Characteristics of the enzymes involved in the assimilation of NO3− and NH4+, in particular the nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, glutamate decarboxylase, and asparagine synthetase, are described. The cellular organization of these enzymes in root and leaf tissues are assessed in view of recent research developments that utilize various tissue blotting techniques. Regulation of nitrate assimilation is analyzed at the physiological, biochemical, and molecular levels. Key words: nitrate, ammonium, assimilation, regulation.

NR5 activity is induced by nitrate in barley (2, 24) and other higher plants (5,7,8,13,14,26)

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The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both (8,17,21) for the induction of nitrate reductase. In addition they show that as root cells age the enzyme becomes less stable. MATERIALS AND METHODSThe seeds of Zea mays (Wf9 X 38-1 1) were surface sterilized by a brief treatment with Javex, a commercial bleach containing 6.0% NaOCl. They were planted under sterile conditions on nitrate-free 0.9% agar which contained one-tenth strength Hoagland solution and an additional supplement of molybdenum (0.02 ,ug/ml). The 26 C before transfer to nitrate induction media. At this time the primary root was approximately 5 cm long. Roots growing on the surface of the agar were selected. The nitrate induction solution contained 5 or 10 mm nitrate, levels which gave a good induction of nitrate reductase but which did not saturate the system either with respect to enzyme synthesis or accumulation of nitrate (Wallace and Oaks unpublished results). In addition, in long term experiments, these concentrations of nitrate did not inhibit growth of the primary root (Wallace and Oaks unpublished results). The inhibitor compounds tested were made up in solution just before use and the pH of the induction solutions was adjusted to 5.8. The experiments were conducted at 26 C in the dark. In all cases, intact seedlings were used and the primary root was sectioned only after the experimental treatment had been completed. The 0-to 10-mm and the 25-to 35-mm root sections were harvested at appropriate time intervals.Extraction Procedure. Root tips and mature root sections were routinely frozen in liquid nitrogen, weighed, and stored at -20 C overnight. They were extracted with four volumes relative to weight of 50 mm phosphate buffer at pH 7.5. The buffer contained 0.5 mm EDTA and 5 mm cysteine. For enzyme preparations used in the in vitro inactivation, root tip sections were extracted as above, whereas the mature sections were extracted with two volumes of buffer. This had no effect on the specific activity of the enzyme, but insured that the protein levels in the two extracts were similar. The extracts were centrifuged at 30,000g for 30 min.Assay Methods. The assay mixture for nitrate reductase was as follows: phosphate, pH 7.8, 90 ,moles; KNO,,20 ,umoles; NADH, 1

Addition of nitrogen leads to increased dry matter accumulation in vegetative plant parts and to increased final yields in cereal crops (Hageman and Lambert, 1988). The efficiency with which nitrogen is used varies with plant species and with environmental conditions. For example, plants that possess a C4 pattem of photosynthesis have, in addition to a superior method for trapping COz from the atmosphere, a greater nitrogen use efficiency (g dry matter gain per mg nitrogen utilized) than do C3 plants (Brown, 1978). Although there are many differences in the metabolism of C3 and C4 plants, the major difference between these two pattems of photosynthesis is the contribution of photorespiration to both carbon and nitrogen metabolism. When photorespiration is reduced in C3 plants either by increasing ambient levels of COz or reducing levels of 02, both the yield (vegetative dry matter) and nitrogen use efficiency are enhanced (Evans, 1989). As indicated in Table I, this effect is apparent in wheat, a C3 cereal, but not in maize, a C4 cereal (Hocking and Meyer, 1991).Factors that could be altered by reducing the contribution of photorespiration are the carbon supply necessary to drive the net increase in carbohydrate and protein and the availability of reductant and ATP. In this article I demonstrate that high levels of NO3-seen in barley and wheat relative to maize and sorghum (Martin et al., 1983) are related to a carbon deficiency caused by the inhibition of the mitochondrial PDC by monovalent cations, in particular by the NH4+ produced by photorespiration in C3 plants (Schuller and Randall, 1989; Gemel and Randall, 1992). NH4+ production is lower in C4 than in C3 cereals (Martin et al., 1983). In addition, since NH4+ production is localized in bundle sheath cells in C4 plants, whereas NO3-assimilation is found in mesophyll cells (Edwards, 1986; Becker et al., 1993), its impact on the carbon flow required for NO3-assimilation should be negligible in C4 plants.

The photorespiratory nitrogen cycle was initially thought to be a closed cyclic process. If this were true the loss of glutamate, glutamine, serine or glycine to other processes, such as protein synthesis or export from the leaves, would not be possible in a stoichiometric sense. However, recent studies with [15N]‐labeled amino acids show that there are alternative sources of nitrogen for photorespiration, indicating that the nitrogen cycle is not a closed cyclic system. In addition recent work with 15NH4Cl and [15N]‐glycine and a metabolically competent mitochondria system has shown that glutamate is synthesized in the mitochondria. Hence the glutamate dehydrogenase (GDH, EC 1.4.1.2) in mitochondria could also be active in the reassimilation of NH4. We would like to propose that one function of mitochondrial GDH is to synthesize glutamate from some of the NH4 released by photorespiration and that this glutamate represents a reserve for use in biosynthetic reactions.