Metabolome analysis in rice suspension cells
Biuret treatment was initiated by subculturing 2 mL of seven-day-old
cell suspension into 80 mL of the medium supplemented with or without
0.3 mmol L-1 biuret. Then, cells were harvested three
and five days after subculturing. Cells were collected by suction
filtration, rinsed with distilled water, frozen with liquid nitrogen,
and stored at -80 °C until analysis. Samples were prepared in duplicate.
Metabolomic analysis was done by the Kazusa DNA Research Institute,
Kisarazu, Chiba, Japan. Briefly, rice cells were extracted with
methanol. A 5-µL aliquot of the extract was analyzed using liquid
chromatography-mass spectrometry (LC-MS). LC-MS analysis was conducted
on an Agilent 1200 series LC system (Agilent Technologies, Santa Clara,
CA, USA) equipped with a TSK-GEL ODS-100V column (5µm, 3 × 50 mm; Tosoh,
Tokyo, Japan) and connected to an LTQ ORBITRAP XL mass spectrometer
(Thermo Fisher Scientific, Waltham, MS, US). The gradient mobile phase
consisted of 0.1% formic acid in water (solution A), and acetonitrile
(solution B). MS detection was performed using positive-ion mode
electrospray ionization. The data were converted into text files using
the MSGet software (http://www.kazusa.or.jp/komics/software/MSGet) and
then organized using the PowerGet software (Sakurai et al., 2014).
Peaks detected in both the replicates of at least one of the four sample
groups were used for subsequent analysis. Statistical analyses were
performed using the R software (R Core Team, Vienna, Austria). The peak
intensities were log2 transformed and then normalized to the median of
each sample, and the missing values were replaced by half the value of
the minimum peak intensity. The Welch’s t-test was used to test the
differences between the means of the two groups. The annotation
information provided by Kazusa DNA Research Institute and a web program
Metaboanalyst 5.0 (Pang et al., 2021) was used to identify candidate
compounds for the peaks.