Jms k9

The 1D- and 2D-NMR (COSY, HSQC and HMBC) spectra were obtained using an Avance 400 instrument (Bruker Co. Ltd., Bremen, Germany) with solvent signal as an internal reference. The EI-MS spectra were measured using a JMS-K9 (JEOL Co. Ltd., Tokyo, Japan). A low-vacuum scanning electron microscope (LVSEM) was used for observation of leaf surfaces by SU-1510 (Hitachi High-Technologies, Tokyo, Japan). Optical rotations were measured with a high-sensitivity SEPA 300 polarimeter (Horiba). Flash column chromatography was performed on an Isolera One (Biotage, Uppsala, Sweden).

Lipids were analyzed by a mass spectrometer, JMS-K9 (JEOL), equipped with a DB-1 column (30 m × 0.25 mm ID × 0.25 μm film) (Agilent Technologies, Santa Clara, CA, USA) under the following parameters: He gas flow, 1.5 mL/min; oven temperature, 50–200°C (50°C/min) and then 200–300°C (5°C/min). Separated samples were ionized by electron ionization at 70 eV, and a m/z range of 50–500 was detected. Representative mass spectrum data of each type of wax components were indicated (Supplementary Fig. S17). We used β-amyrin and palmitic acid as endogenous internal standards for the quantification of alkanes in Arabidopsis stem waxes and in BY-2 cells, respectively. For absolute quantification of wax components, amounts were normalized by the levels of exogenously added C15 alkane and were corrected by relative response factors determined in our condition (0.19 for primary alcohols and 1.0 for aldehydes). For statistical analysis, we used the non-parametric U-test, Steel’s test and Steel–Dwass test for alkane quantification in T1 plants because some data did not follow a normal distribution. For the data of T2 plants and BY-2 cells, we used the parametric Tukey’s test or Tukey–Kramer test.

One- and two-dimensional nuclear magnetic resonance (NMR) spectra were obtained using an Avance 400 instrument (Bruker Co., Ltd., Bremen, Germany), with a solvent signal of CDCl₃ as the internal reference. Gas chromatography-mass spectrometry (GC-MS) analyses were performed using an Agilent 7890A-5975C MSD instrument (Agilent Technologies, Santa Clara, CA, USA). Desorption electron ionization (DEI-MS) was measured using JEOL JMS-K9 (JEOL Co. Ltd., Tokyo, Japan). Headspace-gas chromatography (HS-GC) analyses were performed using an Agilent 6890 GC instrument (Agilent Technologies), which was equipped with a pulsed flame photometric detector (PFPD, model 5380, Agilent Technologies). Liquid chromatography-mass spectrometry (LC-MS) analyses were performed using an ultra-performance liquid chromatography-mass spectrometry system (LCMS-8050, Shimadzu, Kyoto, Japan), which was equipped with Lab Solutions Version 5.75. Flash column chromatography was performed on an Isolera One instrument (Biotage, Uppsala, Sweden).