A robust and efficient method for the extraction of plant extracellular surface lipids as applied to the analysis of silks and seedling leaves of maize

Abstract

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.

A multigenotype maize silk expression atlas reveals how exposure‐related stresses are mitigated following emergence from husk leaves

“…Indeed, silks exhibit a high level of phenotypic variability in emergence rates across diverse germplasm under drought (Bolaños & Edmeades, 1996), as well as differences in susceptibility to different pathogens (Lübberstedt, Klein, & Melchinger, 1998) and pests (Abel, Wilson, Wiseman, White, & Davis, 2000;Lopez et al, 2019). Moreover, the composition of many specialized metabolites varies among cultivars, including maysin (Byrne et al, 1996;Szalma, Buckler, Snook, & McMullen, 2005) and cuticular lipids (Dennison et al, 2019;Loneman et al, 2017;Perera et al, 2010). The emergence of plant tissues, such as during seedling emergence from the ground or monocot leaf emergence from a whorl, introduces microenvironmental changes and therefore new stresses.…”

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition

“…Specifically, in juvenile leaves of maize seedlings, the reductive pathway predominates, and the expression of this metabolic network is under the control of the juvenile-to-adult phase transition, mediated by the transcription factor Gl15 (Moose and Sisco, 1996). By contrast, the fatty acid elongation-decarbonylative pathway is primarily expressed in silks (Perera et al, 2010;Loneman et al, 2017;Dennison et al, 2019). The gl mutations that have been characterized since the early-1900s have been identified via phenotypic screens of seedling leaves (Hayes and Brewbaker, 1928), which therefore primarily affect the fatty acid elongation-reductive pathway.…”

GC-MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum Cultivars

“…While splitless mode can result in large peaks with plateaus and tails, split mode generates metabolite profiles that are better suited for quantitative comparisons. Moreover, the GC oven temperature program is critical for separation of wax compounds [ 24 , 25 , 26 ]. The relatively short temperature ramp used here allowed for quick discrimination of a complex mixture of waxes from a wide range of mass-to-charge ratio values (i.e., 50–650 m / z ) ( Table 1 and Table 2 ), and provided quick sample-to-sample run times, thus reducing analytical error and improving quantitative comparisons among samples.…”

“…Rapid dips in hexane can solubilize wax alkanes, primary alcohols, fatty acids, ketones, and aldehydes that are often known for their agronomic importance [ 10 , 21 , 22 , 23 ]. Further, several studies have compared gas chromatography injection methods (e.g., split ratios) and oven temperatures necessary for wax compounds [ 24 , 25 , 26 ].…”