Veratraldehyde

Antifungal compounds, kresoxim methyl (Kre-Me; strobilurin), octylgallate (octyl 3,4,5-trihydroxybenzoic acid; OG), veratraldehyde (3,4-dimethoxybenzaldehyde; VT), benzohydroxamic acid (BHAM) (Figure 1), were procured from Sigma Co. (St. Louis, MO, USA). Each compound was dissolved in dimethylsulfoxide (DMSO; absolute DMSO amount: <2% in media) before incorporation into culture media (except for those plates used in aflatoxin assays; see below). Throughout this study, control plates (No treatment) contained DMSO at levels equivalent to that of cohorts receiving antifungal agents, within the same set of experiments.

Tyrosinase from Agaricus bisporus, LiP from Phanerochaete chrysosporium, veratryl alcohol (96%), veratraldehyde (99%), 4-phenoxy phenol (99%), benzyl phenyl ether (98%), dimethyl sulfoxide (DMSO, 99%), and benzyl benzoate (99%) were purchased from Sigma-Aldrich (St. Louis, USA). GGE (97%) and trimethylchlorosilane (TMCS, 1%) in N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) [BSTFA + TMCS] were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan) and Supelco analytical (Bellefonte, USA), respectively. To remove trace impurities such as metal salts, tyrosinase was solubilized in phosphate buffer (50 mM, pH 6.5) and then purified with an ultrafiltration device with a cutoff of 10 kDa (Merck Millipore, MA, USA). All other chemicals were used as the highest grade available without further purification.

Leaf, bulb, and inflorescence tissues were collected from adult blooming N. sp. aff. pseudonarcissus and Galanthus sp. plants in St. Louis, MO and Galanthus elwesii in Pullman, WA. Chemicals acquired from Sigma Aldrich include ammonium acetate 97% A.C.S. reagent, HPLC grade ethanol, catalase from bovine liver, and tyramine 99%. Other chemicals purchased include ammonium acetate extra pure 25% solution in water and hydrogen peroxide 35 wt % solution in water from Acros Organics, ampicillin from GoldBio, and vanillin from Merck. Several compounds were obtained from our natural product collection including: veratraldehyde (can be acquired from Sigma), norbelladine, 4′-O-methylnorbelladine, (R)-coclaurine, and (S)-coclaurine (Battersby et al., 1964 (link); Teitel and Brossi, 1968 (link); Park, 2014 (link); Ruiz-Olalla et al., 2015 (link)). Haemanthamine was previously isolated from Narcissus pseudonarcissus. Additional materials are as described previously (Gesell et al., 2009 (link); Kilgore et al., 2014 (link)). Plant alkaloids were extracted with 70% ethanol as previously described (Kilgore et al., 2014 (link)).