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.