Discussion
No biuret was detected in biuret hydrolase overexpressing lines grown with 0.3 mmol L-1 biuret, whereas significant amounts of biuret accumulated in control plants (Fig 1). Combined with the results of previous 15N-labelled biuret uptake experiments (Ochiai et al., 2020), biuret hydrolaseoverexpressing rice plants took up biuret but decomposed most of it in plants. On the other hand, wild-type rice seemingly accumulated biuret without decomposing it. The alleviation of biuret injury in the biuret hydrolase overexpressing lines (Fig 1c, Supplemental Fig. S1) indicates that the excess accumulation of biuret in plants was the cause of injury. Under a uniform distribution of biuret in tissue water, the shoot biuret concentration in wild-type rice seedlings grown with 0.1 and 0.3 mmol L-1 biuret were found to be 0.5 and 1.8 mmol L-1, respectively. This suggests that a relatively high biuret concentration in plants is needed to cause injury, and nonspecific effects of biuret such as hydrogen bonding with polar residues are the cause of injury. The severity of biuret toxicity in wild-type plants, however, differed among trials, even when the biuret concentration in plants was nearly the same (Fig. 1), suggesting the presence of factors that enhance or alleviate biuret injury. To investigate the metabolic steps that are specifically inhibited, we plan to search for binding partners using affinity.
Biuret-injured rice shoots accumulated higher concentrations of allantoin than control plants (Fig. 1c, 1f). This was contrary to our assumption that biuret might inhibit the metabolism of ureide compounds, since biuret in our study did not inhibit ALN activity (Fig. 2). The existing knowledge about factors that affect the allantoin concentration in non-leguminous plants includes growing substrate (Wang et al., 2007), N sufficiency status or C/N ratio (Casartelli et al., 2019; Lee et al., 2018; Lescano et al., 2020), the form of N in the N source (Lescano et al., 2020), and environmental stress (Casartelli et al., 2019; Kaur et al., 2021; Lescano et al., 2016; Nourimand and Todd, 2016; Watanabe et al., 2013). In our study, there were no differences in the conditions around the roots other than the biuret concentration, and there were no differences in the total-N concentration in the shoots, indicating that the allantoin accumulation observed here occurred due to changes in the form of the assimilated N within rice seedlings.
Downregulation of the expression of ALN is often reported in plants under environmental stress, as a mechanism of allantoin accumulation (Casartelli et al., 2019; Lescano et al., 2016). In contrast, an increase in the expression level of OsALN following allantoin accumulation was observed in the shoots of N-deficient rice plants (Lee et al., 2018). In our study, the expression level ofOsALN in rice seedlings under the control condition and those grown with 0.3 mmol L-1 biuret was not different; nevertheless, the allantoin concentration was higher in the biuret-treated plants (Fig. 3d). However, in another independent experiment performed by us, the expression of the OsALN was suppressed by biuret (Fig. S3). It is, therefore, difficult to generalize the relationship between allantoin accumulation andALN expression. It most probably is influenced by the severity of the stress.
Additionally, the expression levels of the genes involved in allantoin synthesis were essentially unchanged by biuret toxicity (Fig. 3a–d). Therefore, one possible mechanism for allantoin accumulation under biuret toxicity could be the increased amount of degraded purine base. Another explanation could be the change in the distribution of allantoin within the plant by controlling transport activity. Biuret, most likely, induced suppression of the putative allantoin transporter geneOsUPS1 in the shoots (Fig. 3e). OsUPS1 is supposedly related to the long-distance transport of allantoin because it is localized to plasma membrane and expressed around vascular tissues (Redillas et al., 2019). Additionally, the expression of OsUPS2had also increased in roots and shoots at several time points because of biuret (Fig. 3f). Given that there are no previous studies on the function of OsUPS2 , the significance of this increase is unknown at this time.
No peaks corresponding to allantoin were detected in the metabolome analysis performed using rice suspension cells, irrespective of the treatment. However, another nitrogenous compound, citrulline, significantly increased in the biuret-treated cells (Fig. 5). Citruline is a non-proteinous amino acid abundant in Cucurbitaceae plants (reference) and accumulates substantially in wild watermelons (Citrullus lanatus ), native to deserts, under drought stress (Kawasaki et al., 2000). Its accumulation have also been observed inArabidopsis thaliana under low CO2 (Blume et al., 2019). Citrulline is considered a scavenger of hydroxyl radicals (Akashi et al., 2001) or NH4+ (Blume et al., 2019; Joshi and Fernie, 2017). Regarding the latter, Blume et al. (2019) suggested that photorespiration is the origin of NH4+ reassimilated as citrulline. However, NH4+ produced by protein degradation or from NH4+ providing N source may also have contributed to the assimilation of citrulline, since the rice suspension-cultured cells were grown in the dark.
To conclude, we demonstrated the accumulation of two N-rich compounds, allantoin in intact rice seedlings, and citrulline in suspension-cultured cells under biuret toxicity. Although these compounds may be involved as functional molecules in the response to biuret excess, rice plants might have to modulate N compounds to assimilate or reduce surplus NH4+ ions produced in cells because of environmental stress. We are currently conducting experiments to investigate the effect of different N supply levels and N sources on biuret injury in rice plants to clarify the significance of accumulating these N-rich compounds under biuret toxicity.