Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi

Abstract

Lipid transfer from host plants to arbuscular mycorrhiza fungi was hypothesized for several years because sequenced arbuscular mycorrhiza fungal genomes lack genes encoding cytosolic fatty acid synthase ( Wewer et al., 2014 ; Rich et al., 2017 ). It was finally shown by two independent experimental approaches ( Jiang et al., 2017 ; Keymer et al., 2017 ; Luginbuehl et al., 2017 ). One approach used a technique called isotopolog profiling ( Keymer et al., 2017 ). Isotopologs are molecules, which differ only in their isotopic composition. For isotopolog profiling an organism is fed with a heavy isotope labelled precursor metabolite. Subsequently, the labelled isotopolog composition of metabolic products is analysed via mass spectrometry. The detected isotopolog pattern of the metabolite(s) of interest yields information about metabolic pathways and fluxes ( Ahmed et al., 2014 ). The following protocol describes an experimental setup, which enables separate isotopolog profiling of fatty acids in plant roots colonized by arbuscular mycorrhiza fungi and their associated fungal extraradical mycelium, to elucidate fluxes between both symbiotic organisms. We predict that this strategy can also be used to study metabolite fluxes between other organisms if the two interacting organisms can be physically separated.

Keywords: Arbuscular mycorrhiza; Inter-organismic lipid transfer; Isotopolog profiling; Lotus japonicus; MSR medium; Nurse plant system; Rhizophagus irregularis; Root organ culture; Stable isotope labelling.

Figure 1.. Cultivation of Lotus japonicus seedlings.

A and C. Lotus japonicus seeds are carefully scarified with sandpaper inside a mortar until the seeds turn slightly grey. Seeds before (B) and after (D) scarification. E. Imbibed seeds on 0.8% Bacto agar plates. F. Cultivation of seedlings in dark-coloured flat boxes (16 h light/8 h dark for 10 days). G. Seedlings ready for transfer to 2-compartement setups.

Figure 2.. Growth system setup.

A. Cultivated Lotus japonicus plants in the split Petri dish growth system at 4 weeks post placement. B. Example of a black carton envelope to keep the plate with roots and fungus in the dark. The red adhesive tape serves to close the opening of the paper envelope. C-D. Completed setup ready for incubation without (C) or with (D) Eppendorf tubes to prevent the seedlings from drying out.

Figure 3.. Schematic representation of the istotopolog profiling pipeline.

Analysis of samples via GC/MS SIM results in chromatograms of derivatized fatty acids. The individual mass spectra of 16:0 and 16:1 ω5 are extracted and the isotopomer distribution as well as the ¹³C overall excess (o.e) of these fatty acids is calculated. The isotopomer patterns can be displayed as stacked bars.