Plant Molecular Biology

, Volume 3, Issue 6, pp 385–391 | Cite as

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

  • Rolf E. Christoffersen
  • Mark L. Tucker
  • George G. Laties


A cDNA library was constructed from poly(A)+RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)+-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.


avocado cDNA-clone cellulase hybrid-select immunoblot poly(A)+RNA 





sodium dodecyl sulfate




base pairs


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  1. 1.
    Awad M, Lewis LN: Avocado cellulase: extraction and purification. J Food Sci 45:1625–1628, 1980.Google Scholar
  2. 2.
    Awad M, Young RE: Postharvest variation in cellulase, polygalacturonase, and pectinmethylesterase in avocado (Persea americana Mill. cv Fuerte) fruits in relation to respiration and ethylene production. Plant Physiol 64:306–308, 1979.Google Scholar
  3. 3.
    Biale JB, Young RE: Respiration and ripening in fruits-retrospect and prospect. In: Friend J, Rhodes MJC (eds) Recent Advances in the Biochemistry of Fruits and Vegetables. Academic Press, London, 1981, pp. 1–39.Google Scholar
  4. 4.
    Boller T, Gehri A, Mauch F, Vogeli V: Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta 157:22–31, 1983.Google Scholar
  5. 5.
    Casadan M, Cohen S: Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207, 1980.Google Scholar
  6. 6.
    Christoffersen RE, Laties GG: Ethylene regulation of gene expression in carrots. Proc Natl Acad Sci USA 79: 4060–4063, 1982.Google Scholar
  7. 7.
    Christoffersen RE, Warm E, Laties GG: Gene expression during fruit ripening in avocado. Planta 155:52–57, 1982.Google Scholar
  8. 8.
    Grunstein M, Hogness D: Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965, 1975.Google Scholar
  9. 9.
    Holmes DS, Quigley M: A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114: 193–197, 1981.Google Scholar
  10. 10.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.Google Scholar
  11. 11.
    Madel M, Higa A: Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162, 1970.Google Scholar
  12. 12.
    Maniatis T, Fritsch EF, Sambrook J: Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1982.Google Scholar
  13. 13.
    Parnes JR, Velan B, Felsenfeld A, Ramanathan L, Ferrini U, Appella E, Sidman JG: Mouse B2 microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rate mRNAs. Proc Natl Acad Sci USA 78:2253–2257, 1981.Google Scholar
  14. 14.
    Pesis E, Fuchs G, Zauberman G: Cellulase activity and fruit softening in avocado. Plant Physiol 61:416–419, 1978.Google Scholar
  15. 15.
    Rigby PWJ, Dieckmann M, Rhodes C, Berg P: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251, 1977.Google Scholar
  16. 16.
    Roychoudary R, Jay E, Wu R: Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res 3:101–116, 1976.Google Scholar
  17. 17.
    Thomas PS: hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci 77:5201–5205, 1980.Google Scholar
  18. 18.
    Towbin H, Staechelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354, 1979.Google Scholar
  19. 19.
    Tucker M, Laties GG: Interrelationship of gene expression, polysome prevalence, and respiration during ripening of ethylene and/ or cyanide-treated avocado fruit. Plant Physiol, in press, 1984.Google Scholar
  20. 20.
    Tucker GA, Robertson NG, Grierson D: Changes in polygalacturonase isoenzymes during the ‘ripening’ of normal and mutant tomato fruit. Eur J Biochem 112:119–124, 1980.Google Scholar
  21. 21.
    Zurfluh LL, Guilfoyle TJ: Auxin- and ethylene-induced changes in the population of translatable messenger RNA in basal sections and intact soybean hypocotyl. Plant Physiol 69:338–340, 1982.Google Scholar

Copyright information

© Martinus Nijhoff/Dr W. Junk Publishers 1984

Authors and Affiliations

  • Rolf E. Christoffersen
    • 1
  • Mark L. Tucker
    • 1
  • George G. Laties
    • 1
  1. 1.Department of Biology and Molecular Biology InstituteUniversity of CaliforniaLAU.S.A.

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