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BioSource amount
For RDRS and Hoshiyutaka, 100 seeds of each variety were selected according to the average weight
and length of seeds. After separating the husks from the seeds, the brown rice seeds obtained were
bulked and crushed by using a Retsch mixer mill MM301 at a frequency of 20 Hz for 2 min at
4 °C. Successively, the obtained powder was divided into three to four pools. For external set of
samples harvested in Akita, 100 seeds of each biological replicate were selected and crushed in the
same way as RDRS.
Sample processing and extraction
Extraction and derivatization for GC-MS One hundred milligrams of each sample was
extracted with extraction buffer [methanol/chloroform/water (3:1:1, v/v/v)] at a concentration of
100 mg/ml containing 10 stable isotope reference compounds as follows:
[2H4]-succinic acid,
[13C5,15N]-glutamic acid,
[2H7]-cholesterol,
[13C3]-myristic acid,
[13C5]-proline,
[13C12]-sucrose,
[13C4]-hexadecanoic acid,
[2H4]-1,4-butanediamine,
[2H6]-2-hydoxybenzoic acid and
[13C6]-glucose.
Each isotope compound was adjusted to a final concentration of 15 ng/µl for each 1-µl injection.
After centrifugation, a 200-µl aliquot of the supernatant (ca. 25 mg of each sample) was drawn and
transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac®
concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA). For
methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the
sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1
h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. Thirty µl of n-heptane was added
following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100
(Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen.
For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml−1 in pyridine) was added
to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated
for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. Thirty µl of n-heptane was
added following silylation. All the derivatization steps were performed in the vacuum glove box
VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen.
GC-TOF-MS conditions
One microliter of extracts (ca. 277.8µg each sample) was injected
in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwin-gen, Switzerland)
into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) equipped
with a 30 m x 0.25 mm inner diameter fused-silica capillary column with a chemically bound
0.25-µl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis.
Helium was used as the carrier gas at a constant flow rate of 1 ml min−1. The temperature
program for metabolome analysis started with a 2-min isothermal step at 80 °C and this was
followed by temperature ramping at 30 °C to a final temperature of 320 °C, which was maintained
for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 °C, respectively.
Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA. The acceleration
voltage was turned on after a solvent delay of 222 and 237 s. Data acquisition was performed on
a Pegasus III and Pegasus IV TOF mass spectrometers (LECO, St. Joseph, MI, USA) with an
acquisition rate of 30 spectra s−1 in the mass range of a mass-to-charge ratio of m/z = 60–800.
Alkane standard mixtures (C8–C20 and C21–C40) were purchased from Sigma–Aldrich (Tokyo,
Japan) and were used for calculating the retention index (RI) . The normalized response
for the calculation of the signal intensity of each metabolite from the mass-detector response
was obtained by each selected ion current that was unique in each metabolite MS spectrum to
normalize the peak response. For quality control, we injected methylstearate in every 6 samples.
Data was normalized using the CCMN algorithm.
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