Metabolite changes in rice suspension cells under biuret toxicity
We performed metabolome analysis using suspension-cultured rice cells to comprehensively investigate the changes in metabolites under biuret toxicity. Metabolites of rice suspension cells at two-time points, day 3 (d3) and day 5 (d5) after subculturing, for two treatments, biuret treatment supplied 0.3 mmol L-1 biuret and a control treatment without biuret, were analyzed using LC-MS technique. Of the 3,566 peaks detected (Supplementary Data S1), 993 peaks detected in replicates of at least one of the four sample groups were used for subsequent analysis.
Principal component analysis (PCA) was performed to compare the metabolic profiles of rice suspension cells. The first principal component (PC1) accounted for 25.3% of the total variance, the second (PC2) for 21.2%, and the third (PC3) for 15.9%. Figure 1 shows the score plots for PC1 and PC2. These two components clearly separated the four sample groups (Fig. 4). Increasing the culture period increased the PC1 scores, and the biuret group had a smaller PC1 score than the control group. Meanwhile, increasing the culture period decreased the PC2 scores, and the biuret treatment accelerated this decrease. PC1 may be a growth component and PC2 an aging component.
We then compared the mean peak intensities between the control and biuret groups. After excluding one peak that was considered an artifact due to the assignment of different ids to the same compound in different samples, 38 peaks showed significantly different intensities between the two groups (Fig. 5).
Only two of these peaks, id 310 and id 326, matched the standard compounds. They were identified as citrulline and citrulline-related compounds, indicating that citrulline accumulated in the biuret-treated rice suspension cells (Fig. 5). Citrulline is a non-proteinaceous amino acid with an ureido group, and thus, it was shown that biuret excess caused accumulation of a N-rich compound in cultured cells.
Additionally, the intensity of the peak id 1202 showed a marked increase in the number of biuret-treated cells (Fig. 5). Two possible compounds responsible for this peak were C14H20N6O5S1and C21H20O7. Peak id 1240, having nearly the same retention time and m/z value of 2.00 greater, indicated that peak 1202 was a sulfur-containing compound. Therefore, it was shown that S -adenosyl homocysteine (SAH) was highly accumulated in biuret-treated cells.
SAH is a byproduct of methylation reactions usingS -adenosylmethionine as a methyl donor and is a potential competitive inhibitor of methylation reactions. SAH is associated with DNA hypomethylation (Huang et al., 2019; Rocha et al., 2005). Thus, the accumulation of SAH observed here could be related to the upregulation of many stress-related genes in rice suspension cells under biuret toxicity (Ochiai et al., 2020).
No peaks that could be attributed to allantoin were detected in this analysis, and no differences between the treatment groups were observed.