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.

Keywords: Catharanthus roseus; Imaging MS; Single-cell MS; idioblast cell; terpenoid indole alkaloid.

Fig. 1.

TIA metabolic pathway in Catharanthus roseus. Purple font represents TIA enzymes localized in ECs. Green represents the TIA enzyme localized in PCs. Blue represents TIA enzymes localized in ICs and LCs. D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-Oacetyltransferase; LAMT, loganic acid O-methyltransferase; NMT, 16-methoxy-2,3-dihydro-3-hydroxy-tabersonine N-methyltransferase; SGD, strictosidine β-glucosidase; SLS, secologanin synthase; STR, strictosidine synthase; T16H, tabersonine 16-hydroxylase; TDC, tryptophan decarboxylase; 16OMT, 16-hydroxytabersonine O-methyltransferase.

Fig. 2.

MS images of C. roseus stem longitudinal section. Most TIA localized in IC and LC; (A) m/z 415.1001 (loganic acid), (B) m/z 429.1157 (loganin), (C) m/z 427.1001 (secologanin), (D) m/z 351.1703 (cathenamine), (E) m/z 353.1859 (ajmalicine), (F) m/z 349.1546 (serpentine), (G) m/z 355.2016 (stemmadenine), (H) m/z 367.2016 (16-methoxytabersonine), (I) m/z 337.1910 (catharanthine), (J) m/z 427.2227 (demethoxyvindoline), (K) MS image control, and (L) longitudinal section excited by UV. Color bar represents MS signal intensity. VB, vascular bundle. (Scale bar, 1 mm.)

Fig. S1.

Sampling of the four types of cells’ contents with nano-electrospray tip. (A) IC emitting blue fluorescence under UV excitation, before sampling. (B) The same cell after contents were sucked up with nano-electrospray tip. (C) IC, (D) LC, (E) PC, (F) EC.

Fig. S2.

MS/MS analysis of m/z 337.19. (A) Catharanthine standard (LC-MS). (B) Catharanthine standard (infusion). (C) Tabersonine standard (LC-MS). (D) Tabersonine standard (infusion). (E) IC sample (Single-cell MS). (F) LC sample (Single-cell MS). (G) PC (Single-cell MS). (H) EC (Single-cell MS). (I) Stem sample m/z 337.19 specific MS/MS fragment (infusion measurement).

Fig. S3.

MS/MS analysis of m/z 349.15. (A) Serpentine standard (LC-MS). (B) Serpentine standard (infusion). (C) IC sample (Single-cell MS). (D) LC sample (Single-cell MS). (E) Stem sample m/z 349.15 specific MS/MS fragment (infusion measurement).

Fig. S4.

MS/MS analysis of m/z 353.18. (A) Ajmalicine standard (LC-MS). (B) Ajmalicine standard (infusion). (C) IC sample (Single-cell MS). (D) LC sample (Single-cell MS). (E) Stem sample m/z 353.18 specific MS/MS fragment (infusion measurement).

Fig. S5.

MS/MS analysis of m/z 427.22. (A) IC sample (Single-cell MS). (B) LC sample (Single-cell MS). (C) Stem sample m/z 427.22 specific MS/MS fragment (infusion measurement).

Fig. S6.

MS/MS analysis of m/z 531.23. (A) Stem sample m/z 531.23 specific MS/MS fragment (LC-MS). (B) IC sample (Single-cell MS). (C) LC sample (Single-cell MS). (D) Stem sample m/z 531.23 specific MS/MS fragment (infusion measurement).

Fig. 3.

PCA of metabolome data according to cell type. (A) PCA derived from intensities of IC, LC, PC, and EC samples in Imaging MS analysis. (B) PC loadings derived from all m/z peaks of IC, LC, PC, and EC samples in Imaging MS analysis. (C) PCA derived from intensities of IC, LC, PC, and EC samples in Single-cell MS analysis. (D) PC loadings derived from all m/z peaks of IC, LC, PC, and EC samples in Single-cell MS analysis. (B and D) Blue points show monoisotopic and isotope data, and pale blue points show undefined data. Many TIA compounds were detected from IC and LC spectra. Red underbar shows TIA related ion peak. Green underbar shows iridoid related ion peak.

Fig. S7.

LC chromatogram of catharanthine specific MS/MS fragment. (A) LC chromatogram of catharanthine standard (LC-MS). (B) Extracted chromatogram of catharanthine standard specific MS/MS fragment ion (m/z 93.07). (C) Extracted chromatogram of catharanthine standard specific MS/MS fragment ion (m/z 133.07). (D) Extracted chromatogram of stem sample (m/z 337.19). (E) Extracted chromatogram of stem sample of catharanthine specific MS/MS fragment ion (m/z 93.07). (F) Extracted chromatogram of stem sample of catharanthine specific MS/MS fragment ion (m/z 133.07).

Fig. 4.

Semiquantitative analysis of TIAs calculated by Single-cell MS analysis data. (A) Serpentine was detected in IC and LC (m/z 349.15). (B) Secologanin was detected in EC (m/z 389.14). (C) Demethoxyvindoline was detected in all cell types (m/z 427.22). (D) Strictosidine was detected in IC and LC (m/z 531.23). The y axis shows percent of intensity normalized by the value of the total ion intensity of each sample. Values are the mean of three measurements (±SEM).

Fig. 5.

Cell specific localization of TIA in C. roseus stem tissue. Most TIA localized in the ICs and LCs.