Xcalibur software suite

The first leaves of 14-d-old plants and spikes from mature plants were dipped twice into 99% HPLC molecular-grade chloroform for 15 s and then 10 s, an internal standard of 0.5 μg.mL^–1 tetracosane (C₂₄n-alkane) was then added. Lemma awns were trimmed from the spikes prior to the analysis. Solvent was removed from the wax extracts under a gentle stream of N₂. Wax residues were derivatised using N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylcholorosilane (BSTFA + 1% TMCS; Sigma-Aldrich) and excess reagent removed again under N₂. The wax constituents were identified by their electron-impact mass spectra (70 eV, m/z 50–950) after capillary GC (Restek Rxi-1HT, 15 m × 0.32 mm, 0.1 μm [Thames Restek Ltd.]) performed on an ISQ-LT GC–MS comprising a 1300 gas chromatograph combined with a single quadrupole mass analyser (Thermo-Fisher Scientific). The samples were injected onto the analytical column at 50 °C, ramping at 0.2 °C s^–1 to a maximum of 380 °C, where it was held for 7 min. The flow rate of the He carrier gas was 5 mL min^–1. All data were acquired using the XCalibur software suite (Thermo Fisher Scientific). Leaf area and spike length were calculated using ImageJ^{87 (link)}.

Nano electrospray-ionization mass spectrometric experiments were performed on an LTQ Orbitrap XL instrument (Thermo Fisher Scientific, Bremen, Germany) with Econo12 Glass PicoTips (New Objective, Littleton, MA, USA) as electrospray emitters. Mass spectrometry was performed in positive ionization mode with spray voltages ranging from 0.8 to 1.3 kV, tube lens voltage of 250 V, capillary voltage of 20 V, and a capillary temperature of 200 °C. The mass spectrometer was used in FTMS mode at a resolution of 100,000. HCD experiments were performed with precursor ion isolation windows in the range of 3 to 8 m/z. Collision energies ranged between 0% and 50% NCE and were converted to eV [45 (link),46 (link)]. Data processing was performed using the Xcalibur software suite (Thermo Fisher Scientific, Bremen, Germany).