Visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging

Abstract

SUMMARYIn comparison with the technology platforms developed to localize transcripts and proteins, imaging tools for visualization of metabolite distributions in plant tissues are less well developed and lack versatility. This hampers our understanding of plant metabolism and dynamics. In this study, we demonstrate that desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of tissue imprints on porous Teflon may be used to accurately image the distribution of even labile plant metabolites such as hydroxynitrile glucosides, which normally undergo enzymatic hydrolysis by specific b-glucosidases upon cell disruption. This fast and simple sample preparation resulted in no substantial differences in the distribution and ratios of all hydroxynitrile glucosides between leaves from wild-type Lotus japonicus and a b-glucosidase mutant plant that lacks the ability to hydrolyze certain hydroxynitrile glucosides. In wild-type, the enzymatic conversion of hydroxynitrile glucosides and the concomitant release of glucose were easily visualized when a restricted area of the leaf tissue was damaged prior to sample preparation. The gene encoding the first enzyme in hydroxynitrile glucoside biosynthesis in L. japonicus leaves, CYP79D3, was found to be highly expressed during the early stages of leaf development, and the hydroxynitrile glucoside distribution in mature leaves reflected this early expression pattern. The utility of direct DESI-MSI of plant tissue was demonstrated using cryo-sections of cassava (Manihot esculenta) tubers. The hydroxynitrile glucoside levels were highest in the outer cell layers, as verified by LC-MS analyses. The unexpected discovery of a hydroxynitrile-derived di-glycoside shows the potential of DESI-MSI to discover and guide investigations into new metabolic routes.

Spatially Resolved Plant Metabolomics: Some Potentials and Limitations of Laser-Ablation Electrospray Ionization Mass Spectrometry Metabolite Imaging

“…Similar metabolic heterogeneity has been observed previously when using lower resolution imaging systems such as MALDI and desorption electrospray ionization (Shroff et al, 2008;Li et al, 2013). Together, these observations indicate how the distribution and abundance of plant metabolites in a tissue are often nonuniform.…”

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

“…Based on LC-MS/MS fragmentation patterns, accurate mass determination as well as (in particular cases) comparisons with authentic standards and NMR, in total 36 structural derivatives of the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin were identified in this study. Five of the derivatives described had been previously found in the same plant species: linamarin-6´-apioside, lotaustralin-6´-apioside, linamarin anitrile-6´-apioside and the (S) stereoisomer of lotaustralin anitrile-6´-apioside (the substitution of the CN-in (R)-lotaustralin with hydrogen results in a formal change of the absolute configuration from R to S, according to CIP conventions) in cassava [18,19,24], and dhurrin-6´-glucoside in sorghum [32]. Linustatin, the diglucoside of linamarin identified in flaxseed (Linum usitatissimum) [21] and in the rubber tree (Hevea brasiliensis) [36,37], had not hitherto been unequivocally identified in cassava although it had been suggested as a transport form of linamarin for its translocation from the site of its biosynthesis in the shoot apex and young leaves downwards to the sink in tuberous roots [16,23,38].…”

Analysis of Lichen Metabolites, a Variety of Approaches

“…Other DESI imaging experiments reported on hydrolysis of hydroxynitrile glucosides along leaves wounds of Lotus japonicus (Li et al 2013). …”