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.

Tomato (Lycopersicon esculentum) seedlings were grown in the presence of cadmium. After 1 week of Cd treatment, a sharp decline in biomass accumulation in the leaves and roots was observed, together with a decrease in the rate of photosynthetic activity due to both Rubisco and chlorophyll degradation and stomata closure. Cadmium induced a significant decrease in nitrate content and inhibition of the activities of nitrate reductase, nitrite reductase, glutamine synthetase (GS) and ferredoxin-glutamate synthase. An increase in NADH-glutamate synthase and NADH-glutamate dehydrogenase activity was observed in parallel. The accumulation of ammonium into the tissues of treated plants was accompanied by a loss of total protein and the accumulation of amino acids. Gln represented the major amino acid transported through xylem sap of Cd-treated and control plants. Cadmium treatment increased the total amino acid content in the phloem, maintaining Gln/Glu ratios. Western and Northern blot analysis of Cd-treated plants showed a decrease in chloroplastic GS protein and mRNA and an increase in cytosolic GS and glutamate dehydrogenase transcripts and proteins. An increase in asparagine synthetase mRNA was observed in roots, in parallel with a strong increase in asparagine. Taken together, these results suggest that the plant response to Cd stress involved newly induced enzymes dedicated to coordinated leaf nitrogen remobilization and root nitrogen storage.

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.

Nitrogen plays an essential role in the nutrient relationship between plants and pathogens. Some studies report that the nitrogen-mobilizing plant metabolism that occurs during abiotic and biotic stress could be a 'slash-and-burn' defence strategy. In order to study nitrogen recycling and mobilization in host plants during pathogen attack and invasion, the Colletotrichum lindemuthianum/Phaseolus vulgaris interaction was used as a model. C. lindemuthianum is a hemibiotroph that causes anthracnose disease on P. vulgaris. Non-pathogenic mutants and the pathogenic wild-type strain were used to compare their effects on plant metabolism. The deleterious effects of infection were monitored by measuring changes in chlorophyll, protein, and amino acid concentrations. It was shown that amino acid composition changed depending on the plant-fungus interaction and that glutamine accumulated mainly in the leaves infected by the pathogenic strain. Glutamine accumulation correlated with the accumulation of cytosolic glutamine synthetase (GS1 alpha) mRNA. The most striking result was that the GS1 alpha gene was induced in all the fungus-infected leaves, independent of the strain used for inoculation, and that GS1 alpha expression paralleled the PAL3 and CHS defence gene expression. It is concluded that a role of GS1 alpha in plant defence has to be considered.

Summary• In wheat the period of grain filling is characterized by a transition for all vegetative organs from sink to source status.• To study this transition, the progression of physiological markers and enzyme activities representative of nitrogen metabolism was monitored from the vegetative stage to maturity in different leaf stages and stem sections of two wheat ( Triticum aestivum ) cultivars grown at high and low levels of N fertilization.• In the two cultivars examined, we found a general decrease of the metabolic and enzyme markers occurred during leaf ageing, and that this decrease was enhanced when plants were N-limited. Both correlation studies and principal components analysis (PCA) showed that there was a strong relationship among total N, chlorophyll, soluble protein, ammonium, amino acids and glutamine synthetase (GS) activity.• The use of a marker such as GS activity to predict the N status of wheat, as a function of both plant development and N availability, is discussed with the aim of selecting wheat genotypes with better N-use efficiency.