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

摘要

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. nnnn--------------------------------------------------------------------------------nPlants utilize nitrate, ammonium, and dinitrogen (N2) molecules as external nitrogen sources. Ammonium is the final form of inorganic nitrogen prior to the synthesis of organic nitrogen compounds. Ammonium is also produced via internal metabolic reactions, including photorespiration, hydrolysis of nitrogen carrying and storage molecules, and amino acid conversion (Ireland and Lea, 1999). In nonleguminous C3 plants, such as tobacco (Nicotiana tabacum), the photorespiratory ammonium production by the oxidative decarboxylation of Gly exceeds by about 10-fold the primary nitrate reduction in the vegetative leaves. In the senescing leaves, a large amount of ammonium is produced as a result of protein hydrolysis (Hörteinsteiner and Feller, 2002). Therefore, it is essential that toxic ammonium be immediately reassimilated into organic molecules for nitrogen cycling. Ammonium is assimilated into the Gln amide group, which is then transferred to the position of 2-oxoglutarate, yielding two molecules of Glu by the concerted reaction of Gln 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). Nitrogen is then incorporated into Asp, Ala, Asn, and other amides and amino acids. Gln-dependent Asn synthetase (AS; EC 6.3.5.4) provides Asn, which serves as a nitrogen carrier together with Gln and Glu. nNumerous studies have been carried out to define the roles of enzymes in nitrogen assimilation and remobilization, tightly interrelated processes during plant growth and development (Miflin and Habash, 2002). It was proposed that ammonium might be directly incorporated into Glu by amination of 2-oxoglutarate via mitochondrial Glu dehydrogenase (NADH-GDH; EC 1.4.1.2) and subsequently into Gln by cytosolic GS1 under particular physiological conditions. Studies on source-sink relations have shown that GDH is induced in old leaves when nitrogen remobilization is maximal (Srivastava and Singh, 1987; Masclaux et al., 2000). This led to the proposal that the physiological role of GDH is to synthesize Glu for translocation in senescing leaves (for review, see Miflin and Habash, 2002). However, there is no evidence to discern a redundant or indispensable role of GDH and GOGAT for Glu synthesis and nitrogen remobilization. In addition, GDH catalyzes the reversible oxidative deamination of Glu to supply 2-oxoglutarate and ammonium (Aubert et al., 2001). nnTo better understand the role of GDH and GOGAT in Glu metabolism in the coordinated reaction with GS, we studied the kinetics of in vivo turnover of [15N]Glu fed to leaf discs during aging of tobacco plants. The time course of [15N]ammonium assimilation into the amino acids was then determined in vivo in young and old leaves. To understand the cellular compartmentation of Glu synthesis and amino acid translocation, we investigated the tissue-specific expression and the cellular localization of Fd-GOGAT in tobacco plants transformed by a fusion between the promoter of the Arabidopsis (Arabidopsis thaliana) Fd-GOGAT gene (GLU1) and a reporter gene.