Fatty Acid Profiling of Brassica Seed Lipids

Twelve seeds from different siliques were harvested at 27, 38 and 45 DAF for each biological repeat. In total, six biological repeats were used. FA extraction was performed as previously described (Woodfield et�al. 2017 (link), Woodfield et al. 2018 (link)). Seed samples were incubated in 1.2 ml of isopropanol at 70�C for 30 min to inactivate any endogenous (phospho-) lipases. Nonadecanoic acid (19:0; Sigma, St. Louis, Missouri, USA) was used as an internal standard. Fatty acid methyl esters (FAMEs) were analyzed by an Agilent GC-MS device (5,973 inert mass spectrometer combined with 6,890 N gas chromatograph) equipped with an Agilent J&W DA-WAX capillary column (30 m � 0.25 mm � 0.25 �m; Lung et�al. 2017 (link)). The oven temperature was set to 170�C for 3 min, increased to 220�C at 4�C�min⁻¹, and held at 220�C for 15 min (Woodfield et�al. 2017 (link)). FAMEs were routinely identified by comparing the retention time of peaks with the Supelco 37 Component FAME MIX standard (Sigma) but had been identified fully in previous work (Woodfield et�al. 2017 (link)).