Plant Molecular Biology

, Volume 21, Issue 3, pp 437–449 | Cite as

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

  • Berta Dopico
  • Alexandra L. Lowe
  • Ian D. Wilson
  • Carmen Merodio
  • Donald Grierson
Research Articles


Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.

Key words

cDNA clones fruit storage low temperatures Persea americana polygalacturonase ripening 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Abe K, Arai S: Purification of a cysteine proteinase-inhibitor from rice, Oryza sativa L. japonica. Agric Biol Chem 49: 3349–3350 (1985).Google Scholar
  2. 2.
    Abe K, Emori Y, Kondo H, Suzuki K, Arai S: Molecularcloning of a cysteine proteinase-inhibitor of rice (oryzacystatin)-homology with animal cystatins and transient expression in the ripening process of rice seeds. J Biol Chem 262: 16793–16797 (1987).PubMedGoogle Scholar
  3. 3.
    Abrahamson M, Ritonja A, Brown MA, Grubb A, Machleidt W, Barret AJ: Identification of the probable inhibitory reactive sites of the cysteine proteinase-inhibitors human cystatin-C and chicken cystatin. J Biol Chem 262: 9688–9694 (1987).PubMedGoogle Scholar
  4. 4.
    Arai S, Watanabe H, Kondo H, Emori Y, Abe K: Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J Biochem 109: 294–298 (1991).PubMedGoogle Scholar
  5. 5.
    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 306–308 (1979).Google Scholar
  6. 6.
    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, pp. 1–39. Academic Press, London (1981).Google Scholar
  7. 7.
    Bozak KR, Yu H, Sirevag R, Christophersen RE: Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit. Proc Natl Acad Sci USA 87: 3904–3908 (1990).PubMedGoogle Scholar
  8. 8.
    Brady C, Meldrum SD, McGlasson WB, Ali ZM: Differential accumulation of the molecular forms of polygalacturonase in tomato mutans. J Food Biochem 7: 7–14 (1983).Google Scholar
  9. 9.
    Brady CJ: Fruit ripening. Annu Rev Plant Physiol 38: 155–178 (1987).Google Scholar
  10. 10.
    Broglie KE, Gaynor JJ, Broglie RM: Ethylene-regulated gene expression: Molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris. Proc Natl Acad Sci USA 83: 6820–6824 (1986).PubMedGoogle Scholar
  11. 11.
    Brown SM, Crouch ML: Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase. Plant Cell 2: 263–274 (1990).CrossRefPubMedGoogle Scholar
  12. 12.
    Cabot EL, Beckenbach AT: Simultaneous editing of multiple nucleic acid and protein sequences with ESEE. Computer Appl Biosci 5: 233–234 (1989).Google Scholar
  13. 13.
    Casas AM, Nelson DE, Raghothama KG, D'Urzo M, Singh NK, Bressan RA, Hasegawa PM: Expression of osmotin-like genes in the halophyte Atriplex nummularia L. Plant Physiol 99: 329–337 (1992).Google Scholar
  14. 14.
    Cass LG, Kirven KA, Christoffersen RE: Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening. Mol Gen Genet 223: 76–86 (1990).CrossRefPubMedGoogle Scholar
  15. 15.
    Christoffersen RE, Warm E, Laties GG: Gene expression during fruit ripening in avocado. Planta 155: 52–57 (1982).CrossRefGoogle Scholar
  16. 16.
    Christoffersen RE, Tucker ML, Laties GG: Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection. Plant Mol Biol 3: 385–391 (1984).Google Scholar
  17. 17.
    Cooper WC, Rasmussen GK, Waldon ES: Ethylene evolution stimulated by chilling in citrus and Persea sp. Plant Physiol 44: 1194–1196 (1969).Google Scholar
  18. 18.
    Cornelissen BJC, Hooft van Huijsduijnen RAM, Bol JF: A Tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321: 531–532 (1986).PubMedGoogle Scholar
  19. 19.
