Skip to main content
SpringerLink
Log in

Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants

  • Genetics and Genomics
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

In an earlier investigation, homologous mutant lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from a callus that had been irradiated with a 50-Gy gamma ray during embryo culture. In order to identify the 5MT-resistant mechanism, we have continued our investigations of these mutant lines and studied the anthranilate synthase activity of the M5 advanced lines by direct fluorometric detection of the anthranilate formed in both control plants and mutant lines grown on 500 μM 5MT. The anthranilate synthase activity of the mutant plants was 2.2- to 3-fold higher than that of the control. In a kinetic analysis with tryptophan, an anthranilate synthase of the mutant lines was insensitive to feedback inhibition. These lines showed an enhanced accumulation of storage proteins and amino acids. The increased rates of protein synthesis in the mutant lines, relative to that of the control seeds, were 17–28.5%. The amino acid contents were 2.4-fold (MRI-40-2) to 2.6-fold (MRI-110-6) higher in the MRI lines than in the control seeds, and 2.4-fold (MRII-12-5) to 3.5-fold (MRII-8-1) higher in the MRII lines than in the control seeds. Significant increases among the amino acids of the MR lines were observed for tryptophan, phenylalanine, and tyrosine, which had been biosynthesized through the shikimate pathway. The transcript levels of putative OASA2, which is one of the key-regulating enzyme subunits in the tryptophan biosynthesis pathway, were studied in the control and 5MT-resistant mutant lines subjected to inhibition by two tryptophan analogs (5MT and αMT) and to other abiotic stresses (ABA, NaCl, and cold). The putative OASA2 gene in the 5MT-resistant mutant lines was highly expressed in at a low 5MT concentration and at an early stage of the 5MT and αMT treatments. However, mRNA accumulation of the putative OASA2 gene in the mutant plants gradually decreased when the plants were subjected to abiotic stresses such as NaCl and cold. These results indicated that the 5MT resistance in the mutant lines is due to altered anthranilate synthase forms.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

ESTs:

Expressed sequence tags

OASA2:

Oryza sativa anthranilate synthase alpha 2

αMT:

α-Methyltryptophan

5MT:

5-Methyltryptophan

References

  • Bernasconi P, Walters EW, Woodworth AR, Siehl DL, Stone TE, Subramanian MV (1994) Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli. Plant Physiol 106:353–358

    Article  CAS  PubMed  Google Scholar 

  • Bohlmann J, De Luca V, Eilert U, Martin W (1995) Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes. Plant J 7:491–501

    Article  CAS  PubMed  Google Scholar 

  • Brotherton JE, Hauptmann RM, Widholm JM (1986) Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L. Planta 168:214–221

    CAS  Google Scholar 

  • Carlson PS (1970) Induction and isolation of auxotrophic mutants in somatic cell cultures of Nicotiana tabacum. Science 168:487–489

    Google Scholar 

  • Carlson PS (1973) Methionine sulfoximine-resistant mutants of tobacco. Science 180:1136–1138

    Google Scholar 

  • Carlson J, Widholm J (1978) Separation of two forms of anthranilate synthetase from S-methyltryptophan susceptible and resistant cultured Solanum tuberosum cells. Physiol Plant 44:251–255

    Google Scholar 

  • Crawfold IP (1989) Evolution of a biosynthetic pathway: the tryptophan paradigm. Annu Rev Microbiol 43:567–600

    Google Scholar 

  • Graf R, Mchmann B, Braus GH (1993) Analysis of feedback-resistant anthranilate synthase from Saccharomyces cerevisiae. J Bacteriol 175:1061–1068

    Google Scholar 

  • Henderson EJ, Zalkin H (1971) On the composition of anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl transferase form Salmonella typhimurium. J Biol Chem 246:6891–6898

    Google Scholar 

  • Hrazdina G, Jensen RA (1992) Spatial organization of enzymes in plant metabolic pathways. Annu Rev Plant Physiol Plant Mol Biol 43:241–267

    Google Scholar 

  • Ishikawa Y, Park JH, Kisaka H, Lee HY, Kanno A, Kameya T (2003) A 5-methyltryptophan resistant mutant of rice has an altered regulation of anthranilate synthase gene expression. Plant Sci 164:1037–1045

    Google Scholar 

  • Kang KK, Kameya T (1995) Characterization of anthranilate synthetase and tryptophan synthase in a 5-methyltryptophan resistant mutant (MR1) of Zea mays L. Breed Sci 45:321–325

