Endogenous Biosynthetic Precursors of (+)-Abscisic Acid. IV. Biosynthesis of ABA from [2Hn] Carotenoids by a Cell-free System from Avocado

Abstract

The supernatant fraction (12 000 g) of a homogenate of avocado mesocarp incorporated 3-R-[2-14C]mevalonate (54 µCi/µmol, 0.9 µCi/5mL) into ABA and was used to examine the incorporation of carotenoids into ABA. [2Hn]carotenoids were isolated by HPLC from mustard (Sinapis alba) seedlings that had germinated in closed flasks in 2H2O (55 atom%). The carotenoids were dissolved in Tween 80 and acetone (1:1 v/v; 100 µL) and added to the cell-free system final vol. 5 mL, 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 [2H] carotenoids were supplied. ‘Cold traps’ of unlabelled carotenoids added to the cell-free system lowered the [14C]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 14C trapped in trans-viola- or trans-neoxanthin but reduced the 14C in ABA to 17% of the control and raised the 14C 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 [14C]mevalonate into ABA by the cell-free system and the incorporation of [14C]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.