Biuret accumulation in rice seedlings
First, biuret concentration was determined in 7-day-old rice (Nipponbare) seedlings grown with 0, 0.1, and 0.3 mmol L-1 biuret supplemented in the culture solution. Visual observation showed chlorosis on the leaves of plants under 0.3 mmol L-1 biuret treatment. The root and shoot dry weights were not significantly different among treatments. However, shoot dry weights tended to decrease with an increasing biuret concentration in the culture solution (Fig. 1a). Biuret concentration in roots and shoots increased with its concentration increase in the culture solution (Fig. 1b). Consistent with the previous15N-labeled biuret uptake experiment (Ochiai et al., 2020), the biuret concentration in the shoots was higher than that in the roots, suggesting that biuret accumulated in the shoots with the transpirational volume flow.
The biuret concentration was also determined for 9-day-old transgenic rice lines overexpressing bacterium biuret hydrolase under the control of a modified CaMV35S promoter. The dry weight of the wild-type plants was significantly reduced by 0.3 mmol L-1biuret in the culture solution (Fig. 1d). In contrast, biuret injury was alleviated in the biuret hydrolase overexpressing lines grown in the same container (Fig. 1d, Supplemental Fig. S1). Biuret was not detected in biuret hydrolase overexpressing plants grown with 0.3 mmol L-1 biuret (Fig. 1f). The results indicated that excess biuret in plants was the cause of biuret injury.