4 DISCUSSION
Although biuret toxicity in crops is a well-known issue, little is known
about the physiology underlying biuret injury. Here, we analyzed for the
first time biuret uptake in rice plants quantitatively using15N-labelled biuret and revealed that a considerable
amount of biuret was taken up by wild-type rice (Table 1). As wild-type
rice plants did not show any biuret decomposing activity (Figure 3),
biuret-derived 15N is considered as biuret in plants.
Therefore, the shoot biuret concentration is approximately equal to 0.4
mmol L-1 when it is expressed on the basis of the
tissue water content. The concentration of biuret was higher in shoots
than in roots, which indicates that the amount of biuret retained in
roots was small, and that biuret was accumulated in shoots through the
transpiration stream. As biuret is a small polar molecule without
lipophilic parts, cellular membranes may be slightly permeable to
biuret. The rate of biuret uptake was calculated from the15N content in whole seedlings, and was found to be
equivalent to 0.5 µmol g-1 root dw
h-1. For comparisons, the rate of urea influx into
roots, which is largely mediated by channels and a high-affinity
transporter, were about 20 µmol g-1 root dw
h-1 in Arabidopsis thaliana (Kojima, Bhner,
Gassert, Yuan & von Wirén, 2007) and about 6 µmol g-1root dw h-1 in rice (Wang et al., 2012), when 0.3 mmol
L-1 urea was supplied as a sole N source. The observed
uptake rate of biuret was one to two orders of magnitudes lower than
that of urea. Similarly, the permeability of biuret could not be
detected at 10ºC in mouse erythrocytes that were permeable to urea
(Zhao, Sonawane, Levin & Yang, 2007). Biuret could possibly move across
membranes via simple diffusion. To evaluate biuret accumulation over a
prolonged period, we need to develop a method to detect biuret directly.
We are currently modifying HPLC methods, to separate biuret from other
UV-absorbing compounds in plants.
The overexpression of bacterial biuret hydrolase conferred biuret
tolerance to rice plants (Figure 4, Table 2). Conversely, biuret was
seemingly not metabolized, or very slowly metabolized in wild-type rice
plants. This is consistent with our enzyme assay results, obtained using
leaf crude extracts (Figure 3), and with the previous report on biuret
in orange leaves, in which biuret was detected by the eight months after
foliar application by a qualitative analysis (Impey & Jones, 1960). The
lack of an efficient decomposition pathway is probably responsible for
biuret accumulation and toxicity in rice plants. Besides, biuret
tolerance conferred by the biuret hydrolase suggested that an
injury in rice plants occurred because of the direct effects of biuret
within plants, but not from the indirect effects of biuret outside
roots.
Additionally, our results on the biuret injury in wild-type rice plants
gave some indications of mechanisms underlying biuret toxicity. In
wild-type rice seedlings, a biuret concentration of 0.1 mmol
L-1 and above in the culture solution caused a
significant reduction in the growth (Figure 1). It was roughly
consistent with the toxic concentration of biuret reported for
hydroponically grown naked barley (Funabiki Ogata & Sakamoto, 1956) and
pot cultured young citrus and avocado plants (Haas & Brusca, 1954). The
rather high dose, together with the significant accumulation of biuret
in rice shoots, suggests that biuret is moderately toxic and that biuret
might have a weak affinity with its target. The occurrence of leaf
chlorosis was observed, along with growth inhibition, in biuret-injured
rice seedlings (Figure 1). The colorless appearance of elongating young
leaves indicates that chloroplast development was impaired by excessive
biuret levels. Closely similar patterns of chlorosis were often observed
in rice seedlings exposed to cold stress (Yoshida, Kanno, Sato &
Kameya, 1996). It has been shown that cold stress especially impairs the
establishment of the plastid genetic system, during chloroplast
development in rice seedlings (Kusumi et al., 2011). Although the
mechanism by which it occurs is yet to be verified, biuret in leaves
might trigger similar downstream cellular responses. On the other hand,
we have found that biuret causes growth inhibition even in heterotrophic
suspension cells of rice (unpublished data). Hence, biuret probably
inhibits other basic metabolic processes as well. To have a better
understanding of the mechanism of biuret injury, we are planning to
analyze changes in transcript and metabolite levels in rice cells under
biuret toxicity.
The findings reported here clearly demonstrate that it is possible to
confer biuret detoxification ability on rice plants by introducing the
microbial biuret hydrolase (Figure 4, Table 2). Moreover, rice
plants overexpressing biuret hydrolase utilize ammonium-N
produced by the hydrolysis of biuret in plant cells as an additional N
source (Table 3). In soil, the decomposition rate, or the mineralization
rate, of biuret is slower than that of urea (Ogata & Funabiki, 1956;
Sahrawat, 1981). Taken together, when biuret is applied as a N
fertilizer to the transgenic rice lines that were generated here, the
fertilizer use efficiency would possibly be improved compared with that
of urea fertilization. We are currently working on soil-culture
experiments to evaluate the effect of biuret as a N fertilizer.