Allantoin accumulation in rice seedlings
The allantoin concentration in rice plants determined by HPLC was
consistent with that measured by the colorimetric method (Supplementary
Fig. S2). The allantoin concentration in 7-day-old wild-type rice was
higher in the roots than in the shoots. However, the allantoin
concentration in roots did not differ among treatments. On the other
hand, the shoot allantoin concentration was significantly higher in
plants under 0.3 mmol L-1 biuret treatment than in
control and 0.1 mmol L-1 biuret-treated plants. The
shoot total N concentrations were 51.7, 49.8, and 49.4 mg
g-1 dw for plants grown with 0, 0.1, and 0.3 mmol
L-1 biuret, and there was no significant difference
among treatments (p < 0.05, Tukey’s test).
The shoot allantoin concentration of the 9-day-old biuret
hydrolase overexpressing plants under 0.3 mmol L-1biuret toxicity was not different from that of the control (Fig. 1f).
This indicated that there was no increase in allantoin accumulation in
the absence of injury. The root allantoin concentration of
biuret-treated plants was lower than that of the control in the
wild-type and line B-2-3-3 plants (Fig. 1f).
In the purine degradation pathway, the primary substrate xanthine is
oxidized to uric acid by xanthine dehydrogenase (XDH). Uric acid is then
oxidized to allantoin via 5-hydroxyisourate and
2-oxo-4-hydroxy-4-carboxy-5-ureido-imidazoline. These steps are
catalyzed by two enzymes, uric acid oxidase (UOX) and bifunctional
allantoin synthase (ALNS). Allantoin is then hydrolyzed to allantoic
acid by allantoinase (ALN).
First, we examined whether biuret inhibited ALN activity. In the assay
using crude extracts prepared from shoots of rice seedlings grown
without biuret and 5 mmol L-1 allantoin as the
substrate, biuret up to 5 mmol L-1 did not inhibit the
allantoic acid-producing activity of ALN (Fig. 2).
The expression levels of genes in the purine degradation pathway were
then analyzed. The relative expression level of OsXDH(Os03g0429800), OsUO (Os01g0865100), OsALNS(Os03g0390700), and OsALN (Os04g0680400) was not changed by the
0.3 mmol L-1 biuret treatment in 4 to 7-day-old
seedlings. However, there was a significant increase in the expression
level of OsUO in the 5-day-old seedlings (Fig. 3a-d).
Additionally, the expression levels of the putative allantoin
transporter gene OsUPS1 (ureide permease 1 , Os12g0503000)
and the two homologous genes OsUPS2 (Os12g0502800) andOsUPS3 (Os12g0503300) were examined. OsUPS1 is a homolog
of Arabidopsis AtUPS1 , which codes the gene of an allantoin
transporter (Desimone et al., 2002). The biuret treatment significantly
reduced the expression level of OsUPS1 in the shoots of 5-to
7-day-old seedlings (Fig. 3e). The expression levels of OsUPS2 in
the roots of 5 and 7-day-old seedlings and in shoots of 7-day-old
seedlings were significantly higher in plants grown with biuret than in
control plants.
Allantoin concentration in 8-day-old seedlings that were grown with
plants used for gene expression analysis was significantly higher in
shoots under biuret treatment than in the control plants (Fig. 3f).