    DeJong AJ, Cordewener J, Lo Schiavo F, Terzi M, Vandekerckhove J, VanKammen A, DeVries SC: A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4: 425–433 (1992).CrossRefPubMedGoogle Scholar
  20. 20.
    Dellapenna D, Alexander DC, Bennett AB: Molecular cloning of tomato fruit polygalacturonase: analysis of polygalacturonase mRNA levels during ripening. Proc Natl Acad Sci USA 83: 20–24 (1986).Google Scholar
  21. 21.
    Dellapenna D, Bennet AB: In vitro synthesis and processing of tomato fruit polygalacturonase. Plant Physiol 86: 1057–1063 (1988).Google Scholar
  22. 22.
    Dunn MA, Hughes MA, Pearce RS, Jack PL: Molecular characterization of a barley gene induced by cold treatment. J Exp Bot 41: 1405–1413 (1990).Google Scholar
  23. 23.
    Grierson D, Tucker GA, Keen J, Ray J, Bird CR, Schuch W: Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucl Acids Res 14: 8595–8603 (1986).PubMedGoogle Scholar
  24. 24.
    Guerrero FD, Jones JT, Mullet JE: Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 15: 11–26 (1990).PubMedGoogle Scholar
  25. 25.
    Guy CL, Kiemi KJ, Brambl R: Altered gene expression during cold acclimation of spinach. Proc Natl Acad Sci USA 82: 3673–3677 (1985).PubMedGoogle Scholar
  26. 26.
    Hamilton AJ, Lycett GW, Grierson D: Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346: 284–287 (1990).CrossRefGoogle Scholar
  27. 27.
    Hatfield R, Nevins DJ: Characterization of the hydrolytic activity of avocado cellulase. Plant Cell Physiol 27: 541–552 (1986).Google Scholar
  28. 28.
    Huang JK, Wen L, Swegle M, Tran HC, Thin TH, Nayler HN, Muthukrishan S, Reeck GR: Nucleotide sequence of a rice genomic clone that encodes a class I endochitinase. Plant Mol Biol 16: 479–480 (1991).PubMedGoogle Scholar
  29. 29.
    Hughes MA, Pearce RS: Low temperature treatment of barley plants causes altered gene expression in shoot meristems. J Exp Bot 39: 1461–1467 (1988).Google Scholar
  30. 30.
    Hughes MA, Dunn MA: The effect of temperature on plant growth and development. Biotechnol Genet Engng Rev 8: 161–188 (1990).Google Scholar
  31. 31.
    Kanellis AK, Solomos T, Mattoo AK: Hydrolytic enzyme activities and protein pattern of avocado fruit ripening in air and low oxygen, with and without ethylene. Plant Physiol 90: 257–266 (1989).Google Scholar
  32. 32.
    Kanellis AK, Solomos T, Roubelakis-Angelakis KA: Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress. Plant Physiol 96: 269–274 (1991).Google Scholar
  33. 33.
    King GJ, Turner VA, Hussey CEJr, Wurtele ES, Lee SM: Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein. Plant Mol Biol 10: 401–412 (1988).Google Scholar
  34. 34.
    Knee M: Development of ethylene biosynthesis in pear fruits at −1°C. J Exp Bot 38: 1724–1733 (1987).Google Scholar
  35. 35.
    Kondo H, Abe K, Nishumira I, Watanabe H, Emori Y, Arai S: Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II. J Biol Chem 265: 15832–15837 (1990).PubMedGoogle Scholar
  36. 36.
    Kurkela S, Franck M, Heino P, Lang V, Palva T: Cold-induced gene expression in Arabidopsis thaliana L. Plant Cell Rep 7: 495–498 (1988).CrossRefGoogle Scholar
  37. 37.
    Lee E, Speirs J, Gray J, Brady CJ: Homologies to the tomato endopolygalacturonase gene in the peach genome. Plant Cell Environ 13: 513–521 (1990).Google Scholar
  38. 38.
    Lyons JM: Chilling injury in plants. Annu Rev Plant Physiol 24: 445–466 (1973).CrossRefGoogle Scholar
  39. 39.
    McGarvey DJ, Sirevag R, Christoffersen RE: Ripening-related gene from avocado fruit. Ethylene-inducible expression of the mRNA and polypeptide. Plant Physiol 98: 554–559 (1992).Google Scholar
  40. 40.
