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.