urine enzyme inhibitor NBPT

Urea as a nitrogen fertilizer is the most important fertilizer in the world's agriculture. However, the urea in the soil is rapidly decomposed by the urease enzyme in the soil to decompose a large amount of nitrogen loss and reduce the utilization efficiency of urea nitrogen. At the same time, due to the hydrolysis of urea, the concentration of ammonia in the soil increases. This is toxic to seed germination and plants. The use of a urinary enzyme inhibitor to inhibit the hydrolysis of urea has been proposed as one of the important methods for solving the above problems. Urine enzyme inhibitors can increase the efficacy of surface fertilization (urea) by reducing the decomposition of ammonia to decompose ammonia.

NBPT urinary enzyme inhibitors have the following characteristics: NBPT has higher urinary enzyme inhibitory activity in common soil and climatic conditions. NBPT can reduce the risk of seed toxicity, reduce ammonia volatilization, and increase the crop yield and protein content to a considerable extent. NBPT has no damaging effects on people, crops, and people who consume and consume crops.

NBPT

Applications: Fine chemical plants.
Production conditions: There must be water, electricity, steam and other public works facilities.

Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and 15N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter 15N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway.

Introduction

Urea is the most frequently used nitrogen (N) fertilizers in the world with annual amount of over 50 million tons accounting for more than 50% of the world N fertilizer consumption (International Fertilizer Industry Association, 2008). The incredible increase in urea fertilizer use during the last decades is mainly due to its competitive price and the high N content (46% of mass), that allow reducing transport and distribution costs (Miller and Cramer, 2004).

Although experimental evidence reported the ability of plants to use urea per se when supplied through leaf application (Wittwer et al., 1963; Nicolaud and Bloom, 1998; Witte et al., 2002), a common agronomic practice is to supply urea to the crops by soil fertilization. Besides using inorganic N sources, plants, including crops, have been shown to be able to take up intact urea (for review, see Kraiser et al., 2011; Nacry et al., 2013). In particular, maize plants possess dedicated transmembrane transport systems in root cells for the acquisition of urea with high and low affinity, mediated by a DUR3 transporter and aquaporins, respectively (Gaspar et al., 2003; Gu et al., 2012; Zanin et al., 2014; Liu et al., 2015; Yang et al., 2015).

In the soil solution the stability of urea is strictly dependent on the activity of the microbial urease, a nickel-dependent enzyme ubiquitously expressed in microorganisms and released into soil (Watson et al., 1994). Moreover urease activity can persist in the soil even after the decay of the microorganisms (Watson et al., 1994). This enzyme catalyzes the hydrolysis of urea into ammonium and carbon dioxide and its activity is proportional to the microbial biomass, which in turn depends on the organic matter amount and the water content of the soil. Ammonium could remain in this form as exchangeable cation or volatilized in form of ammonia; it could also serve as a substrate for nitrification process being transformed into nitrate. Thus, at least for short periods of time, urea fertilization may result in a simultaneous exposure of plant roots to urea, ammonium and nitrate (Mérigout et al., 2008b).

Mainly due to ammonia volatilization and nitrate leaching, the rapid hydrolysis of urea would lead to a decreased N availability for plant nutrition and to a lower use efficiency of urea fertilizers (Zaman et al., 2008). So one of the most used strategies to reduce ammonia emissions from urea fertilizer is to apply urease inhibitors. Besides slowing urea hydrolysis, these molecules allow the diffusion of urea far away from the application site favoring its uptake as an intact molecule by the plant roots.

The most promising and tested soil urease inhibitor is the NBPT(trade name Agrotain®), whose activity is associated with the conversion to its oxidized form (Watson, 2005). NBPT is a structural analog of urea (Medina and Radel, 1988) acting with mixed inhibition on urease activity (increased Km and decreased Vmax; Juan et al., 2009). Molecular dynamic calculations showed that NBPT coordinates both nickel atoms of the urease active site and binds the oxygen atom of the urea-derived carbamate (Manunza et al., 1999).

It is not unusual to find marketing formulations containing urea in combination with urease inhibitor (Watson, 2005). Experimental evidence has been provided showing that the activity of urease inhibitors could be affected by environmental factors such as pH (Hendrickson and Douglass, 1993), temperature (Hendrickson and O’Connor, 1987), and soil moisture content (Sigunga et al., 2002; Clough et al., 2004).

Limited information is available on the physiological effects of NBPT in plants (Watson and Miller, 1996; Cruchaga et al., 2011). It has been reported that some species showed visible symptoms of toxicity when plants were treated with urea and NBPT with the transient development of leaf scorches and necrotic leaf margins (Watson and Miller, 1996; Artola et al., 2011; Cruchaga et al., 2011). Cruchaga et al. (2011) reported that NBPT is taken up by pea and spinach roots and translocated to the leaves; thus NBPT can conceivably inhibit the activity of endogenous leaf and root urease (Watson and Miller, 1996; Artola et al., 2011; Cruchaga et al., 2011; Ariz et al., 2012). Moreover glutamine synthetase activity and amino acid level are reduced in presence of NBPT (Artola et al., 2011; Cruchaga et al., 2011). Altogether these results showed that the urease inhibitor compromised the use of urea as a source of N for plants, but there is still a lack of knowledge on the physiological and molecular aspects of NBPT effects on the acquisition of this N source.

The aim of the current research was to study the short-term effects of NBPT on the capacity of maize plants to acquire urea. Previous studies from our group described in vivo the high affinity transport system of urea in maize roots and showed that urea quickly induce its acquisition (Zanin et al., 2014). Therefore, in the present work the action of NBPT was studied on the functionality of the inducible component of the high affinity influx system. Physiological data were supported by analysis of changes in the transcription of genes known to be modulated by urea.