Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using 15Nammonium and 2-15NGlu tracers. Regardless of leaf age, 15Nammonium 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 15Nammonium and ammonium accumulation patterns support the role of GDH in the deamination of 2-15NGlu to provide 2-oxoglutarate and 15Nammonium. In the dark, excess 15Nammonium 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.