Glutamine Synthetase in the Phloem Plays a Major Role in Controlling Proline Production

Abstract

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...

Removal of Feedback Inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress

“…Recent data have shown that expression of antisense P5CS inhibits Pro production and makes plants hypersensitive to osmotic stress (Najo et al, 1999). This is also consistent with a study on antisense Gln synthetase that reduces the Pro level and renders transgenic plants more sensitive to salt treatment (Brugiere et al, 1999). The present study also suggests that the role of Pro as a free radical scavenger may be more important in overcoming stress than in acting as a simple osmolyte.…”

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

“…1 plants due to a preferential accumulation of Pro and Ser in response to the excess of ammonium (Brugière et al, 1999). An accumulation of Gln was observed in the phloem sap of Fd-GOGAT antisense plants as the result of a lower enzyme activity in these transgenic plants (FerrarioMéry et al, 2000).…”

Unraveling the involvement of ABA in the water deficit-induced modulation of nitrogen metabolism inMedicago truncatulaseedlings

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