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).