Evidences towards deciphering the mode of action of dimethylpyrazole-based nitrification inhibitors in soil and pure cultures of Nitrosomonas europaea

Background: Agriculture relies on the intensive use of synthetic nitrogen (N) fertilizers to maximize crop yields, which has led to the transformation of agricultural soils into high-nitrifying environments. Nevertheless, nitrification inhibitors (Nis) have been developed to suppress soil-nitrifier activity and decrease N losses. The Nis 3,4-dimethylpyrazole phosphate (DMPP) and 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) are able to reduce N2O emissions and maintain soil NH4+ for a longer time. Although both Nls have been proven to be effective to inhibit soil nitrification, their exact mode of action has not been confirmed. We aimed to provide novel insights to further understand the mode of action of DMP-based Nis. We evaluated the performance of DMPP and DMPSA in soil and pure cultures of nitrifying bacteria Nitrosomonas europaea. Results: DMPSA did not inhibit nitrification in pure cultures of N. europaea. In the soil, we evidenced that DMPSA needs to be broken into DMP to achieve the inhibition of nitrification, which is mediated by a soil biological process that remains to be identified. Moreover, both DMPP and DMPSA are thought to inhibit nitrification due to their ability to chelate the Cu2+ cations that the ammonia monooxygenase enzyme (AMO) needs to carry on the first step of NH4+ oxidation. However, the efficiency of DMPP was not altered regardless the Cu2+ concentration in the medium. In addition, we also showed that DMPP targets AMO but not hydroxylamine oxidoreductase enzyme (HAO). Conclusions: The inability of DMPSA to inhibit nitrification in pure cultures together with the high efficiency of DMPP to inhibit nitrification even in presence of toxic Cu2+ concentration in the medium, suggest that the mode of action of DMP-based Nis does not rely on their capacity as metal chelators.This project was funded by the Spanish Government (RTI2018-094623-B-C21 MCIU/AEI/FEDER, UE), by the Basque Government (IT-932-16), and by EuroChem Agro Iberia S.L.U. Dr. Adrian Bozal-Leorri held a grant from the Basque Government (PRE-2020-2-0142)

Dimethylpyrazole-based nitrification inhibitors have a dual role in N2O emissions mitigation in forage systems under Atlantic climate conditions

[EN]Nitrogen fertilization is the most important factor increasing nitrous oxide (N2O) emissions from agriculture, which is a powerful greenhouse gas. These emissions are mainly produced by the soil microbial processes of nitrification and denitrification, and the application of nitrification inhibitors (NIs) together with an ammonium-based fertilizer has been proved as an efficient way to decrease them. In this work the NIs dimethylpyrazole phosphate (DMPP) and dimethylpyrazole succinic acid (DMPSA) were evaluated in a temperate grassland under environmental changing field conditions in terms of their efficiency reducing N2O emissions and their effect on the amount of nitrifying and denitrifying bacterial populations responsible of these emissions. The stimulation of nitrifying bacteria induced by the application of ammonium sulphate as fertilizer was efficiently avoided by the application of both DMPP and DMPSA whatever the soil water content. The denitrifying bacteria population capable of reducing N2O up to N-2 was also enhanced by both NIs provided that sufficiently high soil water conditions and low nitrate content were occurring. Therefore, both NIs showed the capacity to promote the denitrification process up to N-2 as a mechanism to mitigate N2O emissions. DMPSA proved to be a promising NI, since it showed a more significant effect than DMPP in decreasing N2O emissions and increasing ryegrass yield.This work was funded by the Spanish Government (AGL2015-64582-C3-2-R MINECO/FEDER and RTI2018-094623-B-C21 MCIU/AEI/FEDER, UE) , by the Basque Government (IT-932-16) and by EuroChem Agro Iberia S.L.-UPV/EHU 2015.0248 and 2016.0339. Ximena Huerfano was recipient of a specialization fellowship from the UPV/EHU for Ph.D. researchers

