Abstract
Molecular analysis by parallel tandem mass spectrometry (MS/MS) imaging contributes to the in situ characterization of biosynthetic intermediates which is crucial for deciphering the metabolic pathways in living organisms. We report the first use of TOF-SIMS MS/MS imaging for the cellular localization and characterization of biosynthetic intermediates of bioactive γ-lactones rubrynolide and rubrenolide in the Amazonian tree Sextonia rubra (Lauraceae). Five γ-lactones, including previously reported rubrynolide and rubrenolide, were isolated using a conventional approach and their structural characterization and localization at a lateral resolution of ~400 nm was later achieved using TOF-SIMS MS/MS imaging analysis. 2D/3D MS imaging at subcellular level reveals that putative biosynthetic γ-lactones intermediates are localized in the same cell types (ray parenchyma cells and oil cells) as rubrynolide and rubrenolide. Consequently, a revised metabolic pathway of rubrynolide was proposed, which involves the reaction between 2-hydroxysuccinic acid and 3-oxotetradecanoic acid, contrary to previous studies suggesting a single polyketide precursor. Our results provide insights into plant metabolite production in wood tissues and, overall, demonstrate that combining high spatial resolution TOF-SIMS imaging and MS/MS structural characterization offers new opportunities for studying molecular and cellular biochemistry in plants.
Highlights
(LC) coupled to mass spectrometry (MS), together with nuclear magnetic resonance (NMR) spectroscopy, are the most effective methods to analyze and identify plant natural products[4,5,6]
The total synthesis of rubrynolide and rubrenolide has led to stereochemical revision of the stereogenic centers[26,27,28,29], the biosynthesis of these bio-sourced γ-lactones has not been discussed since the original report[30]
Isozuihoenalide (1) shows a molecular formula of C23H40O3 determined by electrospray ionization (ESI) MS ([M + H]+, m/z 365.3051, Δm/z = 0.3 ppm) (Supplementary Fig. S2). 1H and 13C NMR spectra of 1 were similar to those of zuihoenalide[33], indicating the same β-hydroxy-γ-methylene-α,β′-unsaturated-γ-lactone skeleton (Supplementary Table S1, Figs S3 and S4)
Summary
(LC) coupled to mass spectrometry (MS), together with nuclear magnetic resonance (NMR) spectroscopy, are the most effective methods to analyze and identify plant natural products[4,5,6]. Since mass spectrometry imaging (MSI) has been well established for visualizing chemical distributions in biological samples, several studies have employed MSI to reveal the localization of plant metabolites[8,9]. We combined traditional phytochemical analysis, extraction-purification process followed by LC-MS and NMR analyses, with the novel TOF-SIMS tandem MS imaging technique to study the γ-lactone metabolic diversity in different plant organs. This original approach allows us to re-investigate the rubrynolide and rubrenolide biosynthesis in S. rubra. This strategy benefits from high mass-accuracy spectral data of isolated compounds, which facilitates metabolites identification in wood cells in TOF-SIMS imaging. The spatial 2D/3D distribution of bioactive rubrynolide, rubrenolide and their biosynthetic precursors is discussed at cellular and subcellular level in S. rubra wood samples, and in regard to the proposed revised biosynthetic route
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