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

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

“…Enzyme isolation and incubation procedure Aliquots of dried acetone powder obtained from exocarp of mature fruit of Citrus sinensis cv. Midknight, prepared as previously described and stored at 4°C (Richardson and Cowan 1996), were homogenized in 0.1 M Tris-HCl buffer (pH 7.4) containing 10 mM dithiothreitoi (DTT), 5 mAf MgCU and 0.1% (w/v) Tween-20 at 0°C using an Ultra-Turrax top-drive homogenizer The sluiTy was filtered through 2 layers of Miracloth and centrifuged at 23 500 g for 30 min at 2°C in a refrigerated centrifuge. Unless otherwise stated, aliquots of the supernatant equivalent to 2 mg of protein (Bradford 1976) were incuhated in a total volume of 5 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM glutathione, 5 mM ATP, 1.25 mM MgCk, 12.5 M of each of NAD, NADH and NADPH, 1 mM AMO 1618, 5 mM NaF, 1 |iM MOO4", 50 mM DTT and substrate.…”

“…Detection was at 260 nm and compounds of interest were quantified by peak integration following calibration with authentic standards Railton 1986,1987). Where specified, ABA and relied metabolites were quantified by gas chromatogra-phy-electron capture and characterised by combined capillary GC-MS as previously described (Richardson and Cowan 1996).…”

“…Detailed studies describing the properties and cofactor requirements of the above cell-free systems provided frirther evidence to support a precursor/product relationship between ABAdio! and ABA (Richardson and Cowan 1996). Furthermore, in the presence of FAD, label from either MVA or isopentenyl pyrophosphate accumulated in a compound with identical chromatographic and spectrophotometric properties to those of alI-frans-;S-carotene.…”

Some cellular and molecular properties of abscisic acid: its particular involvement in growing plant roots

“…This reaction was stimulated by molybdate and inhibited by the oxidized form of the flavin adenine dinucleotide (FAD). Gel electrophoresis of the enzyme extract revealed the presence of a 53-kDa protein with peroxidase activity characteristic of a cytochrome P-450 [18]. With a similar material and using the gas-chromatography-mass-spectrometry (GC-MS) [19,20], it has been reported [21] that i-carotene is formed from either farnesyl or geranyl pyrophosphates.…”