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Identification of two tungstate-sensitive molybdenum cofactor mutants,chl2 andchl7, ofArabidopsis thaliana

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Summary

The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.

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References

  • Åberg B (1947) On the mechanism of the toxic action of chlorates and some related substances upon young wheat plants. Ann R Agric Coll Sweden 15:37–107

    Google Scholar 

  • Arst HN, Tollervey DW, Sealy-Lewis HM (1982) A possible regulatory gene for the molybdenum-containing cofactor inAspergillus nidulans. J Gen Microbiol 123:1083–1093

    Google Scholar 

  • Braaksma FJ (1982) Genetic control of nitrate reduction inArabidopsis thaliana. PhD Thesis, University of Groningen, Groningen, The Netherlands

    Google Scholar 

  • Braaksma FJ, Feenstra WJ (1973) Isolation and characterization of chlorate resistant mutants ofArabidopsis thaliana. Mutat Res 19:175–185

    Google Scholar 

  • Braaksma FJ, Feenstra WJ (1982) Isolation and characterization of nitrate reductase-deficient mutants ofArabidopsis thaliana. Theor Appl Genet 64:83–90

    Google Scholar 

  • Caboche M, Rouze P (1990) Nitrate reductase: a target for molecular and cellular studies in higher plants. Trends Genet 6:187–192

    Google Scholar 

  • Campbell WH, Kinghorn JR (1990) Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem Sci 15:315–319

    Google Scholar 

  • Chang C, Bowman JL, DeJohn AW, Lander ES, Meyerowitz EM (1988) Restriction fragment length polymorphism linkage map forArabidopsis thaliana. Proc Natl Acad Sci USA 85:6856–6860

    Google Scholar 

  • Cheng C, Dewdney J, Nam H, Den Boer BGW, Goodman HM (1988) A new locus (NIA1) inArabidopsis thaliana encoding nitrate reductase. EMBO J 7:3309–3314

    Google Scholar 

  • Cramer SP, Stiefel El (1985) Chemistry and biology of the molybdenum cofactor. In: Spiro T (ed) Molybdenum enzymes. John Wiley and Sons, New York, pp 411–441

    Google Scholar 

  • Crawford NM, Campbell WH (1990) Fertile fields. Plant Cell 2:829–835

    Google Scholar 

  • Crawford NM, Campbell WH, Davis RW (1986) Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc Natl Acad Sci USA 83:8073–8076

    Google Scholar 

  • Crawford NM, Smith M, Bellissimo D, Davis RW (1988) Sequence and nitrate regulation of theArabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci USA 85:5006–5010

    Google Scholar 

  • De Vries S, Hoge H, Bisseling T (1988) Isolation of total and polysomal RNA from plant tissues. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual, vol B6. Kluwer Academic Publishers, Dordrecht, pp 1–13

    Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabeling restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13

    Google Scholar 

  • Feldmann KA, Marks MD (1987)Agobacterium-mediated transformation of germinating seeds ofArabidopsis thaliana: a nontissue culture approach. Mol Gen Genet 208:1–9

    Google Scholar 

  • Feldmann KA, Marks MD, Christianson ML, Quatrano RS (1989) A dwarf mutant ofArabidopsis generated by T-DNA insertional mutagenesis. Science 243:1351–1354

    Google Scholar 

  • Fu YH, Marzluf GA (1990)nit-2, the major positive acting nitrogen regulatory gene ofNeurospora crassa, encodes a sequencespecific DNA-binding protein. Proc Natl Acad Sci USA 87:5331–5335

    Google Scholar 

  • Guerrero MG, Vega JM, Losada M (1981) The assimilatory nitratereducing system and its regulation. Annu Rev Plant Physiol 32:169–204

    Google Scholar 

  • Heimer YM, Wray JL, Filner P (1969) The effect of tungstate on nitrate assimilation in higher plant tissues. Plant Physiol 44:1197–1199

    Google Scholar 

  • Hewitt EJ (1983) A perspective of mineral nutrition: essential and functional metals in plants. In: Robb D (ed) Metals and micronutrients: Uptake and utilization by plants. Academic Press, London, pp 277–323

    Google Scholar 

  • Higgins ES, Richert DA, Westerfeld WW (1956) Tungstate antagonism of molybdate inAspergillus niger. Proc Soc Exp Biol Med 92:509–511

    Google Scholar 

  • Jacobsen E, Braaksma FJ, Feenstra WJ (1984) Determination of xanthine dehydrogenase activity in nitrate reductase-deficient mutants ofPisum satiaum andArabidopsis thaliana. Z Pflanzenphysiol Bd 113:183–188

    Google Scholar 

  • Kleinhofs A, Warner RL, Narayanam KR (1985) Current progress towards understanding of the genetics and molecular biology of nitrate reductase in higher plants. In: Miflin B (ed) Oxford surveys of plant molecular biology, vol 2. Oxford University Press, New York, pp 91–121

    Google Scholar 

  • Koornneef M, van Eden J, Hanhart CJ, Stam P, Braaksma FJ, Feenstra WJ (1983) Linkage map ofArabidopsis thaliana. J Hered 74:265–272