    Mohapatra SS, Wolfraim L, Poole J, Dhindsa RS: Molecular cloning and relationship to freeze tolerance of cold acclimation-specific genes of alfalfa. Plant Physiol 89: 375–380 (1989).Google Scholar
  41. 41.
    Payne G, Middlestead W, Williams S, Desai N, Parks TD, Dincher S, Carnes M, Ryal J: Isolation and nucleotide sequence of a novel cDNA clone encoding the major form of pathogenesis-related protein R. Plant Mol Biol 11: 223–224 (1988).Google Scholar
  42. 42.
    Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 (1988).PubMedGoogle Scholar
  43. 43.
    Richardson M, Valdes-Rodriguez S, Blanco-Labia A: A possible function for thaumatin and a TMV-induced protein suggested by homology to a maize inhibitor. Nature 327: 432–434 (1987).CrossRefGoogle Scholar
  44. 44.
    Rogers HJ, Allen RL, Hamilton WDO, Lonsdale DM: Pollen specific cDNA clones from Zea mays. Biochim Biophys Acta 1089: 411–413 (1991).PubMedGoogle Scholar
  45. 45.
    Sacher JA: Acid phosphatase development during ripening of avocado. Plant Physiol 55: 382–386 (1975).Google Scholar
  46. 46.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  47. 47.
    Schaffer MA, Fischer RL: Analysis of mRNAs that accumulate in response to low temperature identifies a thiol protease gene in tomato. Plant Physiol 87: 431–436 (1988).Google Scholar
  48. 48.
    Schlumbaum A, Mauch F, Vogeli U, Boller T: Plant chitinases are potent inhibitors of fungal growth. Nature 324: 365–367 (1986).Google Scholar
  49. 49.
    Sheehy RE, Pearson J, Brady CJ, Hiatt WR: Molecular characterization of tomato fruit polygalacturonase. Mol Gen Genet 208: 30–36 (1987).CrossRefGoogle Scholar
  50. 50.
    Shinshi H, Mohnen D, Meins FJr: Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissue by auxin and cytokinin. Proc Natl Acad Sci USA 84: 89–93 (1987).Google Scholar
  51. 51.
    Singh NK, Bracker CA, Hasegawa PM, Handa AK, Buckel S, Hermodson MA, Pfankoch E, Regnier FE, Bressan RA. Characterization of Osmotin. A thaumatinlike protein associated with osmotic adaptation in plant cells. Plant Physiol 85: 529–536 (1987).Google Scholar
  52. 52.
    Smith CJS, Watson CF, Morris PC, Bird CR, Seymour GB, Gray JE, Arnold C, Tucker JA, Schuch W, Harding S, Grierson D: Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14: 369–379 (1990).PubMedGoogle Scholar
  53. 53.
    Swegle M, Huang JK, Lee G, Muthukrishnan S: Identification of an endochitinase cDNA clone from barley aleurone cells. Plant Mol Biol 12: 403–412 (1989).CrossRefGoogle Scholar
  54. 54.
    van derWel H, Loewe K: Isolation and characterization of thaumatin I and II. The sweet-tasting protein from Thaumatococcus daniellii. Eur J Biochem 31: 221–225 (1972).PubMedGoogle Scholar
  55. 55.
    vanKan JAL, van deRhee MD, Zuidema D, Cornelissen BJC, Bol JF: Structure of tobacco genes encoding thaumatin-like proteins. Plant Mol Biol 12: 153–155 (1989).Google Scholar
  56. 56.
    Wang CY, Adams DO: Chilling-induced ethylene production in cucumbers (Cucumis sativus L.). Plant Physiol 69: 424–427 (1982).Google Scholar
  57. 57.
    Watkins CB, Picton S, Grierson D: Stimulation and inhibition of expression of ripening-related mRNAs in tomatoes as influenced by chilling temperatures. J Plant Physiol 136: 318–323 (1990).Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Berta Dopico
    • 1
  • Alexandra L. Lowe
    • 1
  • Ian D. Wilson
    • 1
  • Carmen Merodio
    • 2
  • Donald Grierson
    • 1
  1. 1.Department of Physiology and Environmental ScienceUniversity of Nottingham, Faculty of Agricultural and Food SciencesLoughboroughUK
  2. 2.Instituto del Frío, Consejo Superior de Investigaciones Científicas (CSIC)Ciudad UniversitariaMadridSpain

Personalised recommendations