Determination and quantification of surface lipid metabolites in maize silks: A pathway model for surface lipid biosynthesis based on simultaneous profiling of polar and non-polar metabolites
The elongated stigmas in maize (i.e. silks) are a vital organ for the reproductive success of a major U.S. agricultural crop. These silks possess an extractible coat of surface lipids that is predominantly composed of a complex array of hydrocarbons that function to protect the silks against abiotic and biotic stresses. While hydrocarbons are most likely produced from the conversion of fatty acids, the exact biochemical mechanisms for their synthesis remain unknown. Our team is leveraging genetic variation and morphological gradients during silk development to dissect the metabolic network responsible for surface lipid accumulation.
These metabolomic efforts require a series of large-scale parallel investigations that rely on metabolite extraction and gas chromatography mass spectrometry (GC-MS) for metabolite identification and quantification. To extract and quantify the intermediate- (e.g. fatty acids and aldehydes) and end-point (e.g. hydrocarbons) surface lipid metabolites in a single extraction procedure, we have undertaken a methods development study to identify the best extraction solvent, the best incubation time, the consequences of filtering through silica, and the best GC-MS protocol. We found that a 9:1 mixture of hexanes to diethyl ether, a long incubation time, and the omission of silica performed best as judged by 1) throughput efficiency, 2) the breadth of identified metabolites, 3) the degree to which the quantifications of subsets of metabolites matched results from our previous best methods, and 4) the efficacy and efficiency of the GC-MS protocol required to differentiate the metabolites.
From this study alone, new insights into maize hydrocarbon biosynthesis have already been gained. For example, we have identified homologous series of saturated and unsaturated fatty acids, aldehydes and hydrocarbons, which suggest a decarbonylation mechanism that produces even- and odd-chain hydrocarbons from odd- and even-chain fatty acids, respectively. Moreover, we have demonstrated that a homologous series of alkenes with double bonds situated at the 7th and 9th positions are most abundant, and we have identified a corresponding series of unsaturated fatty acids with double bonds positioned at ω--7 and ω--9. Together, these data suggest that the reduction of even- and odd-numbered fatty acids (saturated or unsaturated) followed by the decarbonylation of their corresponding aldehydes represent a key pathway for hydrocarbon synthesis on maize silks.