Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS

Abstract

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue. A lkaloids constitute one of the largest groups of specialized metabolites, many of which have biological functions that are indispensable, not only for plants themselves but also for human health. Approximately 20% of plant species are known to contain alkaloids (1). The significant value of alkaloids as medicines or luxury items in human life has attracted widespread interest from researchers in a range of scientific fields. These researchers have extensively studied how plant-specialized metabolites are produced at cellular and tissue levels (2). The reports indicate that biosynthetic pathways of plant specialized metabolites often involve multiple cell types that are biochemically and morphologically distinct (3,4

The complexity of intercellular localisation of alkaloids revealed by single‐cell metabolomics

“…While in situ RNA hybridisation and immunocytochemical localisation support this model, cellular localisation of actual metabolites, however, has not yet been realised. Recently we have reported the cell‐specific localisation of intermediates of TIA metabolism in the stem tissue in C. roseus using Imaging mass spectrometry (Imaging MS) and live single‐cell mass spectrometry (single‐cell MS) (Yamamoto et al ., ). In our measurements, loganin and secologanin are localised in epidermal cells, consistent with the reported localisation of iridoid biosynthetic enzymes (LAMT, loganate O ‐methyltransferase; SLS, secologanin synthase) (Murata et al ., ; Guirimand et al ., ).…”

High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

“…Likewise, tissue-specific localization of free flavonoids in rhizome of liquorice was visualized in the cork layer with 10 μm laser pixel resolution6 and at 5 μm pixel resolution metabolites in wheat seed were visualized21. Recently, cell-specific localization of terpenoid indole alkaloids in the stem tissues of Catharanthus roseus was demonstrated with MALDI imaging and single-cell MS22. High spatial resolution alone is not sufficient to generate high quality ion images.…”

Single‐Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity

“…[90][91][92] Fore xample,t his technique was applied to the identification and quantitation of plant hormones in single plant cells by introducing stable isotope-labeled standards in the electrospray solution. [94] Capillary microsampling ESI-IMS-MS utilizes custom tailored capillaries and micromanipulators to extract 5-10 % of the contents from cells of interest. [94] Capillary microsampling ESI-IMS-MS utilizes custom tailored capillaries and micromanipulators to extract 5-10 % of the contents from cells of interest.…”