Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cD NA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn super oxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium- mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GSI mRNA and GS activity in midribs, stems, and roots were obtai ned. Immunogold labeling experiments allowed us to verify that the GS prote in content was markedly decreased in the phloem companion cells of transfor med plants. Moreover, a general decrease in proline content in the transgen ic plants in comparison with wild-type tobacco was observed when plants wer e forced to assimilate large amounts of ammonium. In contrast, no major cha nges in the concentration of amino acids used for nitrogen transport were a pparent. A (NH4+)-N-15-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was dire ctly related to glutamine availability. After 2 weeks of salt treatment, th e transgenic plants had a pronounced stress phenotype, consisting of wiltin g and bleaching in the older leaves. We conclude that GS in the phloem play s a major role in regulating proline production consistent with the functio n of proline as a nitrogen source and as a key metabolite synthesized in re sponse to water stress.