Natural variation in the adjustment of primary metabolism determines ammonium tolerance in the model grass Brachypodium distachyon.

Nitrogen (N) fertilization is essential to maximize crop production. However, around half of the applied N is lost to the environment causing water and air pollution and contributing to climate change. Understanding the natural genetic and metabolic basis underlying plants N use efficiency is of great interest to reach an agriculture with less N demand and thus, more sustainable. The study of ammonium (NH4+) nutrition is of particular interest, because it mitigates N losses due to nitrate (NO3-) leaching or denitrification. In this work, we studied Brachypodium distachyon, the model plant for C3 grasses, grown with NH4+ or NO3- supply. We performed gene expression analysis in the root of the B. distachyon reference accession Bd21 and examined the phenotypic variation across 52 natural accessions through analysing plant growth and a panel of 22 metabolic traits in leaf and root. We found that the adjustment of primary metabolism to ammonium nutrition is essential for the natural variation of NH4+ tolerance, notably involving NH4+ assimilation and PEPC activity. Additionally, genome-wide association studies (GWAS) indicated several loci associated with B. distachyon growth and metabolic adaptation to NH4+ nutrition. For instance, we found that the GDH2 gene was associated with the induction of root GDH activity under NH4+ nutrition and that two genes encoding malic enzyme were associated with leaf PEPC activity. Altogether, our work underlines the value of natural variation and the key role of primary metabolism to improve NH4+ tolerance.

An optimized hydroponic pipeline for large-scale identification of wheat genotypes with resilient biological nitrification inhibition activity.

Several plant species have been reported to inhibit nitrification via their root exudates, the so-called biological nitrification inhibition (BNI). Given the potential of BNI-producing plants to sustainably mitigate N losses in agrosystems, identification of BNI activity in existing germplasms is of paramount importance. A hydroponic system was combined with an optimized Nitrosomonas europaea-based bioassay to determine the BNI activity of root exudates. The pipeline allows collecting and processing hundreds of root exudates simultaneously. An additional assay was established to assess the potential bactericide effect of the root exudates. The pipeline was used to unravel the impact of developmental stage, temperature and osmotic stress on the BNI trait in selected wheat genotypes. Biological nitrification inhibition activity appeared consistently higher in wheat at the pretillering stage as compared to the tillering stage. While low-temperatures did not alter BNI activities in root exudates, osmotic stress appeared to change the BNI activity in a genotype-dependent manner. Further analysis of Nitrosomonas culture after pre-exposure to root exudates suggested that BNI activity has no or limited bactericide effects. The present pipeline will be instrumental to further investigating the dynamics of BNI activity and to uncover the diversity of the BNI trait in plant species.

New Insights on Arabidopsis thaliana Root Adaption to Ammonium Nutrition by the Use of a Quantitative Proteomic Approach.

Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H⁺-consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways.

Elevated CO2 Induces Root Defensive Mechanisms in Tomato Plants When Dealing with Ammonium Toxicity.

An adequate carbon supply is fundamental for plants to thrive under ammonium stress. In this work, we studied the mechanisms involved in tomato (Solanum lycopersicum L.) response to ammonium toxicity when grown under ambient or elevated CO2 conditions (400 or 800 p.p.m. CO2). Tomato roots were observed to be the primary organ dealing with ammonium nutrition. We therefore analyzed nitrogen (N) and carbon (C) metabolism in the roots, integrating the physiological response with transcriptomic regulation. Elevated levels of CO2 preferentially stimulated root growth despite the high ammonium content. The induction of anaplerotic enzymes from the tricarboxylic acid (TCA) cycle led to enhanced amino acid synthesis under ammonium nutrition. Furthermore, the root transcriptional response to ammonium toxicity was improved by CO2-enriched conditions, leading to higher expression of stress-related genes, as well as enhanced modulation of genes related to signaling, transcription, transport and hormone metabolism. Tomato roots exposed to ammonium stress also showed a defense-like transcriptional response according to the modulation of genes related to detoxification and secondary metabolism, involving principally terpenoid and phenolic compounds. These results indicate that increasing C supply allowed the co-ordinated regulation of root defense mechanisms when dealing with ammonium toxicity.

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

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 N2 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 N2 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.

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).

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 (15NH4+, 15NO3-, or [13C]Pyruvate) to follow the incorporation of 15N or 13C into amino acids and organic acids. Roots of plants adapted to ammonium nutrition presented higher capacity to incorporate both 15NH4+ and 15NO3- into amino acids, thanks to the previous induction of the NH4+ assimilative machinery. The 15N label was firstly incorporated into [15N]Gln vía glutamine synthetase; ultimately leading to [15N]Asn accumulation as an optimal NH4+ storage. The provision of [13C]Pyruvate led to [13C]Citrate and [13C]Malate accumulation and to rapid [13C]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.

CO2 enrichment modulates ammonium nutrition in tomato adjusting carbon and nitrogen metabolism to stomatal conductance.

Ammonium (NH4(+)) toxicity typically occurs in plants exposed to high environmental NH4(+) concentration. NH4(+) assimilating capacity may act as a biochemical mechanism avoiding its toxic accumulation but requires a fine tuning between nitrogen assimilating enzymes and carbon anaplerotic routes. In this work, we hypothesized that extra C supply, exposing tomato plants cv. Agora Hybrid F1 to elevated atmospheric CO2, could improve photosynthetic process and thus ameliorate NH4(+) assimilation and tolerance. Plants were grown under nitrate (NO3(-)) or NH4(+) as N source (5-15mM), under two atmospheric CO2 levels, 400 and 800ppm. Growth and gas exchange parameters, (15)N isotopic signature, C and N metabolites and enzymatic activities were determined. Plants under 7.5mM N equally grew independently of the N source, while higher ammonium supply resulted toxic for growth. However, specific stomatal closure occurred in 7.5mM NH4(+)-fed plants under elevated CO2 improving water use efficiency (WUE) but compromising plant N status. Elevated CO2 annulled the induction of TCA anaplerotic enzymes observed at non-toxic NH4(+) nutrition under ambient CO2. Finally, CO2 enrichment benefited tomato growth under both nutritions, and although it did not alleviate tomato NH4(+) tolerance it did differentially regulate plant metabolism in N-source and -dose dependent manner.

Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato.

Plant ammonium tolerance has been associated with the capacity to accumulate large amounts of ammonium in the root vacuoles, to maintain carbohydrate synthesis and especially with the capacity of maintaining high levels of inorganic nitrogen assimilation in the roots. The tricarboxylic acid cycle (TCA) is considered a cornerstone in nitrogen metabolism, since it provides carbon skeletons for nitrogen assimilation. The hypothesis of this work was that the induction of anaplerotic routes of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (NAD-ME) would enhance tolerance to ammonium nutrition. An experiment was established with tomato plants (Agora Hybrid F1) grown under different ammonium concentrations. Growth parameters, metabolite contents and enzymatic activities related to nitrogen and carbon metabolism were determined. Unlike other tomato cultivars, tomato Agora Hybrid F1 proved to be tolerant to ammonium nutrition. Ammonium was assimilated as a biochemical detoxification mechanism, thus leading to the accumulation of Gln and Asn as free amino acids in both leaves and roots as an innocuous and transitory store of nitrogen, in addition to protein synthesis. When the concentration of ammonium in the nutrient solution was high, the cyclic operation of the TCA cycle seemed to be interrupted and would operate in two interconnected branches to provide α-ketoglutarate for ammonium assimilation: one branch supported by malate accumulation and by the induction of anaplerotic PEPC and NAD-ME in roots and MDH in leaves, and the other branch supported by stored citrate in the precedent dark period.