Leaf cuticular waxes of wild-type Welsh onion (Allium fistulosum L.) and a wax-deficient mutant: Compounds with terminal and mid-chain functionalities

Plant cuticles cover aerial organs to limit non-stomatal water loss and protect against insects and pathogens. Cuticles contain complex mixtures of fatty acid-derived waxes, with various chain lengths and diverse functional groups. To further our understanding of the chemical diversity and biosynthesis of these compounds, this study investigated leaf cuticular waxes of Welsh onion (Allium fistulosum L.) wild type and a wax-deficient mutant. Leaf waxes were extracted with chloroform, separated using thin layer chromatography (TLC), and analyzed using gas chromatography-mass spectrometry (GC-MS). The extracts contained typical wax compound classes found in nearly all plant lineages but also two uncommon compound classes. Analyses of characteristic MS fragmentation patterns followed by comparisons with synthetic standards identified the latter as very-long-chain ketones and primary ketols. The ketols were minor compounds, with chain lengths ranging from C₂₈ to C₃₂ and carbonyls mainly on C-18 and C-20 in wild type wax, and a C₂₈ chain with C-16 carbonyl in the mutant. The ketones made up 70% of total wax in the wild type, consisting mainly of C₃₁ isomers with carbonyl group on C-14 or C-16. In contrast, the mutant wax comprised only 4% ketones, with chain lengths C₂₇ and C₂₉ and carbonyls predominantly on C-12 and C-14, respectively. A two-carbon homolog shift between wild type and mutant was also observed in the primary alcohols (a major wax compound class), whilst alkanes exhibited a four-carbon shift. Overall, the compositional data shed light on possible biosynthetic pathways to wax ketones that can be tested in future studies.