The metabolic, biochemical and molecular events occurring during tobacco (Nicotiana tabacum) leaf ageing are presented, with a particular emphasis on nitrogen metabolism. An integrated model describing the source/sink relationship existing between leaves of different developmental stages along the main plant axis is proposed. The results of our study show that a tobacco plant can be divided into two main sections with regards to sink/source relationships. Sink-to-source transition occurs at a particular leaf stage in which a breakpoint corresponding to an accumulation of carbohydrates and a depletion of both organic and inorganic nitrogen is observed. The sink/source transition is also marked by the appearence of endoproteolytic activities and the induction of both cytosolic glutamine synthetase and NAD(H)-dependent glutamate dehydrogenase transcripts, proteins and activities. The role of the newly induced enzymes and the nature of the potential metabolic and developmental signals involved in the regulation of their expression during leaf senescence are discussed.

Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3- reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

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.

C and N metabolism are co-regulated in higher plants. Energy and C skeletons required for N assimilation are provided either directly or indirectly (via Suc) by photosynthesis. A high rate of CO 2 assimilation favors a high rate of N assimilation and vice versa (Ferrario et al., 1995). Molecular and metabolic controls are implicated in the C to N interaction, involving reciprocal regulation between the pathways of C and N assimilation (Champigny and Foyer,

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/senescent leaves. In order to investigate diurnal regulation of gdh1 in leaves, the content in gdh1 mRNA was measured every 3 h over a 48 h period and compared to nia and gs2 mRNA levels, encoding, respectively, nitrate reductase (NR) and chloroplastic glutamine synthetase (GS2). In source leaves, gdh1 mRNA levels exhibit diurnal fluctuations. A 12 h shift was observed between the daynight rhythms of gdh1 and nia expression. Metabolite contents were also measured and a shift in the day-night fluctuations of both glutamate (GLU) and g g g g -aminobutyric acid (GABA) was observed between sink and source leaves, whereas the diurnal rhythm of a a a a -ketoglutarate showed no change. A possible role of GDH in the shift of GLU and GABA contents is discussed. Leaf disc experiments showed that gdh1 expression is enhanced in conditions of continuous darkness. This trend is inhibited 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 genes is suggested.