Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Mesnard, François; Azaroual, Nathalie; Marty, Danielle; Fliniaux, Marc‐André; Robins, Richard J.; Vermeersch, G.; Monti, Jean‐Pierre

doi:10.1007/pl00022919

Cited by 23 publications

(1 citation statement)

References 32 publications

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“…Polyamine acetylation can also create compounds with new functions and, despite being widely viewed as accumulative products, acetylated polyamines can influence plant metabolism by acting as pathway intermediates that facilitate turnover and translocation of polyamines (Tavladoraki et al, 2012; Moschou et al, 2012). Acetylated polyamines, suggestive of SSAT activity, have been identified in several plant species (Mesnard et al, 2000; Bagni and Tassoni, 2001; Fliniaux, 2004; Hennion et al, 2012; Tavladoraki et al, 2012; Lou et al, 2016). In Arabidopsis, N 1 -acetylspermidine was reported to be as abundant as spermidine in both roots and above-ground tissue (Kamada-Nobusada et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Hameed,

Lou,

Yan

et al. 2024

Preprint

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Polyamines have vital functions in organisms, including bacteria, plants, and animals, with key roles in growth, development, and stress responses. Spermine/spermidine N1-acetyl transferases (SSATs) regulate polyamine abundance by catalysing their N-acetylation, thereby reducing the pool of polyamines and producing other bioactive components. The regulatory mechanisms controlling SSAT enzymes are incompletely understood. Here, we investigate the biological role and regulation of the two SSAT isoforms present in Arabidopsis thaliana, N-ACETYLTRANSFERASE ACTIVITY (NATA) 1 and 2. We show that NATA2 is a heat-stable isoform, induced in response to heat. Intriguingly, a nata2 knockout mutation proved beneficial for growth and pathogen defence under heat stress in Arabidopsis, aligning with the stress-mitigating effect of polyamines. In contrast, the double knockout of nata1 and nata2 was lethal, highlighting the essential role of basal SSAT activity. Our numerous crystal structures of both NATAs, supported by functional assays, revealed that stress-produced acidic metabolites can selectively inhibit polyamine acetylation by occupying the NATA substrate-binding pocket. This environment-responsive regulation mechanism may allow Arabidopsis to adjust the deleterious action of NATAs under stress conditions, without eliminating the enzyme. More generally, metabolite-ensemble inhibition may be a novel paradigm for non-genetic feedback regulation of plant enzymes.

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Section: Introductionmentioning

confidence: 99%

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Hameed,

Lou,

Yan

et al. 2024

Preprint

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Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Arroo

Bhambra

Hano

et al. 2020

Phytochemical Analysis

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Introduction Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improved and became more widely available, preference shifted the use stable ‘heavy’ isotopes like deuterium (2H)‐, carbon‐13 (13C)‐ and nitrogen‐15 (15N)‐atoms as labels. Incorporation of the labels could be monitored by mass spectrometry (MS), as part of a hyphenated tool kits, e.g. Liquid chromatography (LC)–MS, gas chromatography (GC)–MS, LC–MS/MS. MS offers great sensitivity but the exact location of an isotope label in a given metabolite cannot always be unambiguously established. Nuclear magnetic resonance (NMR) can also be used to pick up signals of stable isotopes, and can give information on the precise location of incorporated label in the metabolites. However, the detection limit for NMR is quite a bit higher than that for MS. Objectives A number of experiments involving feeding stable isotope‐labelled precursors followed by NMR analysis of the metabolites is presented. The aim is to highlight the use of NMR analysis in identifying the precise fate of isotope labels after precursor feeding experiments. As more powerful NMR equipment becomes available, applications as described in this review may become more commonplace in pathway analysis. Conclusion and Prospects NMR is a widely accepted tool for chemical structure elucidation and is now increasingly used in metabolomic studies. In addition, NMR, combined with stable isotope feeding, should be considered as a tool for metabolic flux analyses.

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Implications of ¹⁵N‐metabolic labeling for automated peptide identification in Arabidopsis thaliana

et al. 2007

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We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.

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Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Cited by 23 publications

References 32 publications

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Implications of ¹⁵N‐metabolic labeling for automated peptide identification in Arabidopsis thaliana

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Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Cited by 23 publications

References 32 publications

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Regulation of Arabidopsis polyamine acetylation by NATA1 and NATA2

Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Implications of 15N‐metabolic labeling for automated peptide identification in Arabidopsis thaliana

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Implications of ¹⁵N‐metabolic labeling for automated peptide identification in Arabidopsis thaliana