Gene expression analysis
Rice plants were grown with and without 0.3 mmol L-1biuret supplementation in the culture solution. Four-to seven-day-old
seedlings were harvested during the light period. Two to four plants
were combined into a single sample. Total RNA was extracted from roots
and shoots using the Plant Total RNA Extraction Miniprep System
(Viogene, Taipei, Taiwan). First-strand cDNA was synthesized from total
RNA using oligo dT primers and ReverTra Ace polymerase (Toyobo, Osaka,
Japan). Quantitative real-time RT-PCR was performed using the TP850
Thermal Cycler Dice Real Time System Single (Takara Bio, Shiga, Japan)
and THUNDERBIRD SYBR qPCR MIX (Toyobo, Osaka, Japan). Primer pairs used
for target genes were 5´-CTGGAGCGTGCTATGTTTCA-3´ and
5´-AGCTTGTGGACCACCAAAAC-3´for xanthine oxidase ,
5´-TCAGCTAAGGATGCCGAACC-3´ and 5´-ATGGTCCCGTGTGGTTCATC-3´ forurate oxidase , 5´-TAACGTCGCTCCTGGTTTCT-3´ and
5´-ACAGAGGGACATGGAAATGC-3´ for allantoin synthase ,
5´-AACGCATACCCGATGTTCAG-3´ and 5´- TGTCTTTCATCGCACGTTGC-3´ forallantoinase , and 5´-TGAACCACAGCAACACCAAT-3´ and
5´-GCCACTTCAGGCTCTTGTTC-3´ for ureide permease 1 ,
5´-CTCTGCTGTACATGCCTCCA-3´ and 5´-TGTTTGGTTCCACACTTCCA-3´ forureide permease 2 , 5´-GAGAAGAGGCGATCCATCAA-3´ and
5´-CGAGAAGAGGGAGAAGCAGA-3 for ureide permease 3 . The expression
levels of the genes were normalized to the mean expression level ofUbiquitin (primer pairs: 5´-AGAAGGAGTCCACCCTCCACC-3´ and
5´-GCATCCAGCACAGTAAAACACG-3´; Yamaji & Ma, 2009) and Actin 1(primer pairs: 5´-ATCCTTGTATGCTAGCGGTCGA-3´ and
5´-ATCCAACCGGAGGATAGCATG-3´; Caldana et al., 2007).