Summary
Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M2 populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M1) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F2 progeny showed that one quarter lacked shoot nitrate reductase activity. These F2 plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.
We conclude that: (a) the nar-2 gene locus encodes a step in molybdopterin biosynthesis; (b) the mutant R11301 represents a further locus involved in the synthesis of a functional molybdenum cofactor; (c) mutant Rl2202 is also defective in molybdopterin biosynthesis; and (d) the nar-2 gene locus and the gene locus defined by R11301 govern molybdenum cofactor biosynthesis in both shoot and root.
Similar content being viewed by others
References
Arst HN Jr, MacDonald DW, Cove DJ (1970) Molybdate metabolism in Aspergillus nidulans. I. Mutations affecting nitrate reductase and/or xanthine dehydrogenase. Mol Gen Genet 108:129–145
Bright SWJ, Norbury PB, Franklin J, Kirk DW, Wray JL (1983) A conditional-lethal enx-type nitrate reductase-deficient barley mutant. Mol Gen Genet 189:240–244
Buchanan RJ, Wray JL (1982) Isolation of molybdenum cofactor-defective lines of Nicotiana tabacum. Mol Gen Genet 188:228–234
Cardenas J, Mortensen LE (1974) Determination of molybdenum and tungsten in biological materials. Anal Biochem 60:372–381
Dailey FA, Kuo T, Warner RL (1982a) Pyridine nucleotide specificity of barley nitrate reductase. Plant Physiol 69:1196–1199
Dailey FA, Warner RL, Somers DA, Kleinhofs A (1982b) Characteristics of a nitrate reductase in a barley mutant deficient in NADH nitrate reductase. Plant Physiol 69:1200–1204
Gewitz H-S, Piefke J, Vennesland B (1981) Purification and characterization of demolybdonitrate reductase (NADH cytochrome c oxidoreductase) of Chlorella vulgaris. J Biol Chem 256:11527–11531
Hewitt EJ, Notton BA, Rucklidge GJ (1977) Formation of nitrate reductase by recombination of apoprotein fraction from molybdenum deficient plants with a molybdenum containing complex. J Less Common Metals 54:537–545
Hoagland DR, Arnon DI (1938) The water-culture method of growing plants without soil. Calif. Agric Exp Sta Circ 347
Johnson JL (1980) The molydenum cofactor common to nitrate reductase, xanthine dehydrogenase and sulphite oxidase. In: Coughlan MP (ed) Molybdenum and molybdenum containing enzymes. Pergamon Press, New York, pp 345–383
Johnson JL, Rajagopalan KV (1982) Structural and metabolic relationship between the molybdenum cofactor and urothione. Proc Natl Acad Sci USA 79:6856–6860
Johnson JL, Hainline BE, Rajagopalan KV, Arison BH (1984) Pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives. J Biol Chem 259:5414–5422
Johnson ME, Rajagopalan KV (1987) Involvement of chlA, E, M and N loci in Escherichia coli molybdopterin biosynthesis. J Bacteriol 169:117–125
Ketchum PA, Cambier HY, Frazier WA III, Madansky CH, Nason A (1970) In vitro assembly of Neurospora mutant assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidising of aldehyde oxidase systems of higher plants. Proc Natl Acad Sci USA 66:1016–1023
Kleinhofs A, Kuo T, Warner RL (1980) Characterization of nitrate reductase-deficient barley mutants. Mol Gen Genet 177:421–425
Kleinhofs A, Taylor J, Kuo TM, Somers DA, Warner RL (1983) Nitrate reductase genes as selectable markers for plant cell transformation. In: Lurquin PF, Kleinhofs A (eds) Genetic Engineering in Eukaryotes. Plenum Press, New York, pp 215–231
Kleinhofs A, Warner RL, Narayanan KR (1985) Current progress towards an understanding of the genetics and molecular biology of nitrate reductase in higher plants. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, vol 2. Oxford University Press, Oxford, pp 91–121
Kleinhofs A, Warner RL, Lawrence JM, Melzer JM, Jeter JM, Kudrna DA (1989) Molecular genetics of nitrate reductase in barley. In: Wray JL, Kinghorn JR (eds) Molecular and genetic aspects of nitrate assimilation. Oxford University Press, Oxford, pp 197–211
Kramer S, Hageman RV, Rajagopalan KV (1984) In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit1: specific incorporation of molybdopterin. Arch Biochem Biophys 233:821–829
