Development of an abscisic acid biosynthesizing cell-free system from flavedo of Citrus sinensis fruit

Cell-free extracts prepared from an acetone powder derived from the flavedo of mature, coloured fruits of Citrus sinensis L. cv. Midknight transformed mevalonic acid, isopentenyl pyrophosphate, all-trans-beta-carotene and 1',4'-trans-ABAdiol into ABA, This is the first report of a cell-free system capable of producing ABA from both a terpenyl pyrophosphate and carotenoid origin, ABA biosynthesizing activity was stimulated by reduced nicotinamide nucleotides, molybdate, AMO1618 and a cold-pool trap of (+/-)-ABA, but was inhibited by FAD, Addition of FAD caused accumulation of label from isopentenyl pyrophosphate in a compound with similar chromatographic and spectrophotometric properties to those of beta-carotene, 1',4'-Trans-ABAdiol, ABA and PA were unequivocally characterized as products of beta-carotene metabolism in this cell-free system, a process that was markedly improved by extraction of enzyme in the presence of detergents. Dithiothreitol enhanced ABA biosynthesis from all-trans-beta-carotene, stigmasterol, and to a lesser extent cholesterol reduced conversion of beta-carotene to ABA, but did not influence transformation of 1',4'-trans-ABAdiol to ABA, AMO1618 stimulated formation of ABA and appeared to exert its effect at the level of conversion of 1',4'-trans-ABAdiol to ABA, Ancymidol and zeatin reduced incorporation of label from all-trans-beta-carotene into ABA suggesting involvement of a mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kDa protein with peroxidase activity characteristic of a cytochrome P-450.