Analysis of the Compositional Variation in the Epicuticular Wax Layer of Wheat (Triticum aestivum)

Abstract

Epicuticular waxes form a layer on the outer surface of all land plants and play a fundamental role in their interaction with the environment. Development of a rapid and accurate method for the characterization of these waxes could allow the use of wax composition as a novel phenotype during plant breeding and selection. In this study, the suitability of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the characterization of epicuticular waxes was investigated. This method provides a “fingerprint” of the relative abundance of all constituents in the analyte based on mass, is suitable for very high throughput, requires minimal sample preparation, and is able to provide for the characterization of even complex biological mixtures. Herein, the suitability of DART-MS for analysis of epicuticular wax was investigated through analysis of samples of extracted wax collected from the flag leaves of a 279 line association mapping population grown across four environments with two replicates in both irrigated and drought treatments. Additionally, for a subset of samples, wax was collected from glumes in order to test for differences in wax composition between tissue types. In all, a total of 3,454 wax extracts were analyzed with three technical replicates. The above analysis generated a total of 13,164 mass spectra (“fingerprints”), made at an average rate of 30 seconds each. Multivariate analyses including random forest, principal component analysis, and linear discriminant analysis, were used to identify the presence of differences between the spectra of wax from different tissues (leaves/glumes) and treatment types (irrigated/drought). The peaks best serving as predictors of sample class for each comparison were examined, and tentative identifications were made through comparison of the associated mass with literature and publicly available databases. As a separate test of concept, the ability to distinguish between the epicuticular wax compositions of individual varieties was determined through analysis of a group of closely related lines developed at CIMMYT which differed in agronomic performance.