    Google Scholar 

  • Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF, Sibley S, Davies RW, Arst HN (1990) The regulatory geneareA mediating nitrogen metabolite repression inAspergillus nidulans: mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–1364

    Google Scholar 

  • LaBrie ST, Wilkinson JQ, Crawford NM (1991) Effect of chlorate treatment on nitrate reductase and nitrite reductase gene expression inArabidopsis thaliana. Plant Physiol 97:873–879

    Google Scholar 

  • Marks MD, Wes J, Weeks DP (1987) The relatively large betatubulin gene family ofArabidopsis contains a member with an unusual transcribed 5′ noncoding sequence. Plant Mol Biol 10:91–104

    Google Scholar 

  • Mendel RR, Alikulov ZA, Lvov NP, Muller AJ (1981) Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines ofNicotiana tabacum. Mol Gen Genet 181:395–399

    Google Scholar 

  • Mendel RR, Kirk DW, Wray JL (1985) Assay of molybdenum cofactor of barley. Phytochemistry 24:1631–1634

    Google Scholar 

  • Nakagawa H, Yamashita N (1986) Chlorate reducing activity of spinach nitrate reductase. Agric Biol Chem 50:1893–1894

    Google Scholar 

  • Nason A, Lee K, Pan S, Ketchum PA, Lamberti A, DeVries J (1971) In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from aNeurospora mutant and a component of molybdenum enzymes. Proc Natl Acad Sci USA 68:3242

    Google Scholar 

  • Notten BA, Hewitt EJ (1971) The role of tungsten in the inhibition of nitrate reductase activity in spinach leaves. Biochem Biophys Res Commun 44:702–710

    Google Scholar 

  • Pouteau S, Chérel I, Vaucheret H, Caboche M (1989) Nitrate reductase mRNA regulation inNicotiana plumbaginifolia nitrate reductase-deficient mutants. Plant Cell 1:1111–1120

    Google Scholar 

  • Rajagopalan KV (1989) Chemistry and biology of the molybdenum cofactor. In: Wray JL, Kinghorn JR (eds) Molecular aspects of nitrate assimilation. Oxford Science Publications, Oxford, pp 212–226

    Google Scholar 

  • Redinbaugh MG, Campbell WH (1983) Purification of squash NADH:nitrate reductase by zinc chelate affinity chromatography. Plant Physiol 71:205–207

    Google Scholar 

  • Solomonson LP, Barber MJ (1990) Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41:225–253

    Google Scholar 

  • Steven B, Kirk DW, Bright S, Wray JL (1989) Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional lethal mutants of barley. Mol Gen Genet 219:421–428

    Google Scholar 

  • Takahashi H, Nason A (1957) Tungstate as a competitive inhibitor of molybdate in nitrate assimilation and in N2 fixation byAzotobacter. Biochim Biophys Acta 23:433–435

    Google Scholar 

  • Theologis A, Huynh T, Davis RW (1985) Rapid induction of specific mRNAs by auxin in pea epicotyl tissue. J Mol Biol 183:53–68

    Google Scholar 

  • Vogel HJ (1956) A convenient growth medium forNeurospora. Microbiol Genet Bul 13:42–43

    Google Scholar 

  • Vunkova-Radeva R, Schiemann J, Mendel RR, Salcheva G, Georgieva D (1988) Stress and activity of molybdenum-containing complex (molybdenum cofactor) in winter wheat seeds. Plant Physiol 87:533–535

    Google Scholar 

  • Walker-Simmons M, Kudrna DA, Warner RL (1989) Reduced accumulation of ABA during water stress in a molybdenum cofactor mutant of barley. Plant Physiol 90:728–733

    Google Scholar 

  • Wilkinson JQ, Crawford NM (1991) Identification of theArabidopsis CHL3 gene as the nitrate reductase structural geneNIA2. Plant Cell 3:461–471

    Google Scholar 

  • Wray JL (1988) Molecular approaches to the analysis of nitrate assimilation. Plant Cell Environ 11:369–382

    Google Scholar 

  • Wray JL, Kinghorn JR (1989) Molecular and genetic aspects of nitrate assimilation. Oxford Science Publications, Oxford

    Google Scholar 

Download references

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Authors and Affiliations

  1. Department of Biology, University of California, 92093-0116, San Diego, La Jolla, CA, USA

    Samuel T. LaBrie, Jack Q. Wilkinson, Yi-Fang Tsay & Nigel M. Crawford

  2. Department of Plant Sciences, University of Arizona, 85721, Tucson, AZ, USA

    Kenneth A. Feldmann

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  1. Samuel T. LaBrie
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  2. Jack Q. Wilkinson
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  3. Yi-Fang Tsay
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  4. Kenneth A. Feldmann
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  5. Nigel M. Crawford
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Communicated by E. Meyerowitz

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LaBrie, S.T., Wilkinson, J.Q., Tsay, YF. et al. Identification of two tungstate-sensitive molybdenum cofactor mutants,chl2 andchl7, ofArabidopsis thaliana . Molec. Gen. Genet. 233, 169–176 (1992). https://doi.org/10.1007/BF00587576

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  • DOI: https://doi.org/10.1007/BF00587576

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