Aerial plant organs possess a diverse array of extracellular surface lipids, including both non-polar and amphipathic constituents that collectively provide a primary line of defense against environmental stressors. Extracellular surface lipids on the stigmatic silks of maize are composed primarily of saturated and unsaturated linear hydrocarbons, as well as fatty acids, and aldehydes. To efficiently extract lipids of differing polarities from maize silks, five solvent systems (hexanes; hexanes:diethyl ether (95:5); hexanes:diethyl ether (90:10); chloroform:hexanes (1:1) and chloroform) were tested by immersing fresh silks in solvent for different extraction times. Surface lipid recovery and the relative composition of individual constituents were impacted to varying degrees depending on solvent choice and duration of extraction. Analyses were performed using both silks and leaves to demonstrate the utility of the solvent- and time-optimized protocol in comparison to extraction with the commonly used chloroform solvent. Overall, the preferred solvent system was identified as hexanes:diethyl ether (90:10), based on its effectiveness in extracting surface hydrocarbons and fatty acids as well as its reduced propensity to extract presumed internal fatty acids. Metabolite profiling of wildtype and glossy1 seedlings, which are impaired in surface lipid biosynthesis, demonstrated the ability of the preferred solvent to extract extracellular surface lipids rich in amphipathic compounds (aldehydes and alcohols). In addition to the expected deficiencies in dotriacontanal and dotriacontan-1-ol for gl1 seedlings, an unexpected increase in fatty acid recovery was observed in gl1 seedlings extracted in chloroform, suggesting that chloroform extracts lipids from internal tissues of gl1 seedlings. This highlights the importance of extraction method when evaluating mutants that have altered cuticular lipid compositions. Finally, metabolite profiling of silks from maize inbreds B73 and Mo17, exposed to different environments and harvested at different ages, revealed differences in hydrocarbon and fatty acid composition, demonstrating the dynamic nature of surface lipid accumulation on silks.

Background Simple non-isoprenoid hydrocarbons accumulate in discrete regions of the biosphere, including within bacteria and algae as a carbon and/or energy store, and the cuticles of plants and insects, where they may protect against environmental stresses. The extracellular cuticular surfaces of the stigmatic silks of maize are rich in linear hydrocarbons and therefore provide a convenient system to study the biological origins and functions of these unique metabolites. Results To test the hypotheses that genetics and environment influence the accumulation of surface hydrocarbons on silks and to examine the breadth of metabolome compositions across diverse germplasm, cuticular hydrocarbons were analyzed on husk-encased silks and silks that emerged from the husk leaves from 32 genetically diverse maize inbred lines, most of which are commonly utilized in genetics experiments. Total hydrocarbon accumulation varied ~ 10-fold among inbred lines, and up to 5-fold between emerged and husk-encased silks. Alkenes accounted for 5-60% of the total hydrocarbon metabolome, and the majority of alkenes were monoenes with a double bond at either the 7th or 9th carbon atom of the alkyl chain. Total hydrocarbon accumulation was impacted to similar degrees by genotype and husk encasement status, whereas genotype predominantly impacted alkene composition. Only minor differences in the metabolome were observed on silks that were emerged into the external environment for 3- versus 6-days. The environmental influence on the metabolome was further investigated by growing inbred lines in 2 years, one of which was warmer and wetter. Inbred lines grown in the drier year accumulated up to 2-fold more hydrocarbons and up to a 22% higher relative abundance of alkenes. In summary, the surface hydrocarbon metabolome of silks is primarily governed by genotype and husk encasement status, with smaller impacts of environment and genotype-by-environment interactions. Conclusions This study reveals that the composition of the cuticular hydrocarbon metabolome on silks is affected significantly by genetic factors, and is therefore amenable to dissection using quantitative genetic approaches. Such studies will clarify the genetic mechanisms responsible for the accumulation of these metabolites, enabling detailed functional investigations of the diverse and complex protective roles of silk surface lipids against environmental stresses.

The hydrophobic cuticle is the first line of defense between aerial portions of a plant and the external environment. On maize silks, the cuticular cutin matrix is infused with cuticular lipids, consisting of a homologous series of very-long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons that serve as precursors, intermediates, and end-products of the elongation, reduction, and decarbonylation reactions of the hydrocarbon-producing pathway. To deconvolute the potentially confounding impacts of the silk microenvironment and silk development on the hydrocarbon-producing pathway, spatio-temporal cuticular lipid profiling was conducted on the agronomically important inbreds B73 and Mo17, and their reciprocal hybrids. Statistical interrogation via multivariate analyses of the metabolite abundances of the hydrocarbon-producing pathway demonstrate that the cellular VLCFA pool is positively correlated with the cuticular lipid metabolome, and this metabolome is primarily affected by the silk microenvironment and the plant genotype. Moreover, genotype has a major effect on the pathway, with increased cuticular hydrocarbon and concomitant reduction of cuticular VLCFA accumulation on B73 silks, suggesting that conversion of VLCFAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals the complexity of the product-precursor ratio relationship, demonstrating a significant role of precursor chain length. Longer-chain VLCFAs are preferentially utilized as precursors for hydrocarbon biosynthesis. Collectively, these findings demonstrate maize silks as an effective and novel system for dissection of the complex dynamics of cuticular lipid accumulation in plants.