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

Abstract

High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 μm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids and monoterpenes, indicating the capability of untargeted localization of metabolites by using high-resolution MSI platform. The employed AP-SMALDI MSI system provides significant technological advancement in the visualization of individual molecular species at the cellular level. In contrast to previous histochemical studies of tannins using unspecific staining reagents, individual gallotannin species were accurately localized and unequivocally discriminated from other phenolic components in the root tissues. High-quality ion images were obtained, providing significant clues for understanding the biosynthetic pathway of gallotannins and monoterpene glucosides and possibly helping to decipher the role of tannins in xylem cells differentiation and in the defence mechanisms of plants, as well as to investigate the interrelationship between tannins and lignins.

Figure 1

Chemical structures of selected metabolites from root extracts of P. lactiflora, (a) gallotannins and (b) monoterpene glucosides.

Figure 2

Mass spectrum acquired from a single 30 μm pixel for mass range m/z = 400–600 (a) and m/z = 950–1600 (b) from the root cross-section of P. lactiflora. Identified compounds are labeled with measured mass, compound name, and mass deviation. See Table S1 for more details.

Figure 3. MALDI images of gallotannins in the P. lactiflora root, recorded with a scanning step size ( = pixel size) of 30 μm and 10 μm, respectively.

(a) Optical image of the ½ root and (c) confined region of interest. (b) Ion images of gallotannins at 30 μm step size and 260 × 140 pixels per image, and at (d) 10 μm step size and 360 × 140 pixels per image. All ions are displayed using the same intensity scale (Gray: 0–255). The mass accuracy was better than 2 ppm (RMSE), and a bin width of m/z =  ± 5 ppm was used for image generation. Images represent the potassium adducts of the compounds listed in Table 1.

Figure 4. MALDI images of monoterpene glucosides in the P. lactiflora root, recorded with a scanning step size of 30 μm and 10 μm, respectively.

(a) Optical image of the ½ root and (c) confined region of interest. (b) Ion images of monoterpene glucosides at 30 μm step size and 260 × 140 pixels per image, and at (d) 10 μm step size and 360 × 140 pixels per image. All ions are displayed using the same intensity scale (Gray: 0–255). The mass accuracy was better than 2 ppm (RMSE), and a bin width of m/z =  ± 5 ppm was used for image generation. Each individual image represents the K⁺ adducts of the compounds listed in Table 2.

Figure 5. MALDI images of selected primary and other secondary metabolites in the P. lactiflora root, recorded with a spatial resolution of 10 μm and 360 × 140 pixels per image.

All ions are displayed using the same intensity scale (Gray: 0–255). The mass accuracy was better than 1 ppm (RMSE), and a bin width of m/z =  ± 5 ppm was used. Each individual image represents the H⁺/Na⁺/ K⁺ adducts of the compounds included in Table 3.

Figure 6. Correlation of histology and ion image in a P. lactiflora root cross section at a scanning step size of 10 μm.

(a) Optical image of a region of interest. (b–d) Overlay of optical image and individual ion images including (a) m/z 519.12632 ([PA/AL + K]⁺), (b) m/z 979.08134 ([5GG + K]⁺) and (c) m/z 1435.11422 ([8GG + K]⁺). (e) Overlay of ion images for m/z 519.12632 (blue, [PA/AL + K]⁺) and m/z 979.08134 (red, [5GG + K]⁺). (f) Overlay of ion images for 519.12632 (blue, [PA/AL + K]⁺) and m/z 1435.11422 (green, [8GG + K]⁺). (g) Overlay of ion images for m/z 979.08134 (red, [5GG + K]⁺) and m/z 1435.11422 (green, [8GG + K]⁺). The regions presenting subtle differences in xylem were marked with a white line. All ion images were generated with a bin width of ± 5 ppm.

Figure 7. PCA of 10 μm AP-MALDI MSI data from P. lactiflora root section.

(a) Optical image and 6 regions selected for PCA. (b) PCA derived from intensities of 6 regions in AP-MALDI MSI. (c) PC loadings derived from all m/z peaks of 6 regions in AP-MALDI MSI.