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.
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Abbreviations
- ACC:
-
1-aminocyclopropane-1-carboxylic acid
- AdoMet:
-
S-adenosyl-l-methionine
- HPLC:
-
high-pressure liquid chromatography
- kDa:
-
kilodalton
- kb:
-
kilobase
- mAb:
-
monoclonal antibody
- Met:
-
methionine
- PCR:
-
polymerase chain reaction
- poly(A)+ RNA:
-
polyadenylated RNA
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Abeles, F.B. (1973) Ethylene in plant biology. Academic Press, New York
Adams, D.O., Yang, S.F. (1979) Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acat. Sci. USA 76, 170–174
Alexander, D.C. (1987) An efficient vector-primer cDNA cloning system. Methods Enzymol. 154, 41–64
Bleecker, A.B., Kenyon, W.H., Somersville, S.C., Kende, H. (1986) Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1-carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA 83, 7755–7759
DellaPenna, D., Christoffersen, R.E., Bennett, A.B. (1986) Biotinylated proteins as molecular weight standards on Western blots. Anal. Biochem. 152, 329–332
Dong, J.G., Yip, W.K., Yang, S.F. (1991) Monoclonal antibodies against apple 1-aminocyclopropane-1-carboxylate synthase. Plant Cell Physiol. 32, 25–31
Hanahan, D., Meselson, M. (1980) Plasmid screening at high colony density. Gene 10, 63–67
Kawasaki, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Champlin, R., Witte, O.N., McCormick, F.P. (1988) Diagnosis of chronic myeloidi and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc. Natl. Acad. Sci. USA 85, 5698–5720
Kim, W.T., Okita, T.W. (1988) Structure, expression, and heterogeneity of the rice seed prolamines. Plant Physiol. 88, 649–655
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lay-Yee, M., DellaPenna, D., Ross, G.S. (1990) Changes in mRNA and protein during ripening in apple fruit (Malus domestica) Borkh. cv. Golden Delicious. Plant Physiol. 94, 850–853
Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., Davis, M.M. (1989) Polymerase chain reaction with single-sided specificity: analysis of T cell receptor chain. Science 243, 217–220
Nakagawa, N., Nakajima, N., Imaseki, H. (1988) Immunochemical difference of wound-induced 1-aminocyclopropane-1-carboxylate synthase from the auxin-induced enzyme. Plant Cell Physiol 29, 1255–1259
Nakajima, N., Nakagawa, N., Imaseki, H. (1988) Molecular size of wound-induced 1-aminocyclopropane-1-carboxylate synthase from Cucurbita maxima Duch. and change of translatable mRNA of the enzyme after wounding. Plant Cell Physiol. 29, 989–998
Nakajima, N., Mori, H., Yamazaki, K., Imaseki, H. (1990) Molecular cloning and sequence of a complementary DNA encoding 1-aminocyclopropane-1-carboxylate synthase induced by tissue wounding. Plant Cell Physiol. 31, 1016–1021
Olson, D.C., White, J.A., Edelman, L., Harkins, R.N., Kende, H. (1991) Differential expression of two genes from 1-aminocyclopropane-1-carboxylate synthase in tomato fruits. Proc. Natl. Acad. Sci. USA (in press)
O'Neill, S.D. (1989) Molecular analysis of floral induction in Pharbitis nil. In: Floral induction to pollination, pp. 19–28, Lord E., Bernier, G., eds. Academic Press, New York, pp. 19–28
Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Sato, T., Theologis, A. (1989) Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants. Proc. Natl. Acad. Sci. USA 86, 6621–6625
Satoh, S., Yang, S.F. (1988) S-adenosylmethionine-dependent inactivation and radiolabeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits. Plant Physiol. 88, 109–114
Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M., Van Montagu, M. (1989) Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp. Eur. J. Biochem. 182, 639–647
Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M., Van Montagu, M. (1990) Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase. Proc. Natl. Acad. Sci. USA 87, 4859–4863
Yang, S.F., Hoffman, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155–189
Yip, W.K., Dong, J.G., Kenny, J.W., Thompson, G.A., Yang, S.F. (1990) Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase. Proc. Natl. Acad. Sci. USA 87, 7930–7934
Yip, W.K., Dong, J.G., Yang, S.F. (1991) Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits. Plant Physiol. 95, 251–257
Zimmern, D., Kaesherg (1978) 3′-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 75, 4257–4261
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This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.
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Dong, J.G., Kim, W.T., Yip, W.K. et al. Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit. Planta 185, 38–45 (1991). https://doi.org/10.1007/BF00194512
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DOI: https://doi.org/10.1007/BF00194512