Analyses of barley spike mutant waxes identify alkenes, cyclopropanes and internally branched alkanes with dominating isomers at carbon 9

Abstract

SummaryAbout 15% of the epidermal wax on Hordeum vulgare cv. Bonus barley spikes is n-alkanes. Longer homologues are greatly reduced in the eceriferum mutants, cer-a 6 , cer-e 8 , cer-n 26 , cer-n 53 , cer-n 985 , cer-x 60 , cer-yc 135 and cer-yl 187 . Simultaneously hydrocarbons accounting for only traces in the wild-type become prominent in the mutants, although their chain-length distributions remain unchanged. Accordingly several new hydrocarbon series were identified. The two major ones were C 23 -C 35 cis monoenoic alkenes (the major 9-ene isomer was part of a homologous series including 11, 13 and 15-enes), and the novel C 27 -C 31 cyclopropanes (the ring carbons of major isomers were 9,10 and 11,12 with lesser amounts of 13,14). Three minor series included 2-and 3-methylalkanes plus C 25 -C 33 internally branched alkanes (methyls on carbons 9, 11, 13, 15 or 17; shorter homologues dominated by the 9 isomer, longer homologues by 11, 13 or 15 isomers). Acyl chains destined for spike waxes are synthesized via acyl and polyketide elongase systems plus associated reductive and decarbonylative/decarboxylative enzyme systems. Both elongation systems are defective in synthesizing C 32 acyl chains in all nine mutants. The similarities in the position of the chemical groups (primarily on carbon 9, secondarily on carbon 11) of the alkenes, cyclopropanes and internally branched methyl alkanes imply an origin from a common, hitherto unrecognized associated pathway in barley, designated the enoic pathway. The elongation system leading to the enoic derived hydrocarbons differs from the known elongation systems by inclusion of a mechanism for introducing a double bond.

Identification of In-Chain-Functionalized Compounds and Methyl-Branched Alkanes in Cuticular Waxes of Triticum aestivum cv. Bethlehem

“…Series G comprised six compounds in the least polar fraction of wheat wax, tentatively assigned as internally methyl-branched alkanes by analogy with spectra of 15-methyl-alkanes [48]. Accordingly, compounds G did not exhibit MS fragments indicative of silylation, and other derivatization reactions (such as acetylation, methoximation, transesterification or LAH reduction) did not alter the compounds in any way, thus confirming the absence of functional groups.…”

“…Finally, alkanes with an in-chain methyl branch had been described before as constituents of insect cuticular waxes [60], of wool wax [61], but also of plant cuticular waxes, namely leaf waxes of walnut tree [62] and spike waxes of barley [48]. It is important to note that, different from previous reports, the in-chain-branched alkane regiomers identified here in wheat wax had methyl groups separated by two carbons, located on odd-numbered carbons in the chain of homologs with odd chain lengths (even total carbon numbers) and on even-numbered carbons in even-numbered homologs (with odd total carbon numbers).…”

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar

“…In contrast, it is much less clear how alkenes, cyclopropanes, and internally branched alkanes are synthesized, but this is proposed to occur via an uncharacterized enoic pathway (von WettsteinKnowles, 2007). Previous studies describing alkenes in maize (Zea mays) silk and barley (Hordeum vulgare) spike waxes eluded to this independent pathway, but it is currently unclear which genes or enzymes are involved in this process in planta (Perera et al, 2010;von Wettstein-Knowles, 2007). Thus, the biosynthesis of alkenes in plants has remained obscure.…”

“…To determine the position of the double bond, osmium tetroxide was used to hydroxylate alkenes (von Wettstein-Knowles, 2007;Capella and Zorzut, 1968). Roughly 1 mg of the alkene-enriched fraction was oxidized by the addition of 2 mg of osmium tetroxide in 200 mL of dioxane.…”

Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf

“…These VLCFAs are further modified in two major pathways, the acyl-reduction and the decarbonylation pathway, into the finally deposited wax molecules. [The existence of an additional elongation system in barley that leads to synthesis of enoic lipids in spike waxes has recently been implied by the study of von Wettstein-Knowles 2007)]. FAE activity is present in the microsomal fraction of cell extracts and is thought to be localized to the endoplasmic reticulum (Xu et al 2002).…”