To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA ( Gln1-5 ) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A 15 NH 4 ؉ -labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress. INTRODUCTIONIn higher plants, glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen into organic forms (O'Neal and Joy, 1973; Lea and Miflin, 1974). GS catalyzes the ATP-dependent condensation of ammonium with glutamate to yield glutamine, which then provides nitrogen groups, either directly or via glutamate for the biosynthesis of all nitrogenous compounds in the plant (Lea et al., 1989).Two groups of GS isoenzymes, plastidic (GS2) and cytosolic (GS1), have been identified in higher plants (McNally et al., 1983;Hirel et al., 1993). In the majority of higher plants, GS2 is predominant in most chlorophyllous tissue and is localized in the chloroplast stroma (Botella et al., 1988; Brangeon et al., 1989; Dubois et al., 1996). In a limited number of species, such as legumes, GS2 was shown to be present also in plastids of either roots (Vézina and Langlois, 1989) or root nodules (Lightfoot et al., 1988; Brangeon et al., 1989), representing ف 5% of total GS protein content. More recently, isolation of cDNAs encoding GS2 allowed the demonstration that in most plant species, this isoenzyme is encoded by a single nuclear gene per haploid genome (Lightfoot et al., 1988;Becker et al., 1992). Using photorespiratory mutants of barley that were deficient in leaf GS2 activity, Blackwell et al. (1987) and Wallsgrove et al. (1987) demonstrated that GS2 is indispensable for the reassimilation of ammonium released from the photorespiratory nitrogen cycle. Interestingly, these mutants possess levels of GS1 protein and activity comparable to the wild ty...
Glutathione (GSH), c-glutamylcysteine (c-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula´P. alba) and poplars expressing Escherichia coli genes for c-glutamylcysteine synthetase (c-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing c-ECS, foliar c-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of c-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and c-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate c-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, c-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, c-EC was negligible in the light but increased in the dark. Cysteine content was little aected by light in any of the poplar types. No light-dependent changes in the extractable activities of c-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing c-ECS at elevated CO 2 or low O 2 largely abolished the inverse light-dependent changes in c-EC and GSH. Low O 2 did not aect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for c-EC synthesis is augmented.
Mitochondrial NAD-dependent (IDH) and cytosolic NADPdependent isocitrate dehydrogenases have been considered as candidates for the production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The increase in IDH transcripts in leaf and root tissues, induced by nitrate or NH 4 ؉ resupply to short-term N-starved tobacco (Nicotiana tabacum) plants, suggested that this enzyme could play such a role. The leaf and root steady-state mRNA levels of citrate synthase, acotinase, IDH, and glutamine synthetase were found to respond similarly to nitrate, whereas those for cytosolic NADP-dependent isocitrate dehydrogenase and fumarase responded differently. This apparent coordination occurred only at the mRNA level, since activity and protein levels of certain corresponding enzymes were not altered. Roots and leaves were not affected to the same extent either by N starvation or nitrate addition, the roots showing smaller changes in N metabolite levels. After nitrate resupply, these organs showed different response kinetics with respect to mRNA and N metabolite levels, suggesting that under such conditions nitrate assimilation was preferentially carried out in the roots. The differential effects appeared to reflect the C/N status after N starvation, the response kinetics being associated with the nitrate assimilatory capacity of each organ, signaled either by nitrate status or by metabolite(s) associated with its metabolism.
The terminal step of glutathione (GSH) synthesis is the condensation of γ‐glutamyl‐cysteine (γ‐EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ‐EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ‐EC and GSH was studied in excised leaves and leaf discs from untransformed poplar (Populus tremula×P. alba) and poplar overexpressing γ‐glutamylcysteine synthetase (γ‐ECS; EC 6.3.2.2). Poplars strongly overexpressing γ‐ECS (ggs28) had enhanced levels of γ‐EC and GSH compared to untransformed poplars. The relationship between γ‐EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50‐fold higher than γ‐EC. On darkening, γ‐EC accumulated markedly and GSH declined, so that the GSH:γ‐EC ratio was close to 1. These dark‐induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ‐EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ‐EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ‐EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.
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