cells, we have re-examined the GDH isoenzyme composition. By analyzing the NADH-GDH isoenzyme composition of single, double and triple mutants deficient in the expression of the three genes encoding the enzyme, we have found that the α, ß and γ polypeptides that comprise the enzyme can be assembled into a complex combination of heterohexamers in roots. Moreover, we observed that when one or two of the three root isoenzymes were missing from the mutants, the remaining isoenzymes compensated for this deficiency. The significance of such complexity is discussed in relation to the metabolic and signaling function of the NADH-GDH enzyme. Although it has been shown that a fourth gene encoding a NADPH-dependent enzyme is present in Arabidopsis, we were not able to detect corresponding enzyme activity, even in the triple mutant totally lacking NADH-GDH activity.

Glutamate dehydrogenase isoenzyme 3 (GDH3) of Arabidopsis thaliana is regulated by a combined effect of nitrogen and cytokinin.

In higher plants, NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant enzyme that exists in different isoenzymic forms. In Arabidopsis thaliana, three genes (Gdh1, Gdh2 and Gdh3) encode three different GDH subunits (ß, α and γ) that randomly associate to form a complex array of homo- and heterohexamers. The modification of the GDH isoenzyme pattern and its regulation was studied during the development of A. thaliana in the gdh1, gdh2 single mutants and the gdh1-2 double mutant, with particular emphasis on GDH3. Investigations showed that the GDH3 isoenzyme could not be detected in closely related Arabidopsis species. The induction and regulation of GDH3 activity in the leaves and roots was investigated following nitrogen deprivation in the presence or absence of sucrose or kinetin. These experiments indicate that GDH3 is likely to play an important role during senescence and nutrient remobilization.

Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.).

We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle, glumes and roots. Expression of GS genes in leaves was developmentally regulated, with both GS2 and GS1 assimilating or recycling ammonia in leaves during the period of grain development and filling. During leaf senescence the cytosolic isozymes, GS1 and GSr, were the predominant forms, suggesting major roles in assimilating ammonia during the critical phases of remobilisation of nitrogen to the grain. A preliminary analysis of three different wheat genotypes showed that the ratio of leaf GS2 protein to GS1 protein was variable. Use of this genetic variation should inform future efforts to modulate this enzyme for pre-breeding efforts to improve nitrogen use in wheat.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production.

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene (GS15) promoter.

In soybean (Glycine max L.) ammonium provided externally or as the result of symbiotic nitrogen fixation stimulates the transcription of GS15, a gene encoding cytosolic glutamine synthetase. Strong constitutive positive expression (SCPE), silencer-like and organ-specific elements, located respectively in the distal, the central and the proximal region of the promoter are required to control the ammonium responsiveness of the gene expression [Tercé-Laforgue et al. (1999) Plant Mol. Biol. 39: 551]. It was hypothesized that the correct spatial conformation of the promoter permitted the cooperative action of these three cis-acting elements. Further investigations were therefore required to ascertain this hypothesis. A nodule nuclear protein, binding to a 66 bp AT-rich DNA fragment containing a 13 bp AT-rich repeated sequence (AT-1) and located just downstream of the SCPE element, was identified using a gel retardation assay. A cDNA clone likely to code for this protein was isolated using the yeast one-hybrid system. It encodes a novel DNA binding protein (AT-1SNBP) similar to HMG A proteins but exhibiting a higher molecular weight. AT-1SNBP appears to be encoded by a single gene that is expressed in roots, root nodules and leaves of soybean. Since two other 13 bp AT-rich repeated sequences (AT-2 and AT-3) were localized in the organ-specific element, we have quantified the binding affinity of AT-1SNBP to these sequences. We demonstrate that AT-1SNBP binds differentially to DNA fragments containing AT-1, AT-2 and AT-3 and that its binding affinity depends on the presence of adjacent sequences. This result suggests that AT-1SNBP may be an architectural protein involved in maintaining the spatial conformation of the GS15 promoter, thus facilitating the interaction between the distal and proximal regulatory elements.

Glutamate dehydrogenase of tobacco is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration.

Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of companion cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Terce-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565-576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of companion cells. Taken together, our results suggest that the enzyme plays a dual role in companion cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.