Abstract
Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.
Similar content being viewed by others
References
Aberg B (1947) On the mechanism of the toxic action of chlorates and some related substances upon young wheat plants. Royal Agric Coll Sweden 15:37–107
Anderson S (1981) Shotgun DNA sequencing using cloned DNaseI-generated fragments. Nucleic Acids Res 9:3015–3027
Barber MJ, Neame PJ (1990) A conserved cysteine in molybdenum oxotransferases. J Biol Chem 265:20912–20915
Braaksma F, Feenstra W (1982) Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana. Theor Appl Genet 64:83–90
Caboche M, Rouze P (1990) Nitrate reductase: a target for molecular and cellular studies in higher plants. Trends Genet 6:187–192
Calza R, Huttner E, Vincentz M, Touze P, Galangau F, Vaucheret H, Cherel I, Meyer C, Kronenberger J, Caboche M (1987) Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants. Mol Gen Genet 209:552–562
Campbell WH, Kinghorn JR (1990) Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem Sci 15:315–319
Cheng C-L, Dewdney J, Nam H, Den Boer BGW, Goodman HM (1988) A new locus (NIA1) in Arabidopsis thaliana encoding nitrate reductase. EMBO J 7:3309–3314
Cheng C-L, Adeco GN, Dewdney J, Goodman HM, Cankling MA (1991) Differential expression of the two Arabidopsis nitrate reductase genes. Plant Physiol 96:275–279
Choi HK, Kleinhofs A, An G (1989) Nucleotide sequence of rice nitrate reductase genes. Plant Mol Biol 13:731–733
Crawford NM (1992) Study of chlorate resistant mutants of Arabidopsis: insights into nitrate assimilation and ion metabolism of plants. In: Setlow JK, Hollander A (eds) Genetic engineering principles and methods. New York, Plenum Press. pp 89–98
Crawford NM, Campbell WH (1990) Fertile fields. Plant Cell 2:829–835
Crawford NM, Campbell WH, Davis RW (1986) Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc Natl Acad Sci USA 83:8073–8076
Crawford NM, Smith M, Bellissimo D, Davis RW (1988) Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci USA 85:5006–5010
Crawford NM, Wilkinson JQ, LaBrie ST (1992) Metabolic Control of nitrate reduction in Arabidopsis thaliana. Aust J Plant Physiol 19:377–385
Dailey FA, Warner RL, Somers DA, Kleinhofs A (1982) Characteristics of a nitrate reductase in a barley mutant deficient in NADH nitrate reductase. Plant Physiol 69:1200–1204
Daniel-Vedele F, Dorbe MF, Caboche M, Rouze P (1989) Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. Gene 85:371–380
Dean-Drummond CE, Glass AD (1982) Nitrate uptake into barley (Hordeum vulgare) plants. A new approach using 36ClO3 − as an analog for nitrate. Plant Physiol 70: 50–54
Deng M, Moureaux T, Caboche M (1989) Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene. Plant Physiol 91:304–309
Dorbe M-F, Caboche M, Daniel-Vedele F (1992) The tomato nia gene complements a Nicotiana plumbaginifolia nitrate reductase-deficient mutant and is properly regulated. Plant Mol Biol 18:363–375
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Friemann A, Brinkmann K, Hachtel W (1991) Sequence of a cDNA encoding the bi-specific NAD(P)H nitrate reductase from the tree Betula pendula and identification of conserved protein domains. Mol Gen Genet 227:97–105
Gabard J, Marion-Poll A, Cherel I, Meyer C, Muller A, Caboche M (1987) Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group. Mol Gen Genet 209:596–606
Guerrero MG, Vega JM, Losada M (1981) The assimilatory nitrate reducing system and its regulation. Annu Rev Plant Physiol 32:169–204
Guiard B, Lederer G (1979) The cytochrome b5 fold: structure of a novel protein superfamily. J Mol Biol 135:639–650
Harker AR, Narayanan KR, Warner RL, Kleinhofs A (1986) NAD(P)H bispecific nitrate reductase in barley leaves: partial purification and characterization. Phytochemistry 25:1275–1279
Hoff T, Stummann BM, Henningsen KW (1991) Cloning and expression of a gene encoding a root-specific nitrate reductase in bean (Phaseolus vulgaris). Physiol Plant 82:197–204
Hoff T, Stummann BM, Henningsen KW (1992) Structure, function and regulation of nitrate reductase in higher plants. Physiol Plant 84:616–624
Hyde GE, Crawford NM, Campbell WH (1991) The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. J Biol Chem 266:23542–23547
Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Cole GE, Brow MAD, Unkles SE, Clutterbuck AJ, Kinghorn JR, Innis MA (1990) Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90:181–192
Kleinhofs A, Warner RL (1990) Advances in nitrate assimilation. In: The Biochemistry of Plants. Academic Press, San Diego, pp 89–120
Kleinhofs A, Warner RL, Hamat HB, Juricek M, Huang C, Schnorr K (1988) Molecular genetics of barley and rice nitrate reductases. In: Current Topics in Plant Biochemistry and Physiology. Columbia, University of Missouri. pp 35–42
