Endogenous biosynthetic precursors of (+)-abscisic acid.: IV.: Biosynthesis of ABA from [2Hn]carotenoids by a cell-free system from avocado

The supernatant fraction (12 000 g) of a homogenate of avocado mesocarp incorporated 3-R-[2-C-14]mevalonate (54 mu Ci/mu mol, 0.9 mu Ci/5mL) into ABA and was used to examine the incorporation of carotenoids into ABA. [H-2(n)]carotenoids were isolated by HPLC from mustard (Sinapis alba) seedlings that had germinated in closed flasks in (H2O)-H-2 (55 atom%). The carotenoids were dissolved in Tween 80 and acetone (1:1 v/v; 100 mu L) and added to the cell-free system final vol. 5 mt, pH 7.1, together with cofactors. After 16 h incubation in darkness the ABA was isolated by HPLC and converted into the pentafluorobenzyl ester. The samples were analysed by capillary gas-liquid chromatography and the CH4 chemical ionisation, negative ion mass spectra recorded. Deuterium-labelled ABA was detected when [H-2] carotenoids were supplied. 'Cold traps' of unlabelled carotenoids added to the cell-free system lowered the [C-14]mevalonate incorporated into ABA to 33% of the control value (1195 dpm) (trans-viola, 33%; trans-neo, 32%) but with 9'-cis-neoxanthin the value fell to 29%. The addition of naproxen (an inhibitor of lipoxygenases) hardly increased the C-14 trapped in trans-viola-or trans-neoxanthin but reduced the C-14 in ABA to 17% of the control and raised the C-14 trapped by the 9'-cis-neoxanthin by 179%. These changes support earlier suggestions that it is 9'-cis-neoxanthin that is cleaved to give the future ABA residue. Other metabolic inhibitors (bisulfite, AMO 1618, tungstate, CO, piperonyl butoxide) affected the incorporation of [C-14]mevalonate into ABA by the cell-free system and the incorporation of [C-14]mevalonolactone into ABA formed in slices of avocado fruit in a closely similar way. This provides strong support for ABA's being biosynthesised in the cell-free system by the same reactions as those by which it is made in vivo.