Identification of a Naturally Occurring Inhibitor of the Conversion of 1-Aminocyclopropane-1-Carboxylic Acid to Ethylene by Carnation Microsomes

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Abstract
During cell-free experiments with membranes isolated from carnation petals (Dianthus caryophillus L. cv White Sim), the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene was blocked by a factor derived from the cytosol. Subsequent characterization of the inhibitor revealed that its effect was concentration dependent, that it was water soluble, and that it could be removed from solution by dialysis and addition of polyvinylpolypyrrolidone. Activity profiles obtained after solvent partitioning over a range of pH values and after chromatography on silica gel, size exclusion gel, and ion exchange resins revealed that the inhibitor was a highly polar, low molecular weight species that was nonionic at low pH and anionic at pH values above 8. Use of selected solvent systems during paper and thin layer chromatography combined with specific spray reagents tentatively identified the compound as a hydroxycinnamic acid derivative. Base hydrolysis and subsequent comparison with known standards by high performance liquid chromatography, gas-liquid chromatography, and ultraviolet light spectroscopy established that the inhibitor was a conjugate with a ferulic acid moiety. Release of ferulic acid following treatment with .beta.-glucosidase also indicated the presence of a glucose moiety, and unequivocal identification of the inhibitor as 1-O-feruloyl-.beta.-D-glucose was confirmed by gas chromatography-mass spectroscopy and by ultraivolet light, 1H-, and 13C-nuclear magnetic resonance spectroscopy. Feruloylglucose constituted about 0.1% of the dry weight of stage III (preclimacteric) carnation petals, but concentrations fell sharply during stage IV (climacteric), when ethylene production peaks and the flowers senesce. In a reaction mixture containing microsome-bound ethylene forming enzyme system, 98% of all ethylene production was abolished in the presence of 50 .mu.m concentrations of the inhibitor.