Role of the Ferredoxin- and NADH-Dependent GOGAT Isozymes in Plant Glutamate Biosynthesis

Since the discovery of the GS/GOGAT-catalyzed pathway for glutamate biosynthesis, extensive studies have unequivocally shown that this pathway is the main route of soil nitrogen assimilation as well as photorespiratory ammonium ion reassimilation in plants (see for reviews Hirel and Lea, 2001; Ireland and Lea, 1999; Lam et al., 1995; Lea and Ireland, 1999; Miflin and Habash, 2002; Stitt et al., 2002). Plants possess two types of ferredoxin- and NADPH-dependent GOGAT isozymes (Fd-GOGAT and NADPH-GOGAT). Genes encoding Fd- and NADHGOGAT isozymes and their regulation of expression have been extensively discussed in other reviews (Hirel and Lea, 2001; Ireland and Lea, 1999; Lam et al., 1995; Lea and Ireland, 1999; Miflin and Habash, 2002; Stitt et al., 2002). The Fd-GOGAT isozymes (two isoforms encoded by two different genes in Arabidopsis) constitute the majority of the GOGAT activity in plants, accounting for over 90% and ~70% of total GOGAT activity in Arabidopsis leaves and roots, respectively (Ireland and Lea, 1999; Somerville and Ogren, 1980; Suzuki et al., 2001). The significant role of Fd-GOGAT in ammonium ion assimilation, particularly of photorespiratory ammonium ion, was demonstrated by a number of genetic and molecular approaches. Many plant mutants, defective in growth under photorespiratory conditions, were based on mutations in genes encoding Fd-GOGAT (Ireland and Lea, 1999; Somerville and Ogren, 1980). Notably, although Arabidopsis possesses two Fd-GOGAT isozymes, mutations in one are sufficient to cause sensitivity to enhanced photorespiration (Somerville and Ogren, 1980). This nonredundant function was explained by two contrasting patterns of expression of the genes encoding these isozymes (Coschigano et al., 1998). The significant role of Fd-GOGAT in reassimilating photorespiratory ammonium ion was also demonstrated in transgenic tobacco plants with reduced Fd-GOGAT due to antisense expression (Ferrario-Mery et al., 2000). When transferred from CO₂-rich conditions to ambient air to enhance photorespiration, the plants accumulated significantly higher levels of ammonium ion as well as the two GOGAT substrates, glutamine and α-ketoglutarate, than control plants (Ferrario-Mery et al., 2000). This suggests that glutamine and α-ketoglutarate were less efficiently converted into glutamate in the transgenic plants, causing a less-efficient incorporation of photorespiratory ammonium ion into glutamine. In addition, the reduced Fd-GOGAT expression was also associated with altered levels of leaf amino acids, implying that a number of amino acid biosynthesis pathways are affected and may be regulated in response to changes in ammonium ion and/or glutamine levels (Ferrario-Mery et al., 2000).

Constituting a minor proportion of the total plant GOGAT activity, NADPHGOGAT received less attention than the Fd-GOGAT. However, several lines of evidence indicate that, despite being a minor isozyme, the NADPH-GOGAT activity in plants is not redundant. NADPH-GOGAT is unable to compensate for Fd-GOGAT shortage, implying a distinct metabolic function (Ireland and Lea, 1999; Somerville and Ogren, 1980). Moreover, plant genes encoding NADPH-GOGAT generally exhibit contrasting expression patterns compared to Fd-GOGAT genes. While Fd-GOGAT is abundantly produced in photosynthetic leaves, NADPH-GOGAT is produced in nonphotosynthetic organs, such as roots, senescing leaves, and nodules formed in legume roots (see Lancien et al., 2002 and references therein). This suggests that in contrast to the major function of Fd-GOGAT in reassimilation of photorespiratory ammonium ion, NADPH-GOGAT functions mainly in primary nitrogen assimilation and in nitrogen transport from source to sink.

To study the function of NADH-GOGAT, its activity was reduced by up to 87% in transgenic alfalfa plants, using antisense constructs controlled either by an AAT-2 promoter with enhanced expression in nodules, or by a nodule-specific leghemoglobin promoter (Cordoba et al., 2003; Schoenbeck et al., 2000). The transgenic plants were chlorotic and exhibited altered symbiotic phenotypes compared to controls. In addition, nodule amino acids and amides levels were lower, while sucrose levels were higher in the transgenic plants than in control plants, implying thatNADPH-GOGAT represents a major rate-limiting enzyme for the incorporation of ammonium ion and sugars into amino acids in nodules.

The functional role of NADPH-GOGAT was also studied in an Arabidopsis T-DNA insertion within the single Arabidopsis gene encoding this enzyme that abolished expression of the gene (Lancien et al., 2002). In contrast to plants with reduced levels of Fd-GOGAT, which exhibited metabolic and growth defects under conditions of enhanced photorespiration (see above), the Arabidopsis T-DNA mutant lacking NADPH-GOGAT exhibited metabolic and growth defects when photorespiration was repressed. Based on these results, NADPH-GOGAT and Fd-GOGAT appear to play nonredundant roles in the assimilation of nonphotorespiratory ammonium (derived from soil nitrogen or nitrogen fixation) and photorespiratory ammonium into glutamate, respectively.

The metabolic function of NADPH-GOGAT was also studied by constitutive expression of the alfalfa enzyme in transgenic tobacco plants (Chichkova et al., 2001). Shoots of the transgenic plants contained higher total carbon and nitrogen than wild-type plants when administered either nitrate or ammonium ion as sole nitrogen sources. In addition, the transgenic plants contained higher dry weight than control plants upon entering flowering. These results are consistent with the rate-limiting role of NADPH-GOGAT in nitrogen assimilation and also with the importance of nitrogen assimilation for plant growth (Chichkova et al., 2001).