A Multi-Species Analysis Defines Anaplerotic Enzymes and Amides as Metabolic Markers for Ammonium Nutrition

Nitrate and ammonium are the main nitrogen sources in agricultural soils. In the last decade, ammonium (NH4+), a double-sided metabolite, has attracted considerable attention by researchers. Its ubiquitous presence in plant metabolism and its metabolic energy economy for being assimilated contrast with its toxicity when present in high amounts in the external medium. Plant species can adopt different strategies to maintain NH4+ homeostasis, as the maximization of its compartmentalization and assimilation in organic compounds, primarily as amino acids and proteins. In the present study, we report an integrative metabolic response to ammonium nutrition of seven plant species, belonging to four different families: Gramineae (ryegrass, wheat, Brachypodium distachyon), Leguminosae (clover), Solanaceae (tomato), and Brassicaceae (oilseed rape, Arabidopsis thaliana). We use principal component analysis (PCA) and correlations among metabolic and biochemical data from 40 experimental conditions to understand the whole-plant response. The nature of main amino acids is analyzed among species, under the hypothesis that those Asn-accumulating species will show a better response to ammonium nutrition. Given the provision of carbon (C) skeletons is crucial for promotion of the nitrogen assimilation, the role of different anaplerotic enzymes is discussed in relation to ammonium nutrition at a whole-plant level. Among these enzymes, isocitrate dehydrogenase (ICDH) shows to be a good candidate to increase nitrogen assimilation in plants. Overall, metabolic adaptation of different carbon anaplerotic activities is linked with the preference to synthesize Asn or Gln in their organs. Lastly, glutamate dehydrogenase (GDH) reveals as an important enzyme to surpass C limitation during ammonium assimilation in roots, with a disparate collaboration of glutamine synthetase (GS).The design of the study, analysis, and interpretation of data and writing of the manuscript was supported by the Basque Government [IT932-16] or GIC15/179, the Spanish Ministry of Economy and Competitiveness [AGL2015-64582-C3-2-R] and [BIO2017-84035-R]. IVM held a postdoctoral grant from the Basque Government (conv. 2018) and MDLP held a PhD grant by COLCIENCIAS (conv. 672)

Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition

Proper carbon (C) supply is essential for nitrogen (N) assimilation especially when plants are grown under ammonium (NH4+) nutrition. However, how C and N metabolic fluxes adapt to achieve so remains uncertain. In this work, roots of wheat (Triticum aestivum L.) plants grown under exclusive NH4+ or nitrate (NO3-) supply were incubated with isotope-labelled substrates ((NH4+)-N-15, (NO3-)-N-15, or [C-13]Pyruvate) to follow the incorporation of N-15 or C-13 into amino acids and organic acids. Roots of plants adapted to ammonium nutrition presented higher capacity to incorporate both (NH4+)-N-15 and (NO3-)-N-15 into amino acids, thanks to the previous induction of the NH4+ assimilative machinery. The N-15 label was firstly incorporated into [N-15]Gln via glutamine synthetase; ultimately leading to [N-15]Asn accumulation as an optimal NH4+ storage. The provision of [C-13]Pyruvate led to [C-13]Citrate and [C-13] Malate accumulation and to rapid [C-13]2-OG consumption for amino acid synthesis and highlighted the importance of the anaplerotic routes associated to tricarboxylic acid (TCA) cycle. Taken together, our results indicate that root adaptation to ammonium nutrition allowed efficient assimilation of N thanks to the promotion of TCA cycle open flux modes in order to sustain C skeleton availability for effective NH4+ detoxification into amino acids.The research leading to these results has received funding from the Basque Government (IT-932-16), the Spanish Government (AGL2015-64582-C3-2-R MINECO/FEDER) and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 334019. The authors thank the technical and human support provided by Phytotron Service (SGIker, UPV/EHU)