The physiological implications of urease inhibitors on N metabolism during germination of Pisum sativum and Spinacea oleracea seeds

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Abstract

The development of new nitrogen fertilizers is necessary to optimize crop production whilst improving the environmental aspects arising from the use of nitrogenous fertilization as a cultural practice. The use of urease inhibitors aims to improve the efficiency of urea as a nitrogen fertilizer by preventing its loss from the soil as ammonia. However, although the action of urease inhibitors is aimed at the urease activity in soil, their availability for the plant may affect its urease activity. The aim of this work was therefore to evaluate the effect of two urease inhibitors, namely acetohydroxamic acid (AHA) and N-(n-butyl) thiophosphoric triamide (NBPT), on the germination of pea and spinach seeds. The results obtained show that urease inhibitors do not affect the germination process to any significant degree, with the only process affected being imbibition in spinach, thus also suggesting different urease activities for both plants. Our findings therefore suggest an activity other than the previously reported urolytic activity for urease in spinach. Furthermore, of the two inhibitors tested, NBPT was found to be the most effective at inhibiting urease activity, especially in pea seedlings.

Introduction

The pollution arising from agricultural activities is currently one of the most important problems faced by many European countries. Indeed, the environmental problems associated with the use of nitrogen fertilizers have led to a compromise between the need to achieve higher productivity and a quality crop and their environmental impact. Sustainable agricultural production requires an in-depth understanding of the processes associated with the transformation of nitrogen in the soil and the different forms in which it is absorbed by the plant, and how these factors affect plant growth and development.

The use of urea as a nitrogen fertilizer has increased to such an extent in the last few years that it is currently the most widely used nitrogen fertilizer in agriculture (Prud’homme and Heffer, 2010). The main advantages arising from the use of urea as a fertilizer include its high N content (46%), its relatively low price (it is the cheapest fertilizer per unit N) and its ease of use. However, it is well known that the hydrolysis of urea in the soil due to the urease activity found therein results in a significant loss of up to 50% of the N applied due to its volatilization as ammonia (Terman, 1979).

The development of new fertilizers and new application methods, especially the use of products that can inhibit the activity of those microorganisms responsible for transformation of the N present in the soil, is therefore of great current interest. One such means of improving the efficiency of urea fertilization involves the use of urease activity inhibitors, a group of compounds that delay the hydrolysis of urea, and therefore its volatilization as ammonia, thereby prolonging the time during which it is available in the soil (Bremner and Krogmeier, 1988). However, the application of both urea and urease inhibitors may have adverse effects on the crops themselves. In previous studies (Artola et al., 2011, Cruchaga et al., 2011), we reported that the urease inhibitor NBPT can be absorbed by roots and inhibit the urease activity of the plant.

The seed, which contains the embryo of a new plant in miniature, is structurally and physiologically equipped for its role as a dispersal unit and is well provided with food reserves to sustain the growing seedling until it establishes itself as self-sufficient, autotrophic organism. Urease has been proposed to act together with arginase in the use of the seed's protein reserves during germination (Thompson, 1980). In this respect, Zonia et al. (1995) observed that urease activity increased during the germination and subsequent growth of Arabidopsis seedlings, with the urea generated by catabolism of the nitrogen reserves being recycled by the activity of arginase. It could therefore be expected that if urease plays an important role in recycling the N obtained from the seed's nitrogen reserves, the presence of a compound that inhibits its activity may have adverse effects on crop growth. Urease inhibitors can broadly be grouped into two categories: (1) substrate-like inhibitors, such as hydroxyurea (Uesato et al., 2002) and acetohydroxamic acid (AHA) (Odake et al., 1994), and (2) mechanism-based inhibitors, such as phosphorodiamidates (Faraci et al., 1995), inside this category, the N-(n-butyl) thiophosphoric triamide (NBPT) stands out as urease inhibitor more effective (Carmona et al., 1990). Likewise, the presence of more than one urease, with different functions, has been described in plants (Follmer et al., 2004) and, in a previous study our group reported that the effect of the inhibitor NBPT in pea and spinach is different (Cruchaga et al., 2011).

The effect of urease activity inhibitors on germination has mainly been studied with a view to eliminating the adverse effects produced by urea-based fertilizers when they decompose and release NH4+/NH3 as a result of the action of soil microorganisms (Dawar et al., 2010, Karamanos et al., 2004). In contrast, the effect of urease inhibitors on urea and nitrogen metabolism in seed tissues during the germination process has received very little attention. The aim of the present study was therefore to evaluate the possible effects of the urease inhibitors AHA and NBPT on germination by analysing the processes, enzymatic activity and metabolite content resulting from N metabolism in pea and spinach seeds.

Section snippets

Experimental design

Pea (Pisum sativum L., “Snap-pea”) and spinach seeds (Spinacea oleracea L., “Winter giant”) were germinated in Petri dishes (92 mm × 15 mm) sterilized previously following the procedure described by Labhilili et al. (1995). Two circular filter papers with a diameter of 9 cm soaked with 3 mL of a 0.5 μM NiCl2·6H2O solution, with or without urease inhibitor, were placed inside each dish. The NiCl2·6H2O was added to ensure maximum urease activity. Two urease inhibitors, namely N-(n-butyl) thiophosphoric

Results

The percentage of germination, root length, dry weight and DW/FW ratio obtained at day 7 post-germination are listed in Table 1. Application of the inhibitors NBPT and AHA had no significant effects in pea for any parameters analysed at any doses tested. In contrast, a significant decrease in the DW/FW ratio was observed in spinach for both inhibitors at all concentrations tested. Likewise, the root length was also found to differ, although only for treatment with AHA at 200 μM.

The effect of

Discussion

Germination involves a series of events that commences with the uptake of water by the quiescent dry seed and terminates with elongation of the embryonic axis (Bewley and Black, 1994). The urease inhibitors tested did not affect the germination percentage of either both species. Therefore, the mechanism of action of these compounds is not involved in these initial stages of the imbibition process. In contrast, the spinach DW/FW ratio was affected by the urease inhibitors. This effect may

Acknowledgements

This work was supported by the Spanish MICIIN (Grant No. AGL2009-13339-CO2-02 [to P.A.T.]). S.C. was supported by a doctoral fellowship from the Public University of Navarre.

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