The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv. phaseolicola in tobacco.

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 NO3⁻ or NH4⁺. The speed of cell death was faster in NO3⁻-fed compared with NH4⁺-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 NH4⁺-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 NH4⁺ feeding. PR1a is a biomarker for the defence signal salicylic acid (SA), and expression was reduced in NH4⁺-fed compared with NO3⁻ 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 NO3⁻ nutrition, polyamine biosynthesis was predominant, whilst after NH4⁺ 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 NH4⁺, which also increases the availability of nutrients to pathogens, are discussed.

Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.

The 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 high-resolution 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.

Ultra performance liquid chromatography and high resolution mass spectrometry for the analysis of plant lipids.

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.

The emerging roles of nitric oxide (NO) in plant mitochondria.

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O(2), is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.