Kramer SP, Johnson JL, Ribeiro AA, Millington DS, Rajagopalan KV (1987) The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl) molybdopterin from sulfite oxidase and xanthine oxidase. J Biol Chem 262:16357–16363
Kuo T, Kleinhofs A, Somers D, Warner RL (1981) Antigenicity of nitrate reductase deficient mutants in Hordeum vulgare L. Mol Gen Genet 181:20–23
Kuo TM, Kleinhofs A, Somers DA, Warner RL (1984) Nitrate reductase-deficient mutants in barley: enzyme stability and peptide mapping. Phytochemistry 23:229–232
Marton L, Dung TM, Mendel RR, Maliga P (1982) Nitrate reductase deficient cell lines from haploid protoplast cultures of Nicotiana plumbaginifolia. Mol Gen Genet 182:301–304
Mendel R-R, Alikulov ZA, Lvov NP, Müller AJ (1981) Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum. Mol Gen Genet 181:395–399
Mendel R-R, Kirk DW, Wray JL (1985) Assay of molybdenum-cofactor of barley. Phytochemistry 24:1631–1634
Müller AJ, Grafe R (1978) Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol Gen Genet 161:67–76
Narayanan KR, Somers DA, Kleinhofs A, Warner RL (1983) Nature of cytochrome c reductase in nitrate reductase-deficient mutants of barley. Mol Gen Genet 190:222–226
Narayanan KR, Müller AJ, Kleinhofs A, Warner RL (1984) In vitro reconstitution of NADH: nitrate reductase in nitrate reductase deficient mutants of barley. Mol Gen Genet 197:358–362
Nason A, Antoine A-D, Ketchum PA, Frazier WA III, Lee DK (1970) Formation of assimilatory nitrate reductase by in vitro intercistronic complementation in Neurospora crassa. Proc Natl Acad Sci USA 65:137–144
Nason A, Lee K-Y, Pan S-S, Ketchum PA, Lamberti A, DeVries J (1971) In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes. Proc Natl Acad Sci USA 68:3242–3246
Rajagopalan KV (1989) Chemistry and biology of the molybdenum cofactor. In: Wray JL, Kinghorn JR (eds) Molecular and genetic aspects of nitrate assimilation. Oxford University Press, Oxford, pp 212–226
Shumney VK, Tokarev BI (1981) Genetic control of nitrate reductase activity in barley. In: Asher MJC, Ellis RP, Hayter AM, Whitehouse RNH (eds) Barley genetics IV. Fourth International Barley Genetics Symposium. Edinburgh University Press, Edinburgh, pp 881–885
Small IS, Wray JL (1980) NADH nitrate reductase and related NADH cytochrome c reductase species in barley. Phytochemistry 19:387–394
Somers DA, Kuo TM, Kleinhofs A, Warner RL (1983) Nitrate reductase deficient mutants in barley. Immuno-electrophoretic characterisation. Plant Physiol 71:145–149
Steven BJ, Kirk DW, Wray JL, Bright SWJ (1987) Isolation and characterisation of chlorate resistant mutants of barley. In: Abstracts 2nd Intl Symp “Nitrate assimilation — molecular and genetic aspects”. St. Andrews, UK, G6
Tokarev BI, Shumney UK (1977) Clarification of barley mutants with lowered nitrate reductase activity after treatment of the grain with ethylmethanesulphonate. Genetika 13:2097–2103
Wahl RC, Hageman RV, Rajagopalan KV (1984) The relationship of Mo, molybdopterin, and the cyanolyzable sulphur in the Mo cofactor. Arch Biochem Biophys 230:264–273
Warner RL, Lin CJ, Kleinhofs A (1977) Nitrate reductase-deficient barley. Nature 269:406–407
Wray JL (1986) The molecular genetics of higher plant nitrate assimilation. In: Blonstein A, King PJ (eds) A genetic approach to plant biochemistry. Springer-Verlag, Vienna, pp 101–157
Wray JL, Filner P (1970) Structural and functional relationships of enzyme activities induced by nitrate in barley. Biochem J 119:715–725
Wray JL, Steven B, Kirk DW, Bright SWJ (1985) A conditional-lethal molybdopterin-defective mutant of barley. Mol Gen Genet 201:462–466
Author information
Authors and Affiliations
Additional information
Communicated by H. Böhme
Rights and permissions
About this article
Cite this article
Steven, B.J., Kirk, D.W., Bright, S.W. et al. Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley. Mol Gen Genet 219, 421–428 (1989). https://doi.org/10.1007/BF00259615
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00259615