Language：English   Publishing type：Research paper (scientific journal)  

To understand how the plant regulates metabolism, it is important to determine where metabolites localize in the tissues and cells. Single-cell level omics approaches in plants have shown remarkable development over the last several years, and this data has been instrumental in gene discovery efforts for enzymes and transporters involved in metabolism. For metabolomics, Imaging Mass Spectrometry (IMS) is a powerful tool to map the spatial distribution of molecules in the tissue. Here, we describe the methods which we used to reveal where secondary metabolites, primarily alkaloids, localize in Catharanthus roseus stem and leaf tissues.

DOI： 10.1007/978-1-0716-2349-7_2

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Language：English   Publishing type：Research paper (scientific journal)  

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

DOI： 10.1093/plphys/kiab285

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The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

DOI： 10.1021/acs.orglett.1c00206

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Language：English   Publishing type：Research paper (scientific journal)  

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

DOI： 10.1111/nph.16138

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Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.56.70

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Language：English   Publishing type：Research paper (scientific journal)  

We have examined the changes due to Cd treatment in the vacuolar form in root tip cortical cells in Arabidopsis thaliana employing a transformant with GFP fused to a tonoplast protein. A Cd-induced enhancement in complexity with general expansion of vacuolar system within 24 h was evident. The changes in the vacuolar form were dependent on the applied Cd concentrations. Concomitantly, as revealed through dithizone staining, Cd accumulated in the seedling roots exhibiting abundance of Cd-dithizone complexes in root tip, root hairs and vasculature. To get insight into the involvement of SNARE protein-mediated vesicle fusion in Cd detoxification, the magnitude of Cd toxicity in a couple of knock out mutants of the vacuolar Qa-SNARE protein VAM3/SYP22 was compared with that in the wild type. The Cd toxicity appeared to be comparable in the mutants and the wild type. In order to analyze the Cd effects at cellular level, we treated the Arabidopsis suspension-cultured cells with Cd. Cd, however, did not induce a change in the vacuolar form in suspension-cultured cells although Cd measured with ICP-MS was obviously taken up into the cell. The V-ATPase activity in the microsomal fractions from vacuoles isolated from A. thaliana suspension cultured cells remained unaffected by Cd. Changes in the levels of certain metabolites of Cd-treated cells were also not so distinct except for those of glutathione. The significance of findings is discussed.

DOI： 10.1016/j.plaphy.2017.02.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Significance

Terpenoid indole alkaloids are known to be valuable bioactive compounds. In situ RNA hybridization of gene expression of the terpenoid indole alkaloid (TIA) synthetic enzymes has suggested that the TIA metabolic pathway in Catharanthus roseus stem tissue involves the successive metabolic flow of four types of cells: internal phloem-associated parenchyma, epidermal, idioblast, and laticifer cells. It has never been directly determined in which of these cells these TIA intermediates are localized. The present study showed, using both Imaging mass spectrometry (MS) and Single-cell MS, that many kinds of TIA intermediates, including catharanthine and serpentine, were accumulated in idioblast and laticifer cells. The developed methods should prove useful for studying other aspects of secondary metabolism in plants.

DOI： 10.1073/pnas.1521959113

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Other Link： https://pnas.org/doi/pdf/10.1073/pnas.1521959113

Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Plant Morphology  

DOI： 10.5685/plmorphol.28.23

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