Date Published: July 11, 2017
Publisher: Public Library of Science
Author(s): Derek M. Loneman, Layton Peddicord, Amani Al-Rashid, Basil J. Nikolau, Nick Lauter, Marna D. Yandeau-Nelson, Paula B. Andrade.
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.
The plant cuticle is the outermost chemical barrier between aerial portions of a plant and the external environment. The cuticle is a structure deposited by epidermal cells and consists of an insoluble polyester, cutin, which is embedded and coated with a complex chemical mixture of extracellular, non-polar and amphipathic lipids that are derived from fatty acid precursors , and can include long-chain fatty acids, primary and secondary alcohols, aldehydes, wax esters, ketones and linear hydrocarbons. These lipids are sometimes referred to as “cuticular and epicuticular waxes” and are referred to herein as “extracellular surface lipids”, to avoid confusion with waxes, which are normally very-long chain esters. Collectively, the hydrophobic nature of these surface lipids confers a functional role as a water barrier between the organism and its environment, and is thus important in modulating water status [1, 2]. The concentration and composition of the extracellular surface lipid metabolome has been shown to vary widely across organisms [3–5], as well as among organs and tissues or across stages of development within an individual organism [3, 6].
This study establishes a method for extracting extracellular surface lipids from plant tissues (i.e. maize silks and seedling leaves), with specific focus on 1) the choice of solvent for efficient extraction of non-polar and amphipathic metabolites of different polarities, and 2) extraction time. In a previous characterization of extracellular surface lipids from maize silks, it was demonstrated that hydrocarbons primarily accumulate on the silk surface and internal accumulation was not detectable . Based on this observation, hydrocarbon content reported by Perera and colleagues  was measured from silks that were first lyophilized and ground into a fine powder, and then extracted with hexanes and subsequently purified by treating with silica that would absorb more polar compounds (e.g. sugars and fatty acids). In the current study, we have developed methodology to extract and characterize non-polar and amphipathic metabolite classes that are frequent constituents of extracellular surface lipids in plants (e.g. hydrocarbons, fatty acids, aldehydes, alcohols) using a single and straightforward extraction procedure that is amenable to large sample sizes and can be applied to fresh, intact tissues. All method-development experiments were conducted on tissues from the maize inbred, B73. The solvent- and extraction time-optimized method was applied to the analysis of silks from maize inbreds B73 and Mo17, and on glossy1 seedlings that are impaired in extracellular surface lipid accumulation.
The one-step surface lipid extraction method using hexanes:diethyl ether (90:10) effectively extracts non-polar (i.e., linear hydrocarbons) and amphipathic metabolites (i.e., fatty acids, alcohols, and aldehydes) from plant tissues. Extraction with the hexanes:diethyl ether (90:10) solvent likely provides a better representation of extracellular surface lipids as compared to the traditionally used chloroform solvent, particularly in mutants that impact extracellular surface lipid composition. The optimized method allows for changes in extraction time that are amenable to handling large sample sizes in large-scale experiments without impacting metabolite recovery, and possibly limiting the potential contamination of surface lipid extracts with metabolites from internal lipid pools. Moreover, application of the extraction method further revealed the dynamic nature of the surface lipid metabolome on maize silks. Based on the reported findings, the described extraction method may have broader applicability for large-scale studies of the roles of cuticular lipids in plant systems and their importance in protection against environmental stresses, as well as the dissection of genetic and metabolic networks that underlie cuticular lipid production.