Figure legend
Figure 1 Effects of biuret on dry weight (a, d), biuret concentration (b, e), and allantoin concentration (c, f) in roots and shoots of 7-day-old wild-type rice plants (a–c) and 9-day-oldbiuret hydrolase overexpressing rice lines (d–f). Bars and circles represent mean and each sample, respectively. nd means not detected. (a–c) Wild-type plants were grown with 0, 0,1, and 0.3 mmol L-1 biuret supplemented in the culture solution. Ten seedlings were combined for a single sample. Different alphabets indicate significant difference among treatments in each organ (p< 0.05, Tukey’s test, n = 3). (d–f) Wild-type and two independent transgenic lines (B3-9-1 and B2-3-3) were grown with or without 0.3 mmol L-1 biuret in the culture solution. Four to six plants were combined into one sample. Gray and black bars represent control and biuret-treated plants, respectively. Asterisks indicate significant difference between the treatment (*p< 0.05; **p < 0.01, Welch’s t-test, n = 3).
Figure 2 Inhibitory effect of biuret for allantoinase activity. Crude extracts were prepared from shoots of 9-day-old rice seedlings hydroponically grown without biuret. Extracts were incubated at 30ºC with 10 mmol L-1 allantoin, 50 mmol L-1 Tricine-NaOH (pH8.0), 2mmol L-1MnSO4, and the desired concentration of biuret for 30min. The amount of allantoic acid produced from allantoin was colorimetorically determined. Same shape symbols indicate a same crude extract. Crossbars represent the mean value. The means were not significantly different among treatments at 5% level (One-way ANOVA with blocking, n = 4).
Figure 3 Relative expression of genes related to purine degradation and ureide metabolisms in roots and shoots of 4 to 7-day old rice seedlings. Rice plants were hydroponically grown under the control condition and 0.3 mmol L-1 biuret toxicity. (a-h) Relative expression levels of OsXO (a), OsUO (b),OsALNS (c), OsALN (d), OsUPS1 (e), OsUPS2(f), and OsUPS3 (g). The expression levels were normalized to the expression of Ubiquitin and Actin1 and expressed in log2 scale. Gray and black symbols indicate control and biuret treated samples, respectively. Crossbars indicate means of the three samples. (h) Allantoin concentration in 8-day-old seedlings. Ten plants were combined for a single sample. Boxes indicate mean of three samples, and symbols indicate each sample. Asterisks indicate statistically significant difference between the treatments at the time point (Welch’s t-test). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4 Principal component analysis of metabolomics profile of control and biuret-treated rice suspension cells. Rice cells were transferred into a medium without biuret or with 0.3 mmol L-1 biuret and harvested 3 and 5 days after transfer. Closed symbols indicate control cells, and open symbols indicate biuret-treated cells. Circles indicate day 3 samples, and triangles indicate day 5 samples.
Figure 5 Normalized peak intensities of differentially accumulated metabolites between control and biuret treated rice suspension cells. Peaks with significantly different intensity between the control-group and biuret-group are shown in the list (p< 0.05, Welch’s t-test, n = 4). RT column indicate retention time in second. In the formula column, the formula is shown when the formula is uniquely determined from the m/z value, blank when there are multiple possible candidates, and unknown when there are no candidates. D3C: day 3 control cell sample; D3B: day 3 biuret-treated cell sample; D5C: day 5 control cell sample; D5B: day 5 biuret-treated cell sample.