Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis.

Studies of enzymes involved in nitrogen assimilation in higher plants have an impact on both basic and applied plant research. First, basic research in this area should uncover the mechanisms by which plants regulate genes involved in a metabolic pathway. Second, because nitrogen is a rate-limiting element in plant growth (Hageman and Lambert, 1988), it may be possible to increase the yield or improve the quality of crop plants by the molecular or genetic manipulation of genes involved in nitrogen assimilation. Research on nitrogen assimilation into amino acids has been complicated by the fact that some of these reactions are catalyzed by multiple isoenzymes located in distinct subcellular compartments. With traditional biochemical approaches, it has been impossible to sort out the function of each isoenzyme in plant nitrogen metabolism. The discovery that genes for chloroplastic and cytosolic isoenzymes of glutamine synthetase (GS) are expressed in distinct cell types (Edwards et al., 1990; Carvalhoet al., 1992; Kamachi et al., 1992)suggeststhat traditional biochemical studies, which begin with tissue disruption, artificially mix isoenzymes that may not coexist in the same cell type in vivo. Thus, in vitro biochemical methods commonly used to define the rate-limiting enzyme in a pathway in unicellular microorganisms may lead to erroneous interpretations when employed to study plant metabolic pathways. An alternative way to define the in vivo function of a particular isoenzyme or to define a rate-limiting enzyme in a pathway is by mutant analysis, as shown by studies of Escherichia coli and yeast. Plant mutants defective in particular isoenzymes of GS or ferredoxin-dependent glutamate synthase (Fd-GOGAT) have been identified in screens for photorespiratory mutants in Arabidopsis and barley (Somerville and Ogren, 1980,1982; Wallsgrove et al., 1987). More recently, Arabidopsis mutants with alterations in the activity of additional enzymes of nitrogen assimilation have been identified using a screening method that does not depend on a growth phenotype (Schultz and Coruzzi, 1995). The in vivo role of the mutated isoenzyme