2.3 Determination of biuret uptake in rice plants
15N-labelled biuret (biuret-15N3, ≥ 98 atom% 15N) was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Rice seedlings were raised in the absence of biuret for 17 days. Then, seedlings with uniform sizes were picked from the mesh, and two plants were transferred to a 50-mL glass vial with 40 mL of culture solution, one day before performing the uptake experiments, to eliminate the possible effects of damaged roots. When we used transgenic lines, the presence of the transgene was confirmed beforehand by PCR, using the DNA extracted from leaf blades of the second leaf. Uptake experiments were initiated by the addition of an aliquot of15N-labeled biuret solution into the vial, to achieve a solution with a final biuret concentration of 0.3 mmol L-1. Plants were allowed to take up15N-labelled biuret for 48 h. Control plants, to which biuret was not applied, were also grown similarly.
At harvest, roots were rinsed twice in 100 mL of distilled water (3 minutes each). Plants were separated into shoots and roots, dried in an oven at 70ºC for two days, and ground into a powder using a ball mill. The N concentration in plant samples was determined using a CN analyzer (SUMIGRAPH NC-22F, Sumika Chemical Analysis Service, Osaka, Japan). The atom % of 15N was determined under contract (Shoko Science Co Ltd, Kanagawa, Japan). Biuret-derived N content in plant parts were calculated from the 15N content in samples.