Abstract
Mass spectrometry based imaging is a powerful tool to investigate the spatial distribution of a broad range of metabolites across a variety of sample types. The recent developments in instrumentation and computing capabilities have increased the mass range, sensitivity and resolution and rendered sample preparation the limiting step for further improvements. Sample preparation involves sectioning and mounting followed by selection and application of matrix. In plant tissues, labile small molecules and specialized metabolites are subject to degradation upon mechanical disruption of plant tissues. In this study, the benefits of cryo-sectioning, stabilization of fragile tissues and optimal application of the matrix to improve the results from MALDI mass spectrometry imaging (MSI) is investigated with hydroxynitrile glucosides as the main experimental system. Denatured albumin proved an excellent agent for stabilizing fragile tissues such as Lotus japonicus leaves. In stem cross sections of Manihot esculenta, maintaining the samples frozen throughout the sectioning process and preparation of the samples by freeze drying enhanced the obtained signal intensity by twofold to fourfold. Deposition of the matrix by sublimation improved the spatial information obtained compared to spray. The imaging demonstrated that the cyanogenic glucosides (CNglcs) were localized in the vascular tissues in old stems of M. esculenta and in the periderm and vascular tissues of tubers. In MALDI mass spectrometry, the imaged compounds are solely identified by their m/z ratio. L. japonicus MG20 and the mutant cyd1 that is devoid of hydroxynitrile glucosides were used as negative controls to verify the assignment of the observed masses to linamarin, lotaustralin, and linamarin acid.
Highlights
Plants synthesize a diverse range of specialized metabolites to fend off herbivores and pests and to communicate with the environment (Møller, 2010; Neilson et al, 2013)
The results indicate that a significant amount of the cyanogenic glucosides (CNglcs) content is lost using the freeze-thaw mounting technique compromising to some extent the ability to accurately assess the distribution of linamarin in the tissue section
The defense response is activated when otherwise compartmentalized CNglcs and hydrolytic enzymes are brought into contact by tissue disruption (Morant et al, 2008)
Summary
Plants synthesize a diverse range of specialized metabolites to fend off herbivores and pests and to communicate with the environment (Møller, 2010; Neilson et al, 2013). Some specialized metabolites like cyanogenic glucosides (CNglcs) may serve as storage and transport forms of reduced carbon and nitrogen used to fine tune primary metabolism (Jenrich et al, 2007; Picmanova et al, 2015; Nielsen et al, 2016; Bjarnholt et al, 2018) Understanding these interactions is key to developing robust crop plants for the future. For frozen plant samples containing CNglcs, the cryo-sectioning and freeze-thaw steps disrupt the cellular structures and results in mixing of the CNglcs with β-glucosidase enzymes leading to hydrolysis This lowers signal intensity and impairs the spatial distribution analyses. Cyanogenic glycosides (CNglcs) are specialized metabolites widely distributed throughout the plant kingdom (Gleadow and FIGURE 1 | Structures of hydroxynitrile glucosides and their endogenous turn-over products in Manihot esculenta (Me) and Lotus japonicus (Lj). Localisation of CNglcs and related metabolites with high spatial resolution within a range of tissues may be able to help shed light on some of these areas
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