Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

Jian Guo Dong, Woo Taek Kim, Wing Kin Yip, Gregory A. Thompson, Liming Li, Alan B. Bennett, Shang Fa Yang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

103 Scopus citations

Abstract

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)+ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3′-end was intact, it lacked a portion of sequence at the 5′-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5′-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

Original languageEnglish (US)
Pages (from-to)38-45
Number of pages8
JournalPlanta
Volume185
Issue number1
DOIs
StatePublished - Aug 1991

Keywords

  • 1-Amninocyclopropane-1-carboxylate synthase
  • Ethylene synthesis
  • Fruit ripening
  • Gene expression
  • Malus
  • cDNA

ASJC Scopus subject areas

  • Genetics
  • Plant Science

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