Production and Characterization of Nitrate Reductase Deficient Mutants in Petunia parviflora

  • Lee, Cheol-Hee (Dept. of Horticultural Science & Research Center for Bioresource and Health, Chungbuk National University)
  • Published : 2006.12.30

Abstract

Nitrate reductase deficient (NR) mutant lines were selected indirectly by their resistance to 100mM chlorate in cell cultures of P. parviflora. A total of 585 chlorate resistant lines were confirmed by a second passage on a high concentration of chlorate. Frequency of spontaneous mutation was $9.7{\times}10^{-7}$ in 3 month old suspension-cultured cells, and in non-selective media containing amino acids as sole nitrogen source. The frequency of mutation could be increased up to 11-fold by culture for 12 months. Out of 40 randomly selected calli, 22 were fully deficient in NR. The rest of the clones contained a decreased level of NR activity. Further characterization was carried out in 13 mutant lines which were fully deficient in NR and in 5 mutant lines containing residual (0-7.0%) NR activity, as compared to wild-type cells cultured on the same medium. The $NR^-$ mutants were tentatively classified as defective in the NR apoenzyme (nia-type; 11 mutant lines including the 5 with residual NR activity) or in the molybdenum cofactor (cnx-type; 7 mutant lines) by the XDH activity. The cnx-type could be further classified into two groups. In one group (5 mutant lines) of these, the NR activity could be partially restored by nonphysiologically high (1.0mM) molybdate in the culture medium. Both types of $NR^-$ mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They also proved to be extremely sensitive to the standard medium ($MSP_1$) containing nitrate and ammonium. Shoot regeneration was obtained only in the $NR^-$ mutants, which contained residual NR activity, but they so far have failed to grow into plants.

