An Efficient Method for Medium Throughput Screening of Cuticular Waxes Composition In

An efficient method for medium throughput screening of cuticular waxes composition in different plant species

Metabolomics

Fernandez-Moreno, J.-P., Malitsky, S., Lashbrooke, J., Biswal, A., Racovita, R., Mellerowicz, E.J., Jetter, R., Orzaez, D., Aharoni, A. & Granell, A.

SUPPLEMENTARY FIGURES

Online Resource 1 Typical mass spectrum obtained for the different observed wax classes. The typical fragments (m/z) obtained from the rupture of the molecule are showed for the fatty acid, n-aldehyde, n-alkane, alkyl ester and n-alcohol wax classes exemplified by one wax compound in each class. Also the fragments (m/z) 73 and 75 belonging to the trimethyl silylation (TMS) are shown (see asterisk, *). Molecular mass in the positive ionization mode[M]+ and specific fragments (m/z) (e.g. [M-15]+ or [M-18]+) are also showed.

Online Resource 7Standard protocol for cuticular wax analysis in fleshy fruits. The protocol consisted in five processes: (1) harvesting and tissue sampling; (2) cuticular membrane isolation; (3) cuticular wax extraction; (4) GC separation and MS detection; and (5) identification of cuticular waxes and their quantification. One sample consisted in 12 tissue discs from 3 different fruits belonging to the same plant. It is possible to sampling 50 samples per day (process 1). To isolate the cuticle membrane it is necessary 4 days per sample (apple samples require an extra day: 5 days). It is possible to link both tissue sampling and cuticular membrane isolation in the same day (process 2). Thus, in a week it is possible to process 200 samples. These 200 samples could be extracted in a single morning (process 3). To dry them until the final volume requires 2 days (100 samples per day). Our GC run allows injecting 12 samples per day (including blanks, acid mix and alkane mix injections during the sequence). Thus, we can process 63 samples per week (200 samples in 3 weeks). Finally, identify and quantify the different compounds in the 200 samples requires 2 more days. Therefore, the timing of the process from tissue sampling to the cuticular wax composition is ~1 month for 200 samples (by a single person).EB, enzymatic buffer; TTB, sodium tetraborate decahydrated solution; ITSD, internal standard; CW, cuticular waxes.

Online Resource 8Putative salicylate derivative phenolics in hybrid aspen leaves (Populustremula x tremuloides). (a) Chromatogram view for hybrid aspen leaves. In zooms 1 (a.1) and 2 (a.2) are shown the elution peaks for the nine putative polyphenols. (b) Mass spectrum for the benzoyl salicin standard, also known as tremuloidin, analyzed by our standard method. Putative fragmentation of the molecule is provided. Those active hydroxyl groups (hydrogen in red) are trimethyl silylated (TMS) during derivatization. Red arrow represents the characteristic m/z fragment used to detect other salicylate derivative polyphenols. Typical fragment for TMS is represented by an asterisk (*). (c) Wax composition for the nine salicylate derivative polyphenols in hybrid aspen leaves.

Online Resource 9Pie chart for cuticular wax composition in tomato, apple and hybrid aspen samples. The percentage for each wax class is shown.