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Nitrogen is the most limiting nutrient for plants, and it is preferentially absorbed in the form of nitrate by roots, which adapt to nitrate fluctuations by remodelling their architecture. Although core mechanisms of the response to nitrate availability are relatively well-known, signalling events controlling root growth and architecture have not all been identified, in particular in Legumes. However, the developmental effect of nitrate in Legumes is critical since external nitrate not only regulates root architecture but also N 2 -fixing nodule development. We have previously shown that in barrel medic (Medicago truncatula), the nitrate transporter MtNPF6.8 is required for nitrate sensitivity in root tip. However, uncertainty remains as to whether nitrogen metabolism itself is involved in the MtNPF6.8-mediated response. Here, we examine the metabolic effects of MtNPF6.8-dependent nitrate signalling using metabolomics and proteomics in WT and mtnpf6.8 root tips in presence or absence of nitrate. We found a reorchestration of metabolism due to the mutation, in favour of the branched chain amino acids/pantothenate metabolic pathway, and lipid catabolism via glyoxylate. That is, the mtnpf6.8 mutation was likely associated with a specific rerouting of acetyl-CoA production (glyoxylic cycle) and utilisation (pantothenate and branched chain amino acid synthesis). In agreement with our previous findings, class III peroxidases were confirmed as the main protein class responsive to nitrate, although in an MtNPF6.8-independent fashion. Our data rather suggest the involvement of other pathways within mtnpf6.8 root tips, such as Ca 2+ signalling or cell wall methylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Potassium is a macroelement essential to many aspects of plant life, such as photosynthesis, phloem transport or cellular electrochemistry. Many enzymes in animals or microbes are known to be stimulated or activated by potassium (K + ions). Several plant enzymes are also strictly K + -dependent, and this can be critical when plants are under K deficiency and thus intracellular K + concentration is low. Although metabolic effects of low K conditions have been documented, there is presently no review focusing on roles of K + for enzyme catalysis or activation in plants. In this mini-review, we compile the current knowledge on K + -requirement of plant enzymes and take advantage of structural data to present biochemical roles of K + . This information is instrumental to explain direct effects of low K + content on metabolism and this is illustrated with recent metabolomics data., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Potassium (K) deficiency has consequences not only on cellular ion balance and transmembrane potential but also on metabolism. In fact, several enzymes are K-dependent including enzymes in catabolism, causing an alteration in glycolysis and respiration. In addition, K deficiency is associated with the induction of specific pathways and accumulation of metabolic biomarkers, such as putrescine. However, such drastic changes are usually observed when K deficiency is established. Here, we carried out a kinetic analysis with metabolomics to elucidate early metabolic events when nutrient conditions change from K-sufficiency to K-deficiency in Arabidopsis rosettes from both wild type and mutants affected in both K absorption and low-K signalling (hak5 akt1 cipk23). Our results show that mutants have a metabolomics pattern similar to K-deficient wild-type, showing a constitutive metabolic response to low K. In addition, shifting to low K conditions induces (i) changes in sugar metabolism and (ii) an accumulation of salicylic acid metabolites before the appearance of biomarkers of K deficiency (putrescine and aconitate), and such an accumulation is more pronounced in mutants. Our results suggest that early events in the response to low K conditions involve salicylic acid metabolism., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined 12 C/ 13 C, 14 N/ 15 N and H 2 O/D 2 O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A 14 N/ 15 N isotope effect ( 15 V/K ≈ 1.015, with respect to substrate NH 4 + ) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse ( 15 V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring 15 N. There was no 12 C/ 13 C kinetic isotope effect ( 13 V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water ( D V ≈ 0.5), showing that restructuration of the active site due to displacement of H 2 O by D 2 O facilitates the processing of intermediates., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Intense efforts are currently devoted to improve plant metabolomic analyses so as to describe more accurately the whole picture of metabolic pathways. Analyses based on liquid chromatography/time-of-flight mass spectrometry (LC-TOF) are now widely distributed among plant science laboratories. However, the use of reliable, sensitive LC-TOF methods to identify and quantify micromolar or inframicromolar key metabolites is often impeded by the sensitivity of the technique to sample preparation or chromatographic conditions. Typically, the sample matrix has a substantial influence on ionization efficiency and therefore, on the detectability of such compounds. Here, we describe a new method to analyze simultaneously 23 nucleotides and cofactors from plant extracts, taking advantage of solid-phase extraction (SPE) prior to injection. The influence of common m/z fragments in several metabolites and adducts is considered. We applied this method to characterise metabolic intermediates of NAD biosynthesis in Arabidopsis thaliana, using a wild-type and an engineered transgenic plant line that produces bacterial quinolinate phosphoribosyl transferase (nadc). We show that sample pre-purification with SPE is strictly required not only for compound quantification and identification but also to allow ionization of matrix-sensitive compounds (e.g. nicotinamide) or alleviate fragmentation of others (e.g. NAD). When exogenous substrate quinolinate was infiltrated into Arabidopsis leaves to increase the natural content in downstream metabolites, a clear correlation between intermediates of NAD biosynthesis was seen, showing the accuracy of our method for quantification in biological samples. Nadc plants only showed very modest changes in NAD-related metabolites and furthermore, they were associated with slightly lower photosynthetic performance and ATP production., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)