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.