Koornneef M, Dellaert LWM, van der Veen JH (1982) EMS- and radiation-induced mutation frequencies at individual loci in Arabidopsis thaliana. Mutat Res 93:109–123
LaBrie S, Wilkinson J, Crawford N (1991) Effect of chlorate treatment on nitrate reductase and nitrite reductase gene expression in Arabidopsis thaliana. Plant Physiol 97:873–879
LaBrie ST, Wilkinson JQ, Tsay YF, Feldmann KA, Crawford NM (1992) Identification of two tungstate-sensitive molybdenum cofactor mutants ch12 and chl17 of Arabidopsis thaliana. Mol Gen Genet 233:169–176
Marks MD, West J, Weeks DP (1987) The relatively large betatubulin gene family of Arabidopsis contains a member with an unusual transcribed 5′-noncoding sequence. Plant Mol Biol 10:91–104
Mendel R, Kirk D, Wray J (1985) Assay of molybdenum cofactor of barley. Phytochemistry 24:1631–1634
Meyer C, Levin JM, Roussel JM, Rouze P (1991) Mutational and structural analysis of the nitrate reductase heme domain of Nicotiana plumbaginifolia. J Biol Chem 266:20561–20566
Miyazaki J, Juricek M, Angelis K, Schnorr KM, Kleinhofs A, Warner RL (1991) Characterization and sequence of a novel nitrate reductase from barley. Mol Gen Genet 228:329–334
Müller AJ (1983) Genetic analysis of nitrate reductase-deficient tobacco plants regenerated from mutant cells. Mol Gen Genet 192:275–281
Neame PJ, Barber MJ (1989) Conserved domains in molybdenum hydroxylases. The amino acid sequence of chicken hepatic sulfite oxidase. J Biol Chem 264:20894–901
Okamato PM, Fu Y-H, Marzluf GA (1991) Nit-3, the structural gene of nitrate reductase in Neurospora crassa. nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet 227:213–223
Oostindier-Braaksma FJ (1970) A chlorate-resistant mutant of Arabidopsis thaliana. Arabidopsis Inf Serv 7:24
Pelsy F, Gonneau M (1991) Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia. Genetics 127:199–204
Pouteau S, Chérel I, Vaucheret H, Caboche M (1989) Nitrate reductase mRNA regulation in Nicotiana plumbaginifolia nitrate reductase-deficient mutants. Plant Cell 1:1111–1120
Prosser IM, Lazarus CM (1990) Nucleotide sequence of a spinach nitrate reductase cDNA. Plant Mol Biol 15:187–190
Redinbaugh MG, Campbell WH (1983). Purification of squash NADH:nitrate reductase by zinc chelate affinity chromatography. Plant Physiol 71:205–207
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sato Y, Shiraishi N, Sato T, Ogura N, Nakagawa H (1992) Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach. Phytochemistry 31:2259–2262
Schnorr KM, Juricek M, Huang C, Culley D, Kleinhofs A (1991) Analysis of barley nitrate reductase cDNA and genomic clones. Mol Gen Genet 227:411–416
Scholten HJ, de Vries SE, Feenstra WJ (1985) Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana. Theor Appl Genet 71:556–562
Scholten HJ, Feenstra WJ, Nijdam H, Datema G (1982) The use of cell culture of Arabidopsis thaliana for study of nitrate reductase-less mutants which are lethal as intact plants. Arabidopsis Inf Serv 19:108–110
Solomonson LP, Barber MJ (1990) Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41:225–253
Solomonson LP, Vennesland B (1972) Nitrate reductase and chlorate toxicity in Chlorella beijerinck. Plant Physiol 50:421–424
Steingrover E, Feenstra WJ (1981) Resistance to low concentration of chlorate on nitrate-free medium. Arabidopsis Inf Serv 18:143–145
Unkles SE, Campbell EL, Punt PJ, Hawker KL, Contreras R, Hawkins AR, Van den Hondel CAMJJ, Kinghorn JR (1992) The Aspergillus niger niaD gene encoding nitrate reductase: Upstream nucleotide and amino acid sequence comparisons. Gene 111:149–155
Vaucheret H, Chabaud M, Kronenberger J, Caboche M (1990) Functional complementation of tobacco and Nicotiana plumbaginofolia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes. Mol Gen Genet 220:468–474
Vaucheret H, Kronenberger J, Rouze P, Caboche M (1989a) Complete nucleotide sequence of the two homologous tobacco nitrate reductase genes. Plant Mol Biol 12:597–600
Vaucheret H, Vincentz M, Kronenberger J, Caboche M, Rouze P (1989b) Molecular cloning and characterisation of the two homologous genes coding for nitrate reductase in tobacco. Mol Gen Genet 216:10–15
Warner RL, Kleinhofs A (1981) Nitrate utilization by nitrate reductase-deficient barley mutants. Plants Physiol 67:740–743
Wilkinson JQ, Crawford NM (1991) Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2. Plant Cell 3:461–471
Wray J, Kinghorn J (1989). Molecular and genetic aspects of nitrate assimilation. Oxford Science Publications, Oxford
Author information
Authors and Affiliations
Additional information
Communicated by E. Meyerowitz
Rights and permissions
About this article
Cite this article
Wilkinson, J.Q., Crawford, N.M. Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2 . Molec. Gen. Genet. 239, 289–297 (1993). https://doi.org/10.1007/BF00281630
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00281630