Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO2 environment predicted for the near future will reduce nitrate utilization by C3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate

Xylem sap is the major transport route for nutrients from roots to shoots. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, while nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased whereas salicylic acid and jasmonoyl-isoleucine decreased with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Epitranscriptome changes triggered by ammonium nutrition regulate the proteome response of maritime pine roots

ABSTRACTEpitranscriptomic modifications constitute a gene expression checkpoint in all living organisms. As nitrogen is an essential element for plant growth and development, a reasonable hypothesis is that changes in the epitranscriptome may regulate nitrogen acquisition and metabolism. In this study, epitranscriptomic modifications caused by ammonium nutrition were monitored in maritime pine roots through direct RNA sequencing using Oxford Nanopore Technology. Transcriptomic responses mainly affected transcripts involved in nitrogen and carbon metabolism, defense, hormone synthesis/signaling, and translation. Global detection of epitranscriptomic marks was performed to evaluate this posttranscriptional mechanism in untreated and ammonium-treated seedlings. Increased m6A deposition in the 3’-UTR was observed in response to ammonium, which seems to be correlated with poly(A) lengths and changes in the relative abundance of the corresponding proteins. The results showed that m6A deposition and its dynamics seem to be important regulators of translation under ammonium nutrition. These findings suggest that protein translation is finely regulated through epitranscriptomic marks likely by changes in mRNA poly(A) length, transcript abundance and ribosome protein composition. An integration of multiomics data suggests that the epitranscriptome modulates responses to developmental and environmental changes, including ammonium nutrition, through buffering, filtering, and focusing the final products of gene expression.

Ammonium supply induces differential metabolic adaptative responses in tomato according to leaf phenological stage

Nitrate (NO3 -) and ammonium (NH4 +) are the main inorganic nitrogen sources available to plants. However, exclusive ammonium nutrition may lead to a stress situation characterized by growth inhibition, generally associated with a profound metabolic reprogramming. In this work, we aimed at studying how the metabolism adapts according to leaf position in the vertical axis of tomato (Solanum lycopersicum cv. M82) plants grown with NH4 +, NO3 - or NH4NO3 supply. To do so, we dissected leaf biomass composition and metabolism through an integrative analysis of metabolites, ions and enzyme activities. Under ammonium nutrition, carbon and nitrogen metabolism was more perturbed in mature leaves than in young ones, overall suggesting a trade-off between NH4 + accumulation and assimilation to preserve young leaves from ammonium stress. Moreover, NH4 +-fed plants exhibited a rearrangement of carbon partitioning, accumulating sugars and starch at the expense of organic acids, with respect to plants supplied with NO3 -. We explain such reallocation by the action of the biochemical pH-stat to compensate the differential proton production that depends on the nitrogen source provided. This work also underlines that the regulation of leaf primary metabolism is dependent on both the leaf phenological stage and the nitrogen source provided.

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).