1 INTRODUCTION
Urea, currently the most widely used nitrogen (N) fertilizer worldwide (IFASTAT, https://www.ifastat.org/databases), might contain biuret [(CONH2)2NH], as a common impurity. Biuret is formed by the thermal condensation of urea. It has been known since the 1950s that excessive amounts of biuret in urea fertilizers cause injury in crops (Jones, 1954; Sanford, Gowing, Young & Leeper, 1954). A wide range of crops can be potentially affected by biuret toxicity, which often manifests as leaf chlorosis and stunted growth, especially in the young seedling stage (Mikkelsen, 1990). Earlier studies indicated that biuret inhibited protein synthesis inXanthium pensylvanicum leaves (Webster, Verner & Gansa, 1957) and wheat (Triticum aestivum ) germplasms (Ogata & Yamamoto, 1959). The protein content, however, did not so much decrease in biuret-injured orange (Citrus sinensis ) leaves (Impey & Jones, 1960). It remains uncertain whether biuret has a direct effect on the protein synthetic machinery. Additionally, ultrastructural analyses showed that changes in chloroplast structure in biuret-injured leaves were similar to those in senescent leaves in grapefruit (Citrus paradise ) and orange plants (Achor & Albrigo, 2005). Moreover, biuret seems to remain unmetabolized in plants, because it was still detected in orange leaves, eight months after foliar spraying was performed (Impey & Jones, 1960). The exact mechanism underlying biuret toxicity in plants, however, is still far from being understood.
To avoid this hazard, the biuret content in fertilizers is regulated; for example, the upper limit of biuret-N in urea fertilizer is set at 2% of the total N content in Japanese law. Currently, biuret injury has become less frequent in farmers’ fields, owing to advances in the technology used for manufacturing urea fertilizers. One method for the fertilization of rice (Oryza sativa ) crops involves a single basal application of polymer-coated urea into seedling trays, which improves N use efficiency and labor efficiency. The extremely high density of coated urea fertilizer adjacent to the roots resulted in biuret toxicity, even though fertilizers that met the official standards were used (Tanahashi, Honda, Takahashi & Yano, 2003). This illustrates that the risk of biuret toxicity in crops remains latent.
Certain soil bacteria decompose biuret (Cameron, Durchschein, Richman, Sadowsky & Wackett, 2011; Esquirol et al., 2018; Jensen & Schrøder, 1965; Martinez, Tomkins, Wackett, Wing & Sadowsky, 2001; Robinson, Badalamenti, Dodge, Tassoulas & Wackett, 2018). One molecule of biuret is converted into three ammonium and two bicarbonate ions via the biodegradation pathway. The first step of biuret degradation is the hydrolysis of biuret into ammonium and allophanate, which is catalyzed by biuret hydrolase (Cameron et al., 2011). Then, allophanate could either undergo spontaneous decarboxylation to form urea under neutral and acidic conditions, or be hydrolyzed further by allophanate hydrolase into ammonium and bicarbonate (Cheng, Shapir, Sadowsky & Wackett, 2005).
Homologs of biuret hydrolase were detected in a broad range of microorganisms but remained undetected in animals and land plants (Robinson et al., 2018). Although it is not known yet how much biuret plants would take up and accumulate, the introduction of biuret hydrolase from soil bacteria might contribute to biuret detoxification in plant cells. Furthermore, if we could confer the biuret-detoxifying ability to crop plants, biuret could act as a slow-release N fertilizer and weed controller (Figure S1).
The principal aim of our study is to elucidate the physiological mechanisms underlying biuret toxicity. Additionally, we intend to confer biuret-detoxifying ability to crop plants. Here, we investigated biuret injury in rice plants. We first evaluated biuret uptake in rice plants quantitatively using 15N-labelled biuret. Further, we generated transgenic rice plants that overexpressed bacterialbiuret hydrolase and examined their biuret tolerance.