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Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein

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Abstract

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH2-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5′ signal sequence, an open reading frame of 718 nucleotides, a 3′ AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.

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References

  1. Arnon DI: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 (1949).

    Google Scholar 

  2. Benton WD, Davis RW: Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182 (1977).

    Google Scholar 

  3. Burnett WN: Western blotting: Electrophoretic transfer of proteins from sodium sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203 (1981).

    Google Scholar 

  4. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  5. Chen E, Seeburg PH: Laboratory methods. Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4: 165–170 (1985).

    Google Scholar 

  6. Cornelissen BJC, Hooft van Huijsduinen RAM, Bol JF: A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321: 531–532 (1986).

    Google Scholar 

  7. De Vos AM, Hatada M, Van der Wel H, Krabbendam H, Peerdeman AF, Kim SH: Three-dimensional structure of thaumatin I. An intensely sweet protein. Proc Natl Acad Sci USA 82: 1406–1409 (1985).

    Google Scholar 

  8. Edens L, Van der Wel H: Microbial synthesis of the sweettasting plant protein thaumatin. Trends in Biotechnology 3: 61–64 (1985).

    Google Scholar 

  9. Edman P, Begg G: A protein sequenator. Eur J Biochem 1: 80–91 (1967).

    Google Scholar 

  10. Ericson MC, Alfinito SH: Proteins produced during salt stress in tobacco cell culture. Plant Physiol 74: 506–509 (1984).

    Google Scholar 

  11. Helentjaris T, King G, Slocum M, Siedenstrang C, Wegman S: Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding. Plant Mol Biol 5: 109–118 (1985).

    Google Scholar 

  12. Higgenbotham JD, Hough CAM: Useful taste properties of amino acids and proteins. In: GC Birch, JG Brennan and KJ Parker (eds) Sensory Properties of Foods. Applied Sciences, London (1977) pp. 129–149.

    Google Scholar 

  13. Hill RL, Bradshaw RA: Fumarase, Methods Enzymol 13: 91–99 (1969).

    Google Scholar 

  14. Hooftvan Huijsduijnen, van Loon LC and Bol JF: cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products. EMBO J 5: 2057–2061 (1986).

    Google Scholar 

  15. King G, Hussey C, Turner V: A protein induced by NaCl in suspension cultures of Nicotiana tabacum accumulates in whole plant roots. Plant Mol Biol 7: 441–449 (1986).

    Google Scholar 

  16. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).

    Google Scholar 

  17. Ledebaer AM, Verrips CT, Dekker BMM: Cloning of the natural gene for the sweet-tasting plant protein thaumatin. Gene 30: 23–32 (1984).

    Google Scholar 

  18. Maniatis F, Fritsch EF, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).

    Google Scholar 

  19. Merril CR, Goldman D, Sedman SA, Ebert MH: Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211: 1437–1438 (1981).

    Google Scholar 

  20. Nikolau BJ, Wurtele ES, Stumph PK: Use of streptavidin to detect biotin-containing proteins in plants. Anal Biochem 149: 448–453 (1985).

    Google Scholar 

  21. Ohtsuka E, Matsuki S, Ikehara M, Takahashi Y, Matsubara K: An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem 2605–2608 (1985).

  22. Proudfoot JN, Brownlee GG: 3′ Non-coding region sequences in eukaryotic messenger RNA. 263: 211–214 (1976).

    Google Scholar 

  23. Rave N, Czukehjakov R, Boedtker H: Identification of procallagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. NAR 6: 3559–3567 (1979).

    Google Scholar 

  24. Rigby PWJ, Dieckman M, Rhodes C, Berg P: Labeling deoxyribonucleic acid to high specific in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237–251 (1977).

    Google Scholar 

  25. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  26. Singh NK, Handa AK, Hasegawa PM, Bressan RA: Proteins associated with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79: 126–137 (1985).

    Google Scholar 

  27. Singh NK, LaRosa C, Handa AK, Hasegawa PM, Bressan RA: Hormonal regulation of protein synthesis associated with salt tolerance in plant cells. Proc Natl Acad Sci USA 84: 739–743 (1987).

    Google Scholar 

  28. Takahashi Y, Kato K, Hayashizaki Y, Wakabayashi T, Ohtsuka E, Matsuki S, Ikehara M, Matsubara K: Molecular cloning of the human cholecystokinan gene by use of a synthetic probe containing deoxyinosine. Proc Natl Acad Sci USA 82: 1931–1935 (1985).

    Google Scholar 

  29. Tarr GE, Bucher JF, Bell M, McKean DJ: Polyquarternary amines prevent peptide loss from sequenators. Anal Biochem 84: 622–627 (1978).

    Google Scholar 

  30. Ting IP: CO2 metabolism in corn roots. III. Inhibition of Penolpyruvate carboxylase by L-malate. Plant Physiol 43: 1919–1924 (1968).

    Google Scholar 

  31. Uchimya H, Murashige T: Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells. Plant Phys 54: 936–944 (1974).

    Google Scholar 

  32. Van de Wel H, Loeve K: Isolation and characterization of thaumatin I and II, the sweet tasting proteins from Thaumatococcus danielii Benth. Eur J Biochem 31: 221–225 (1975).

    Google Scholar 

  33. Winge DR, Nielson KB, Zeikus RD, Gray WR: Structural characterization of the isoforms of neonatal and adult rat liver metallothionein. J Biol Chem 259: 11419–11425 (1984).

    Google Scholar 

  34. Wishnick M, Lane MD: Ribulose diphosphate carboxylase from spinach leaves. Methods Enzymol 23: 570–599 (1971).

    Google Scholar 

  35. Zarkower D, Stephenson P, Sheets M, Wickens M: The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro. MCB 6: 2317–2323 (1986).

    Google Scholar 

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King, G.J., Turner, V.A., Hussey, C.E. et al. Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein. Plant Mol Biol 10, 401–412 (1988). https://doi.org/10.1007/BF00014946

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