ISHS Acta Horticulturae 379: International Symposium on Quality of Fruit and Vegetables: Influence of Pre- and Post- Harvest Factors and Technology
MOLECULAR BIOLOGICAL INVESTIGATIONS OF ACC OXIDASE AND ITS EXPRESSION ATTENDING APPLE FRUIT RIPENING |
| Authors: | D.R. Dilley, J. Kuai, I.D. Wilson, Y. Pekker, Y. Zhu, D.M. Burmeister, R.M. Beaudry |
| Abstract:
Two-dimensional isoelectric focusing and SDS-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) was employed to determine protein changes during the development of the ethylene climacteric of apple fruits (Malus domestica Borkh.). l-Aminocyclopropane-1-carboxylate (ACC) oxidase was among several other proteins found to accumulate in postclimacteric apple fruit. Polyclonal antibodies against ACC oxidase were raised in rabbits from the antigen purified by IEF/SDS-PAGE. Western blot analyses revealed that ACC oxidase was not detectable in apples prior to the onset of the ethylene climacteric but increased markedly as the climacteric developed. In vitro translation of poly(A)+ RNA purified from apples at progressive developmental stages of the ethylene climacteric and immunoprecipitation with ACC oxidase antisera indicated that a translatable mRNA encoding ACC oxidase accumulated to high levels over the course of the ethylene climacteric. A quantitative ELISA (Enzyme-Linked Immuno Sorbent Assay) for apple ripening-related ACC oxidase revealed that induction of ACC oxidase preceded by 4 to 7 days an increase in ACC content activity and the onset of the ethylene climacteric in all apple cvs. examined. Cortical tissue levels of ACC oxidase in preclimacteric fruits rose from about 1–2 nmoles g/1 FW about three weeks before the onset of the ethylene climacteric and then increased several-fold before increases were noted in ACC levels and in vivo ACC oxidase activity. The initiation of the ethylene climacteric was closely related to enhanced ACC synthase activity (as measured by ACC accumulation) and in vivo ACC oxidase activity. Results of western analyses and immunoprecipitation of in vitro translation products of poly A+mRNA indicate that the translation of ripening-related ACC oxidase results in accumulation of the enzyme prior to the ethylene climacteric as a consequence of an increase in its translatable mRNA transcript. Escherichia coli BL21 (DE3) pLysS were transformed with the cDNA pAP4 known to encode apple ripening-related ACC oxidase (I.D. Wilson, Y. Zhu, D.M. Burmeister, D.R. Dilley [1993] Plant Physiol. 102:783–788). The cDNA insert of pAP4 was fused to a His-TagTM leader sequence with an intervening thrombin proteolytic site on the N-terminal methionine of ACC oxidase yielding a clone (pETAOEx2a) with acquired ability to convert ACC to ethylene under induction by isopropyl-b-D-thiogalactoside (IPTG). This was correlated with the synthesis of a novel 1.2 kb mRNA that hybridized with the pAP4 cDNA. The His-TagTM-ACC oxidase fusion protein (37.7 kD) was purified to homogeneity on a Ni2+-Sepharose affinity column and proteolytically cleaved with thrombin yielding a recombinant enzyme with a mass of 35.4 kD. comprised of Gly-4-Ser-3-His-2-Met-1-[Met-ACC oxidase]. The His-TagTM-ACC oxidase fusion protein and the recombinant ACC oxidase exhibited enzymatic properties similar to those of the native apple fruit enzyme (35.3 kD) in all respects including: kinetic properties, CO2 activation, catalytic inactivation, substrate and inhibitor specificity and recognition of polyclonal antibodies. Preliminary studies suggest that the His-Tag-ACC oxidase fusion protein binds proteins (of unknown nature) from E. coli and apple fruits through protein/protein interactions and that some of these are subject to becoming phosphorylated in the presence of the fusion protein. | |
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