Tracing the fate and transport of secondary plant metabolites in a laboratory mesocosm experiment by employing mass spectrometric imaging

Abstract

Mass spectrometric imaging (MSI) has received considerable attention in recent years, since it allows the molecular mapping of various compound classes, such as proteins, peptides, glycans, secondary metabolites, lipids, and drugs in animal, human, or plant tissue sections. In the present study, the application of laser-based MSI analysis of secondary plant metabolites to monitor their transport from the grass leaves of Dactylis glomerata, over the crop of the grasshopper Chorthippus dorsatus to its excrements, and finally in the soil solution is described. This plant-herbivore-soil pathway was investigated under controlled conditions by using laboratory mesocosms. From six targeted secondary plant metabolites (dehydroquinic acid, quinic acid, apigenin, luteolin, tricin, and rosmarinic acid), only quinic acid, and dehydroquinic acid, an in-source-decay (ISD) product of quinic acid, could be traced in nearly all compartments. The tentative identification of secondary plant metabolites was performed by MS/MS analysis of methanol extracts prepared from the investigated compartments, in both the positive and negative ion mode, and subsequently compared with the results generated from the reference standards. Except for tricin, all secondary metabolites could be tentatively identified by this approach. Additional liquid-chromatography mass spectrometry (LC-MS) experiments were carried out to verify the MSI results and revealed the presence of quinic acid only in grass and chewed grass, whereas apigenin-hexoside-pentoside and luteolin-hexoisde-pentoside could be traced in the grasshopper body and excrement extracts. In summary, the MSI technique shows a trade-off between sensitivity and spatial resolution. Graphical abstract Monitoring quinic acid in a mesocosm experiment by mass spectrometric imaging (MSI).

Keywords: Laser desorption/ionization; Mass spectrometric imaging; Mesocosm; Secondary plant metabolites; Time-of-flight.

Graphical abstract

Monitoring quinic acid in a mesocosm experiment by mass spectrometric imaging (MSI).

Fig. 1

Laboratory mesocosm experiment: The photographs show a the mesocosms with the grown grass species D. glomerata in the climatic chamber; b a grasshopper from the species C. dorsatus; and c its feces. Scale bar 5 mm (c)

Fig. 2

MALDI-TOF MSI analysis of a D. glomerata leaf. a Optical image taken before the matrix application, and ion images (green color) overlaid onto the optical image showing the distribution of b quinic acid (m/z 215), c apigenin (m/z 271), d luteolin (m/z 309), e tricin (m/z 331), and f rosmarinic acid (m/z 383) Note: The assignments of the secondary plant metabolites are tentative based on the MS and MS/MS data of reference standards, as presented in the ESM, and additional MS and MS/MS experiments of methanol extracts of D. glomerata leaves, as shown in Figs. 3 and 4, respectively. The ion images are normalized using the TIC. Scale bar 5 mm (a)

Fig. 3

LDI-TOF MS and MS/MS spectra of a methanol extract from D. glomerata leaves in the negative ion mode for the identification of the secondary metabolites monitored in the ion images in Fig. 2. The MS/MS spectra of b quinic acid; c apigenin; d luteolin; e tricin; and f rosmarinic acid show the same diagnostic ions as for the reference substances summarized in Table 1

Fig. 4

LDI-TOF MS and MS/MS spectra of a methanol extract from D. glomerata leaves in the positive ion mode for the identification of the secondary metabolites monitored in the ion images in Fig. 2. The ion images in Fig. 2 were as well recorded in the positive ion mode. The MS/MS spectra of b quinic acid; c apigenin; d luteolin; e tricin; and f rosmarinic acid show the same diagnostic ions as for the reference substances summarized in Table 1

Fig. 5

LDI-TOF MSI analysis of the gastrointestinal tract of a female C. dorsatus. The photographs show a the fixation of the partly frozen animal, and b the halfway extracted gastrointestinal tract. Ion images from the LDI-TOF MSI analysis present the distribution of c dehydroquinic acid (m/z 213, [M+Na]⁺, red color) and d quinic acid (m/z 215, [M+Na]⁺, green color), overlaid on the optical image taken before MSI Note: The assignments of the secondary plant metabolites are tentative based on MS and MS/MS experiments of methanol extracts of C. dorsatus, as summarized in Table 2 and compared with reference standards, as presented in the ESM. The ion images are normalized using the TIC. Scale bar 1000 μm (d)

Fig. 6

LDI-TOF MSI analysis of excrements of C. dorsatus. The photographs show a excrements embedded in 10% gelatin in a disposable specimen mold; b view into the cryostat where the embedded excrements are attached with BPS onto the specimen disc, which is mounted on the object head. The ion images generated by LDI-TOF MS of dehydroquinic acid (m/z 213, [M+Na]⁺, red color) and quinic acid (m/z 215, [M+Na]⁺, green color) are overlaid on the optical image in picture c and d, respectively Note: The assignments of the secondary plant metabolites are tentative based on MS and MS/MS experiments of methanol extracts of the excrements, as summarized in Table 2 and compared with reference standards, as presented in the ESM. The ion images are normalized using the TIC. Scale bar 5 mm (c)

Fig. 7

LDI-TOF MS analysis of water extractable SOM in both, control (black line color) and grasshopper (red line color) mesocosms in the positive ion mode. The secondary plant metabolites dehydroquinic acid (m/z 213, [M+Na]⁺) and quinic acid (m/z 215, [M+Na]⁺) are readily visible in both mesocosms and were putative assigned by MS/MS analysis

Fig. 8

LDI-TOF MS analysis of methanol extractable SOM in both, control (black line color) and grasshopper (red line color) mesocosms in the positive ion mode. The secondary plant metabolites dehydroquinic acid (m/z 213, [M+Na]⁺) and luteolin (m/z 309, [M+Na]⁺) are readily visible in both mesocosms and were putative assigned by MS/MS analysis