    Google Scholar 

  • Kim DS, Lee IS, Jang CS, Hyun DY, Seo YW, Lee YI (2004a) Selection of 5-methyltryptophan resistant rice mutants from irradiated calli derived from embryos. Euphytica 135:9–19

    Google Scholar 

  • Kim DS, Lee IS, Jang CS, Kang SY, Song HS, Lee YI, Seo YW (2004b) Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants. Plant Cell Rep 23:71–80

    Google Scholar 

  • Kim DS, Lee IS, Jang CS, Lee SJ, Song HS, Lee YI, Seo YW (2004c) AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes. Plant Sci 167:305–316

    Google Scholar 

  • Kisaka H, Kisaka M, Kameya T (1996) Characterization of interfamilial somatic hybrids 5-methyltryptophan resistant rice (Oryza sativa L.) and 5MT-sensitive carrot (Daucus carota L.); expression of resistance to 5MT by the somatic hybrids. Breed Sci 46:221–226

    Google Scholar 

  • Kutchan TM (1995) Alkaloid biosynthesis-the basis for metabolic engineering of medical plants. Plant Cell 7:1059–1070

    Article  CAS  PubMed  Google Scholar 

  • Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214

    Google Scholar 

  • Moyed HS (1960) False feedback inhibition: inhibition of tryptophan biosynthesis by 5-methyltryptophan. J Biol Chem 235:1098–1102

    Google Scholar 

  • Niyogi KK, Fink GR (1992) Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway. Plant Cell 4:721–733

    Google Scholar 

  • Niyogi KK, Last RL, Fink GR, Keith B (1993) Suppressors of trp1 fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase β subunit. Plant Cell 5:1011–1027

    Google Scholar 

  • Ranch JP, Rick S, Brotherton JE, Widholm JM (1983) Expression of 5-methyltryptophan resistance in plants regenerated from resistant cell lines of Datura innoxia. Plant Physiol 71:136–140

    CAS  Google Scholar 

  • Robinson JA, Gani D (1985) The shikimate pathway. Nat Prod Rep 2:306–319

    Google Scholar 

  • Schaeffer GW, Sharpe FT (1983) Mutations and selection: genetic variation for improved protein in rice. In: Genetic engineering, application to agriculture. Beltsville, Md., pp 237–254

  • Schaeffer GW, Sharpe FT (1997) Electrophoretic profiles and amino acid composition of rice endosperm proteins of a mutant with enhanced lysine and total protein after backcrosses for germplasm improvements. Theor Appl Genet 95:230–235

    Google Scholar 

  • Song HS, Brotherton JE, Gonzale RA, Widholm JM (1998) Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 117:533–543

    Article  CAS  PubMed  Google Scholar 

  • Tozawa Y, Hasegawa H, Terakawa T, Wakasa K (2001) Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1. Plant Physiol 126:1493–1506

    Article  CAS  PubMed  Google Scholar 

  • Widholm JM (1971) Control of tryptophan biosynthesis in plant tissue cultures: lack of repression of anthranilate and tryptophan synthetases by tryptophan. Physiol Plant 25:75–79

    Google Scholar 

  • Widholm JM (1972) Anthranilate synthetase from 5-methyltryptophan-susceptible and resistant cultured Daucus carota cells. Biochim Biophys Acta 279:48–57

    Google Scholar 

  • Widholm JM (1973) Measurement of the five enzymes which convert chorismate to tryptophan in cultured Daucus carota cell extracts. Biochim Biophys Acta 320:217–226

    Google Scholar 

  • Widholm JM (1977) Selection and characterization of amino acid analog resistant plant cell culture. Crop Sci 17:597–600

    Google Scholar 

  • Zhao J, Williams CC, Last RL (1998) Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor. Plant Cell 10:359–370

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Radiation Plant Breeding and Genetics, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong-Gu, Daejeon, 305-600, Korea

    D. S. Kim, I. S. Lee & S.-Y. Kang

  2. Division of Biotechnology and Genetic Engineering, College of Life & Environmental Sciences, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, 136-701, Korea

    C. S. Jang & Y. W. Seo

Authors
  1. D. S. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. S. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. S. Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S.-Y. Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Y. W. Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y. W. Seo.

Additional information

Communicated by I.S. Chung

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, D.S., Lee, I.S., Jang, C.S. et al. Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants. Plant Cell Rep 24, 357–365 (2005). https://doi.org/10.1007/s00299-005-0943-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-005-0943-y

Keywords

Search

Navigation