Primary N-assimilation into Amino Acids in Arabidopsis

Abstract

The assimilation of inorganic nitrogen into organic form has marked effects on plant productivity, biomass, and crop yield (Hageman and Lambert, 1988; Lawlor et al., 1989). As such, a tremendous amount of biochemical and physiological studies have been performed on nitrogen assimilatory enzymes from a variety of plant species, especially crops and legumes. Summaries of such biochemical studies can be found in several comprehensive reviews (Miflin and Lea, 1976, 1977; Miflin, 1980; Miflin and Lea, 1980; Miflin and Lea, 1980, 1982; Miflin and Cullimore, 1984; Poulton et al., 1989). Although these biochemical studies have provided a solid groundwork, a complete picture of the N-assimilation process and its regulation in a single plant is still lacking for a number of reasons. The existence of multiple isoenzymes for each step in nitrogen assimilation has confounded biochemical purification as have the technical difficulties of isoenzyme purification and organelle isolation. As the mechanisms controlling intraand intercellular transport of inorganic and organic nitrogen in plants are still under investigation, it is impossible to predict the in vivo function of nitrogen assimilatory enzymes localized in distinct cells or subcellular compartments based on in vitro biochemistry. Plant mutants have provided a mechanism to dissect the process of N-assimilation in vivo (Lea and Forde, 1994) (Lam et al., 1996). The aim of this chapter is to specifically highlight and update examples where molecular, genetic, and biochemical analyses of Nassimilation genes and mutants in Arabidopsis have begun to define the in vivo roles of individual isoenzymes in plant nitrogen assimilation and to uncover the mechanisms regulating this process. In all higher plants, inorganic nitrogen is first reduced to ammonia prior to its incorporation into organic form (Lea, 1993). For a review of the regulation of Nitrate assimilation and reduction in Arabidopsis see (Crawford and Forde, 2002). Ammonia is assimilated into organic form as glutamine and glutamate, which serve as the nitrogen donors in the biosynthesis of essentially all amino acids, nucleic acids, and other nitrogen-containing compounds such as chlorophyll (Lea, 1993). The individual isoenzymes of GS, GOGAT or GDH have been proposed to play roles in three major ammonium assimilation processes: (i) primary nitrogen assimilation; (ii) reassimilation of photorespiratory ammonia; and (iii) reassimilation of recycled nitrogen. For a review of these processes see (Stewart et al., 1980; Lea, 1993). Glutamine and glutamate can then be used to form aspartate and asparagine, and these four amino acids are used to translocate organic nitrogen from sources to sinks (Lea and Miflin, 1980; Peoples and Gifford, 1993). The enzymes involved in the primary assimilation of ammonium into these four N-transport amino acids Glu/Gln and Asp/Asn are shown in Fig. 1 and include; glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT) and asparagine synthetase (AS). While most studies of nitrogen metabolism have previously been performed in legumes and crop species, HPLC analyses of Arabidopsis has demonstrated that these same four amino acids can account for 60-64% of the total free amino acids present in Arabidopsis leaves and are also transported in the vascular tissues (Fig. 2) (Schultz, 1994; Lam et al., 1995). Thus, Arabidopsis appears to be a suitable model plant for the study of nitrogen assimilation into primary amino acids and the results should have impact on less genetically tractable crop plants. It is noteworthy that each enzyme for GS, GOGAT, AspAT and AS exists as multiple isoenzymes, encoded by multiple genes, even in Arabidopsis (Fig. 1). Table 1 lists the enzymes, the genes, and available mutants for these N-assimilatory enzymes in Arabidopsis. Below, we review how studies of the genes and mutants for these isoenzymes in Arabidopsis have helped to illuminate the role of specific genes in this Nassimilation pathway.