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Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.

Abstract A novel cDNA encoding glutamate dehydrogenase (GDH) from tobacco(Nicotiana tabacum ), named gdh1, was characterized.The gdh1 mRNA was detected in roots, stems and source/senescentleaves. In order to investigate diurnal regulation of gdh1 inleaves, the content in gdh1 mRNA was measured every 3 h overa 48 h period and compared to nia and gs2 mRNAlevels, encoding, respectively, nitrate reductase (NR) and chloroplasticglutamine synthetase (GS2). In source leaves, gdh1 mRNA levelsexhibit diurnal fluctuations. A 12 h shift was observedbetween the day–night rhythms of gdh1 and nia expression.Metabolite contents were also measured and a shift in the day–nightfluctuations of both glutamate (GLU) and γ -aminobutyricacid (GABA) was observed between sink and source leaves, whereasthe diurnal rhythm of α -ketoglutarate showed no change.A possible role of GDH in the shift of GLU and GABA contents isdiscussed. Leaf disc experiments showed that gdh1 expressionis enhanced in conditions of continuous darkness. This trend isinhibited by sucrose feeding. The opposite was observed for nia expression.An important outcome of this work is the reverse regulation of gdh1 and nia genes.A possible role of sugars and amino acids in the co-regulation of gdh1 and nia genesis suggested. [ABSTRACT FROM AUTHOR]