FIGURE LEGENDS
FIGURE 1 Symptoms of biuret toxicity in rice plants. Rice seedlings were hydroponically grown in a culture solution supplemented with 0, 0.1, 0.3, and 1.0 mmol L-1 biuret. (a) Plant heights of 7-day-old seedlings. The data represent means ± SD (n = 22-24). Different alphabets indicate significant differences between groups (p < 0.05, Tukey test). (b) Representative images of 7-day-old seedlings.
FIGURE 2 Relative expression levels of biuret hydrolasein transgenic rice plants.
The expression in the youngest leaf at the vegetative growth stage was determined via real-time PCR. Ubiquitin and Actin1 were used as internal standards for normalization. The values are means of two technical replicates. In names of transgenic lines, the combination of the first and second numbers indicates a callus derived from a single seed, and the third number indicates an individual plant regenerated from the callus.
FIGURE 3 Biuret-decomposing activities of crude extracts of rice plants.
Crude extracts were prepared from shoots of individual 19-day-old wild-type Nipponbare (solid boxes, n = 3) and transgenic B3-9-1 (solid circles, n =2) plants and 16-day-old B2-3-3-3 (open circles, n = 2). Extracts were incubated at 30ºC with 50 mmol L-1sodium phosphate buffer (pH 8) and 3 mmol L-1 biuret. The reaction was stopped by adding 0.5 mol L-1H2SO4. The amount of ammonia released from biuret was determined using indophenol blue. The data represent means ± SD.
FIGURE 4 Biuret tolerance of a transgenic rice line overexpressing exogenous biuret hydrolase . Biuret treatment (0 and 0.3 mmol L-1) was started two days after the sowing process. Photos were taken 9 days after sowing. Wild-type rice plants (left panel) and transgenic rice plants (line B2-3-3-3, right panel) grown without and with biuret.
Table 1 15N-Biuret uptake in rice plants. Nineteen-day-old Nipponbare seedlings were exposed to 0.3 mmol L-1 15N-biuret for 48 hr. Two plants were maintained in a 40-mL vial. Values are expressed as means ± SD (n = 3).
Dry weight Total-N Biuret-derived N
(mg vial-1) (mmol g-1 DW) (µmol g-1 DW) (µmol vial-1)
Shoots 124 ± 3.88 2.43 ± 0.09 13.5 ± 1.02 1.67 ± 0.18
Roots 23.8 ± 1.06 1.75 ± 0.01 5.83 ± 0.55 0.13 ± 0.01