Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting

Carqueijeiro, Inês; Guimarães, Ana Luísa; Bettencourt, Sara; Martínez-Cortés, Teresa; Guedes, João Miranda; Gardner, Rui; Lopes, Telma; Andrade, Claúdia; Bispo, Cláudia; Martins, Nuno P.; Andrade, Paula Bresciani Martins de; Valentão, Patrı́cia; Valente, Inês M.; Rodrigues, José A.; Duarte, Patrícia; Sottomayor, Mariana

doi:10.1104/pp.16.01028

Cited by 17 publications

(32 citation statements)

References 15 publications

(24 reference statements)

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“…b,c). Idioblast cells and laticifer cells were easily distinguishable from parenchyma cells by blue autofluorescence emitted from the chemical compounds accumulated in these cells when the specimen was excited by UV (Mersey & Cutler, ; Carqueijeiro et al ., ). The autofluorescence of laticifer cells, which is derived mainly from the TIA serpentine, was already observed in leaf primordium (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, our single‐cell MS measurements are consistent with a previous report that showed that serpentine, ajmalicine and vindoline accumulated in the fraction of idioblast cells (Mersey & Cutler, ; Carqueijeiro et al ., ; Yamamoto et al ., ). Additionally, we also showed here that a compound of m/z 531.23 (strictosidine) localised not only in the epidermal cells but also in the idioblast cells and laticifer cells (Table ; Figs a, a).…”

Section: Discussionmentioning

confidence: 97%

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

et al. 2019

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Summary 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

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

et al. 2019

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“…So far reports on FACS and post FACS cultivation of plant protoplasts are relatively scarce [30], due to the removal of the rigid and structure providing cell wall, which otherwise stabilize the cell integrity [31–33]. The present study explores the combined use of Agrobacterium -mediated delivery of viral replicons for expression of GFP tagged gRNA/Cas9 in leaves of N. benthamiana with FACS in order to obtain protoplast populations with significantly increased gene editing.…”

Section: Introductionmentioning

confidence: 99%

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

et al. 2019

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Background CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. Results In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA , together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. Conclusions FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations. Electronic supplementary material The online version of this article (10.1186/s12896-019-0530-x) contains supplementary material, which is available to authorized users.

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“…To protect themselves from UV stress, plants biosynthesize a variety of specialized metabolites in specific intracellular compartments. However, the functional characterization of these natural products is often hampered by their low abundance and limited availability in plant tissues [ 40 ], so single-cell analysis based on microfluidics can be an effective alternative tool. In this study, we are interested in the possible role of anthocyanins as part of the complex antioxidant defense system that the plant employs to minimize oxidative damage caused by UV radiation.…”

Section: Resultsmentioning

confidence: 99%

Functional Characterization and Phenotyping of Protoplasts on a Microfluidics-Based Flow Cytometry

et al. 2022

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A better understanding of the phenotypic heterogeneity of protoplasts requires a comprehensive analysis of the morphological and metabolic characteristics of many individual cells. In this study, we developed a microfluidic flow cytometry with fluorescence sensor for functional characterization and phenotyping of protoplasts to allow an unbiased assessment of the influence of environmental factors at the single cell level. First, based on the measurement of intracellular homeostasis of reactive oxygen species (ROS) with a DCFH-DA dye, the effects of various external stress factors such as H2O2, temperature, ultraviolet (UV) light, and cadmium ions on intracellular ROS accumulation in Arabidopsis mesophyll protoplasts were quantitatively investigated. Second, a faster and stronger oxidative burst was observed in Petunia protoplasts isolated from white petals than in those isolated from purple petals, demonstrating the photoprotective role of anthocyanins. Third, using mutants with different endogenous auxin, we demonstrated the beneficial effect of auxin during the process of primary cell wall regeneration. Moreover, UV-B irradiation has a similar accelerating effect by increasing the intracellular auxin level, as shown by double fluorescence channels. In summary, our work has revealed previously underappreciated phenotypic variability within a protoplast population and demonstrated the advantages of a microfluidic flow cytometry for assessing the in vivo dynamics of plant metabolic and physiological indices at the single-cell level.

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Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting

Cited by 17 publications

References 15 publications

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

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

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

Functional Characterization and Phenotyping of Protoplasts on a Microfluidics-Based Flow Cytometry

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