Biosynthesis of Esterified Alkan-2-Ols and β-Diketones in Barley Spike Epicuticular Wax: Synthesis of Radioactive Intermediates

Thirteen different ¹⁴C -and ³H-labelled epicuticular wax precursors have been synthesized and their structuredetermined by gas chromatography-mass spectrometry analyses. The biosyntheses of β-diketones and alkan-2-olcontaining esters were studied by incorporating these intermediates into tissue slices of barley spikes whose awns had been removed. A differential labelling pattern of the alkan-2-ol esters and the β-diketones was observed after feeding three selected mutants blocked in different steps catalyzed by a multifunctional enzyme encoded for by the cer-cqu gene. In cer-u⁶⁹ tissue slices (9,10-³H)-3-oxopalmitoyl-CoA was incorporated into both the esterifiedalkan-2-ols and the β~diketones. Only the former wax component was synthesized by the mutants cer-c ³⁶ and _q⁴².When C,, and C~6 fatty acyl chains were fed to the tissue slices, those of cer-u 6~ and -c ³⁶ readily labelled the esterifiedalkan-2-ols, whereas those of cer-q 42 were totally inactive. In all three mutants (2-¹⁴C)-pentadecan-2-one,(10,11³H)-heptadecan-2-one and (2-3H)-pentadecan-2-ol exclusively labelled the alkan-2-ol moieties of thespecified esters, (9,10-³H)-L-3-hydroxypalmitoyl-CoA and (3-¹⁴C)-labelled DL-3-hydroxy fatty acids having 14, 16and 18 carbon atoms were incorporated with a very low efficiency into the 13-diketones and the esterified aikan-2-ols.(9,10-³H)-3-oxopalmitoyl-CoA is the primer for the enzyme system known as βketoacyl elongase which forms theC₂₉ (nonacosan- 14,16-dione), C₃₁ (hentriacontan- 14,16-dione) and C₃₃ (tritriacontan- 16,18-dione) β-diketones. After protection of the β-dicarbonyl group, 7 or 8 C₂ units are added before the presumed decarboxylation to yieldthe complete ~diketone carbon chain. The alkan-2-ol esters arise from the 3-oxoacyl-CoA derivative by an initialdecarboxylation to form a methyl ketone, followed by a reduction to an alkan-2-ol. The latter is then esterified with a fatty acid to form the alkan-2-ol containing esters. The three steps involved in the alkan-2-ol ester synthesis areaccomplished by the coordinated action of a decarboxylase, reductase and ester synthetase.