SUMMARYThe unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled 13 C, 15 N and 34 S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a highresolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

Holistic analysis of lipids is becoming increasingly popular in the life sciences. Recently, several interesting, mass spectrometry-based studies have been conducted, especially in plant biology. However, while great advancements have been made we are still far from detecting all the lipids species in an organism. In this study we developed an ultra performance liquid chromatography-based method using a high resolution, accurate mass, mass spectrometer for the comprehensive profiling of more than 260 polar and non-polar Arabidopsis thaliana leaf lipids. The method is fully compatible to the commonly used lipid extraction protocols and provides a viable alternative to the commonly used direct infusion-based shotgun lipidomics approaches. The whole process is described in detail and compared to alternative lipidomic approaches. Next to the developed method we also introduce an in-house developed database search software (GoBioSpace), which allows one to perform targeted or un-targeted lipidomic and metabolomic analysis on mass spectrometric data of every kind.

Different forms of nitrogen (N) fertilizer affect disease development; however, this study investigated the effects of N forms on the hypersensitivity response (HR)—a pathogen-elicited cell death linked to resistance. HR-eliciting Pseudomonas syringae pv. phaseolicola was infiltrated into leaves of tobacco fed with either or . The speed of cell death was faster in -fed compared with -fed plants, which correlated, respectively, with increased and decreased resistance. Nitric oxide (NO) can be generated by nitrate reductase (NR) to influence the formation of the HR. NO generation was reduced in -fed plants where N assimilation bypassed the NR step. This was similar to that elicited by the disease-forming P. syringae pv. tabaci strain, further suggesting that resistance was compromised with feeding. PR1a is a biomarker for the defence signal salicylic acid (SA), and expression was reduced in -fed compared with fed plants at 24h after inoculation. This pattern correlated with actual SA measurements. Conversely, total amino acid, cytosolic and apoplastic glucose/fructose and sucrose were elevated in - treated plants. Gas chromatography/mass spectroscopy was used to characterize metabolic events following different N treatments. Following nutrition, polyamine biosynthesis was predominant, whilst after nutrition, flux appeared to be shifted towards the production of 4-aminobutyric acid. The mechanisms whereby feeding enhances SA, NO, and polyamine-mediated HR-linked defence whilst these are compromised with , which also increases the availability of nutrients to pathogens, are discussed.

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