Genetic and environmental variation impact the cuticular hydrocarbon metabolome on the stigmatic surfaces of maize

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

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

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.This article is published as Loneman DM, Peddicord L, Al-Rashid A, Nikolau BJ, Lauter N, Yandeau-Nelson MD (2017) 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. PLoS ONE 12(7): e0180850. doi: 10.1371/journal.pone.0180850.</p

Genetic and environmental variation impact the cuticular hydrocarbon metabolome on the stigmatic surfaces of maize

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.This article is published as Dennison, Tesia, Wenmin Qin, Derek M. Loneman, Samson GF Condon, Nick Lauter, Basil J. Nikolau, and Marna D. Yandeau-Nelson. "Genetic and environmental variation impact the cuticular hydrocarbon metabolome on the stigmatic surfaces of maize." BMC plant biology 19 (2019): 430. doi: 10.1186/s12870-019-2040-3.</p

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

<div><p>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 <i>glossy1</i> 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 <i>gl1</i> seedlings, an unexpected increase in fatty acid recovery was observed in <i>gl1</i> seedlings extracted in chloroform, suggesting that chloroform extracts lipids from internal tissues of <i>gl1</i> 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.</p></div

Evaluation of extracellular surface lipids from wildtype (<i>Gl</i>) and <i>glossy1</i> (<i>gl1</i>) seedlings.

<p>A. The water adherence phenotype in <i>gl1</i> seedlings impaired in surface lipid deposition. B. Extracellular surface lipids (fatty acids, aldehydes and alcohols) extracted from wildtype and <i>gl1</i> seedlings, using chloroform or hexanes:diethyl ether (90:10) solvent systems. C. Individual alcohol constituents of different carbon-chain lengths, extracted from wildtype seedlings with hexanes:diethyl ether (90:10) or chloroform. D. Individual alcohol constituents of different carbon-chain lengths, extracted from <i>gl1</i> seedlings with hexanes:diethyl ether (90:10) or chloroform. Asterisks denote statistically significant differences between different solvents in the recovery of specific constituents, at p<0.05, according to a Tukey’s HSD test. A minimum of two replicates were used per combination of genotype and solvent type, with an N = 13. Averages ± SE are reported.</p

Optimization of extraction times for recovery of extracellular surface lipids from seedlings.

<p>Recovery of hydrocarbons (A), fatty acids (B), aldehydes (C), and alcohols (D) from seedling leaves of inbred B73 treated with hexanes:diethyl ether (90:10) or chloroform for different extraction times. Different letters above data-bars in panel B denote statistically significant differences in recoveries in response to time of extraction, at p<0.05, according to a Tukey’s HSD test. No such differences were observed for the data presented in panels A, C and D. Four replicates were used per combination of extraction period and solvent type, with an N = 48. Averages ± SE are reported.</p

Evaluation of extracellular surface lipids from B73 and Mo17 silks harvested 3- and 6-days post-silk emergence.

<p>A and B. Concentrations of extracellular surface lipid classes (hydrocarbons, fatty acids and aldehydes) from silks harvested at either 3 days (A) or 6 days (B) post-silk emergence (PSE). Surface lipid concentrations are compared between silks that were encased by the husk leaves (black square) and silks that had emerged from the husk leaves (grey square). C and D. Relative abundances (%) of alkanes (saturated), alkenes (unsaturated), and saturated and unsaturated fatty acids relative to total extracellular surface lipids on silks harvested either 3 days (C) or 6 days (D) PSE. Relative abundances are shown as stacked bars and are compared between emerged and husk-encased silks. In all cases, aldehydes comprised <1% of observed metabolites and therefore are not shown. For panels A-D, an asterisk denotes statistically significant differences between these two samples, at p<0.05 according to a Tukey’s HSD test. Four replicates were used per combination of genotype, emerged vs. husk-encased silks and days PSE, with an N = 32. Averages ± SE are reported.</p

Methods development parameters.

<p>Silk tissues were extracted with one of five different solvents for a defined period of time. Solvents are presented in order of increasing polarity, as denoted by the dielectric constants provided in brackets.</p

Optimization of extraction time for the recovery of extracellular surface lipids from silks.

<p>Recovery of total extracellular surface lipids (A), hydrocarbons (B), and fatty acids (C) from emerged maize silks of inbred B73 extracted for different periods of time with hexanes:diethyl ether (90:10), chloroform or hexanes. Aldehydes comprised <1% of observed metabolites, regardless of extraction period and therefore are not shown. The asterisks denote statistically significant differences in recoveries with different solvents, at p<0.05, according to a Tukey’s HSD test. In panels A and C, different letters above data-bars denote statistically significant differences in recoveries in response to time of extraction with a specific solvent, at p<0.05. No such differences were observed in the data presented in panel B. A minimum of three replicates were used per combination of extraction time and solvent type, with an N = 67. Averages ± SE are reported.</p

Comparison of lipid recovery from lyophilized, powderized silks extracted with different solvents.

<p>A. Total extracted lipid recovery (including hydrocarbons, fatty acids, aldehydes) from lyophilized, powderized emerged silks of inbred B73 extracted for ten minutes with the specified solvents: hexanes, hexanes:diethyl ether (95:5 and 90:10), chloroform:hexanes (1:1) and chloroform. B. Relative abundances of the observed lipid classes, hydrocarbons and fatty acids. Aldehydes were not detected above the limit of quantitation in this experiment. C. Relative abundances (%) of saturated (alkanes) and unsaturated (alkenes) relative to total hydrocarbon recovery. D. Relative abundances (%) of unsaturated 18-carbon fatty acids relative to total fatty acid recovery. Different lowercase letters above or within data-bars for a specific lipid class or constituent denote statistically significant differences in recovery with different solvents, at p<0.05, according to a Tukey’s HSD test. In panel A, uppercase letters denote statistically significant differences among total extracellular surface lipid recoveries. A minimum of four replicates were used per solvent, with an N = 28. Averages ± SE are reported.</p