References

  1. Arst,H.N., Jr.,D.W.MacDonald and D.J. Cove. 1970.Molybdatemetabolismin Aspergillus nidulans. I.Mutations affecting nitrate reductase and/or xanthine dehydrogenase. Mol. Gen. Genet. 108: 129-145 https://doi.org/10.1007/BF02430519
  2. Braaksma, F.J. and W.J. Feenstra. 1982. Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis theliana. Theor. Appl. Genet. 64: 83-90 https://doi.org/10.1007/BF00303657
  3. Bright, S.W.J., P.B.Norbury and J.L.Wray. 1982.Aconditional-lethal cnx-type nitrate reductase-deficient barley mutant. In: Nitrate Assimilation.Abst. Intl. Symp.NitrateAssimilation-Molecular and Genetic Aspects. Gatersleben, GDR. pp.34
  4. Brunold, C., S. Krüer-Lebus, M.W Saul, S. Wegmuller and I. Potrykus. 1987. Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants.Mol.Gen.Genet. 208: 469-473 https://doi.org/10.1007/BF00328141
  5. Buchanan, R.J. and J.L. Wray. 1982. Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum. Mol. Gen. Genet. 188: 228-234 https://doi.org/10.1007/BF00332680
  6. Cocking, E.C., D. George, M.J. Price-Jones and J.B. Power. 1977. Selection procedures for the production of inter-species somatic hybrids of Petunia hybrida and Petunia parodii. . Albino II complementation selection. Plant Sci. Lett. 10: 7-12 https://doi.org/10.1016/0304-4211(77)90042-6
  7. Cove, D.J. 1976. Chlorate toxicity in Aspergillus nidulans: studies of mutants altered in nitrate assimilation. Mol. Gen. Genet. 146: 147-159 https://doi.org/10.1007/BF00268083
  8. Cove,D.J. 1979.Genetic studies of nitrate assimilation in Aspergillus nidulans. Biol. Rev. 54: 291-327 https://doi.org/10.1111/j.1469-185X.1979.tb01014.x
  9. De Vries, S.E., W.J. VanEverdink, M.A. Ferwerda, E. Jacobsen, L.P. Pijnacker andW.J. Feenstra. 1988. Studies on genemapping in the potato using somatic hybrids of Solanum tuberosum L. +Nicotiana plumbaginifolia. In: Progress in plant protoplast Reserch. (Eds. Puite, K.J., Dons, J.J.M., Huizing, H.J., Kool, A.J., Koornneef,M. and Krens, F.A.). Kluwer Academic Publishers, Dordrecht. pp. 215-218
  10. Grafe, R.,A.Marion-Poll andM. Cabiche. 1986. Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbarginifolia. Theor. Appl. Genet. 73: 299-304 https://doi.org/10.1007/BF00289289
  11. Grafe, R. and A.J.Muller. 1983. Complementation analysis of nitrate reductase deficient mutants of Nicotiana tabacum by somatic hybridization. Theor. Appl. Genet. 66: 127-130
  12. Jaworski, E.G. 1971. Nitrate reductase assay in intact plant tissues. Biochem. Biophys. Res. Comm. 43: 1274-1279 https://doi.org/10.1016/S0006-291X(71)80010-4
  13. Kleinhofs, A., R.L.Warner and K.R.Narayanan. 1985. Current progress towards an understanding of the genetics and molecular biology of nitrate reductase in higher plants. Oxford Surv. Plant Mol.Cell Biol. 2: 91-121
  14. Khon, H., R. Schieder and O. Schieder. 1985 Somatic hybrids in tobacco mediated by electro-fusion. Plant Sci. 38: 121-128 https://doi.org/10.1016/0168-9452(85)90141-4
  15. Lee, C.H. 1989. Somatic and Gametosomatic hybridization in the family Solanaceae. Ph.D. Thesis, Univ. of Nottigham, England
  16. MacDonald,D.W.,D.J. Cove and A. Coddington. 1974. Cytochrome-C reductases fromwild-type andmutant strains of Aspergillus nidulans. Mol. Gen. Genet. 128: 187-199 https://doi.org/10.1007/BF00267108
  17. Marton, L., T.M. Dung, R.R.Mendel and P.Maliga. 1982. Nitrate reductase deficient cell lines from haploid protoplast cultures of Nicotiana plumbaginifolia. Mol. Gen. Genet. 186: 301-304 https://doi.org/10.1007/BF00729445
  18. Mendel, R.R., Z.A. Alikulov, N.P. Lvov and A.J. Müller. 1981. Presence of themolybdenumcofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum. Mol. Gen. Genet. 181: 395-399 https://doi.org/10.1007/BF00425618
  19. Mendel, R.R., Z.A. Alikulov, N.P. Lvov and A.J. Müller. 1982a. Molybdenum cofactor in nitrate reductase-deficient tabacco mutants. . Release of cofactor by heat treatment. Plant Sci. II Lett. 25: 67-72 https://doi.org/10.1016/0304-4211(82)90208-5
  20. Mendel, R.R., Z.A. Alikulov, N.P. Lvov and A.J. Müller. 1982b. Molybdenum cofactor in nitrate reductase-deficient tabacco mutants. III. Induction of cofactor synthesis by nitrate. Plant Sci. Lett. 27: 95-101 https://doi.org/10.1016/0304-4211(82)90076-1
  21. Mendel, R.R., L. Marton and A.J. Müller. 1986. Comparative biochemical characterization of mutants of the nitrate reductase/ molybdenum cofactor loci cnxA, cnxB and cnxC of Nicotiana plumbaginifolia. Plant Sci. 43: 125-129 https://doi.org/10.1016/0168-9452(86)90152-4
  22. Mendel, R.R. and A.J. Müller. 1979. Nitrate reductase-deficient cell lines Nicotiana tabacum: Further biochemical characterization. Mol. Gen. Genet. 177: 145-153 https://doi.org/10.1007/BF00267264
  23. Muller, A.J. 1981. Nitrate reductase mutants and the cell culture approach to higher plants genetics. In: Problems inGeneral Genetics, vol. II. Proc. 14th Intl. Cong. Genet. (Ed. Beliaev, D.K.). Mir, Moscow. pp. 229-237
  24. Muller, A.J. 1982. Genetic studies on nitrate reduction in Nicotiana and Hordeum vulgare. In: Nitrate Assimilation. Abs. Intl. Symp. NitrateAssimilation-Molecular and GeneticAspects.Gatersleben, GDR. pp. 9
  25. Muller, A.J. 1983. Genetic analysis of nitrate reductase-deficient tobacco plants regenerated from mutant cells : Envidence for duplicate structureal genes. Mol. Gen. Genet. 192: 275-281 https://doi.org/10.1007/BF00327678
  26. Muller, A.J. and R. Grafe. 1978. Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet. 161: 67-76 https://doi.org/10.1007/BF00266616
  27. Negrutiu, I., D. De Brouwer, R. Dirks and M. Jacobs. 1985. Amino acid auxotrophs fromprotoplast cultures of Nicotiana plumbaginifolia, Viviani. 1. BUdR enrichment selection, plant regeneration and general characterisation. Mol. Gen. Genet. 199: 330-337 https://doi.org/10.1007/BF00330276
  28. Oostindier-Braaksma, F.J. and W.J. Feenstra. 1973. Isolation and characterization of chlorate resistant mutants of Arabidopsis thaliana. Mutat. Res. 19: 175-185 https://doi.org/10.1016/0027-5107(73)90076-6
  29. Pelsy, F., J. Gabard, I. Cherel, C.Meyer, A.Marion-poll, A.J.Müller andM. Caboche. 1988. Selection and characterization of nitrate reductase- deficientmutants of Nicotiana plumbaginifolia. Plant Cell Tiss. Org. Cult. 12: 231-233 https://doi.org/10.1007/BF00040093
  30. Pirrie, A. and J.B. Power. 1986. The production of fertile, triploid somatic hybird plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic : somatic protoplast fusion. Theor. Appl. Genet. 72: 48-52
  31. Power, J.B., S.F. Berry, J.V. Chapman, E.C. Cocking and K.C. Sink. 1979. Somatic hybrids between unilateral cross-incompatible Petunia species. Theor. Appl. Genet. 55: 97-99 https://doi.org/10.1007/BF00295433
  32. Power, J.B., S.F. Berry, J.V. Chapman, E.C. Cocking and K.C. Sink. 1980. Somatic hybridization of sexually incompatible petunias: Petunia parodii, Petunia parviflora. Theor. Appl. Genet. 57: 1-4
  33. Scazzocchio, C. 1974. The genetic determination of molybdoflavoenzymes in Aspergillus nidulans. J. Less-common Metals 36: 461-464 https://doi.org/10.1016/0022-5088(74)90130-1
  34. Steffen,A. and O. Schieder. 1984. Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia. Plant Cell Rep. 3: 134-137 https://doi.org/10.1007/BF00270206
  35. Tempelaar,M.J., A. Duyst, S.Y. De Vlas, G. Krol, C. Symmonds and M.G.K. Jones. 1987.Modulation and direction of the electrofusion response in plant protoplasts. Plant Sci. 48: 99-105 https://doi.org/10.1016/0168-9452(87)90136-1
  36. Xuan, L.T., R. Grafe and A.J.Müller. 1983. Complementation of nitrate reductase-deficient mutants in somatic hybrids between Nicotiana Species. In: Protoplasts 1983. (Eds. Potrykus, I.,Harms, C.T., Hinnen, A., Hutter, R., King, P.J. and Shillito, R.D.). Poster Proc. 6th Intl. Protoplast Symp., Basel, Switzerland. pp. 21