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.