Studies of nitrate reductase (NR)-deficient mutants in barley have identified five molybdenum cofactor (MoCo) function genes and one gene each for the two NR apoproteins NADH-NR and NAD(P)H-NR. This is similar to the intensively studied Aspergillus nidulans system where 6 different loci encode MoCo functions and a single locus encodes the NR apoprotein (Kinghorn, 1989). Two loci encode NADH-NR structural genes in Arabidopsis (Wilkinson and Crawford, 1993).

Note that some of the mutants in the nar-1 locus of barley accumulate much more NR mRNA than wildtype suggesting that some of the mutations affect the regulation of mRNA accumulation (Kleinhofs et al, 1989).

Mutants of Nicotiana plumbaginifolia which produce a defective NR apoenzyme also overexpressed NR mRNA, and do not exhibit diurnal fluctuations in NR mRNA (Pouteau et al, 1989).

Nitrate is the most critical factor required for the expression of NR genes in plants. The rapid increase of NR mRNA in response to nitrate is due, at least in part, to increased transcription. The 5' flanking regions of the two NR genes (NR1 and NR2) of Arabidopsis which are necessary for nitrate-dependent transcription have been characterized (Hwang et al, 1997). In Arabidopsis the Nia1 gene is induced by the cytokinin benzyladenine whereas the Nia2 gene is not responsive to this hormone (Yu et al, 1998). This may explain the unexpected observation that Nia2-deficient Arabidopsis seedlings grown in high concentrations of cytokinins have an unusually high NR activity and chlorate sensitivity (Yu et al, 1998).

Transgenes derived from the tobacco Nia and Nii genes have been built to overexpress NR and NiR, respectively. Introduction of these transgenes into transgenic tobacco plants frequently leads to co-suppression resulting in a chlorotic phenotype (Palauqui et al, 1996).

Regulatory mutants and genes putatively involved in regulation of nitrate assimilation are beginning to be identified in higher plants. The cr88 mutant of Arabidopsis is impaired in light regulation of NR2 (Lin and Cheng, 1997). A gene encoding a NIT2-like protein (named NTL1 for NIT2-like), characterized by a single zinc finger domain, has been identified in Nicotiana plumbaginifolia (Daniel-Vedele and Caboche, 1993). The Ntl1 gene is present as a single copy and its expression is compatible with those of a regulator of the nitrate assimilation pathway; it shows weak nitrate inducibility and regulation by light (Daniel-Vedele and Caboche, 1993). The -240 to -110 fragment of the spinach NiR promoter contains two NIT2 consensus core elements, and binds in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. Thus, nitrate-inducible expression of the NiR gene in spinach may be mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants (Rastogi et al, 1997).

Note that the molybdenum cofactor (MoCo) is shared by NR, xanthine dehydrogenase (XDH) and abscisic acid aldehyde oxidase in plants (Leydecker et al, 1995).

MoCo hydroxylases such as xanthine dehydrogenase (XDH) and aldehyde oxidase use a desulfo form of the cofactor, whereas reductive dehydroxylases such as NR utilize a dioxo form.

The lesion in the nar2a mutant must occur at an early step in the pathway because it affects both forms of the cofactor.

The aba3 mutant of Arabidopsis defective in ABA synthesis has a lesion which appears to be in the introduction of S into the MoCo (Schwartz et al, 1997). The aba3 mutants have a normal NR activity. Treatment of aba3 extracts with dithionite restored normal abscisic acid aldehyde oxidase activity (Schwartz et al, 1997).

Daniel-Vedele F, Caboche M 1993 A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi. Mol. Gen. Genet. 240: 365-373.

Hwang CF, Lin Y, D'Souza T, Cheng CL 1997 Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. Plant Physiol. 113: 853-862.

Kinghorn JR 1989 Genetic, biochemical, and structural organization of the Aspergillus nidulans crnA-niiA-niaD gene cluster. In (JL Wray, JR Kinghorn eds) "Molecular and Genetic Aspects of Nitrate Assimilation", Oxford Science Publications, Oxford, pp. 69-87.

Kleinhofs A, Warner RL, Melzer JM 1989 Genetics and molecular biology of higher plant nitrate reductases. In (JE Poulton, JT Romeo, EE Conn eds) "Plant Nitrogen Metabolism", Recent Advances in Phytochemistry, Vol 23, Plenum Press, New York, pp. 117-155.

Leydecker M-T, Moureaux T, Kraepiel Y, Schnorr K, Caboche M 1995 Molybdenum cofactor mutants, specifically impaired in xanthine dehydrogenase activity and abscisic acid biosynthesis, simultaneously overexpress nitrate reductase. Plant Physiol. 107: 1427-1431.

Lin Y, Cheng CL 1997 A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus. Plant Cell 9: 21-35.

Palauqui JC, Elmayan T, de Borne FD, Crete P, Charles C, Vaucheret H 1996 Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiol. 112: 1447-1456.

Pouteau S, Cherel I, Vaucheret, Caboche M 1989 Nitrate reductase mRNA regulation in Nicotiana plumbaginifolia nitrate reductase-deficient mutants. Plant Cell 1: 1111-1120.

Rastogi R, Bate NJ, Sivasankar S, Rothstein SJ 1997 Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants. Plant Mol. Biol. 34: 465-476.

Schwartz SH, Leon-Kloosterziel KM, Koornneef M, Zeevaart JA 1997 Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana. Plant Physiol. 114: 161-166.

Wilkinson JQ, Crawford NM 1993 Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both NIA1 and NIA2 nitrate reductase structural genes. Mol. Gen. Genet. 239: 289-297.

Yu X, Sukumaran S, Marton L 1998 Differential expression of the Arabidopsis Nia1 and Nia2 genes. Cytokinin-induced nitrate reductase activity is correlated with increased Nia1 transcription and mRNA levels. Plant Physiol. 116: 1091-1096.