Distribution of salicifoline in freeze-fixed stems of Magnolia kobus as observed by cryo-TOF-SIMS

Abstract

Alkaloids are basic nitrogen-containing chemicals that have important physiological and pharmacological characteristics. Many vascular plant species contain alkaloids, and their roles in planta are of interest. However, the detailed distribution of alkaloids remains unclear because of their low water solubility and low concentrations in plants. In this study, we visualized the distribution of salicifoline, a water-soluble quaternary ammonium alkaloid, in the freeze-fixed stems of Magnolia kobus by cryo time-of-flight secondary ion mass spectrometry. Most of the salicifoline was distributed in living phloem tissues. In the xylem, salicifoline was detected in ray cells, lignifying wood fibres, and in vessels in the latest annual ring. The salicifoline distribution in the xylem varied with the cell wall formation stage. These results provide new insights into the storage, transportation, and role of the alkaloid salicifoline in M. kobus.

Figure 1

Cryo-TOF-SIMS spectra of (a) salicifoline, (b) sinapyl alcohol, (c) syringin, and (e) phosphatidylcholine. A typical cryo-TOF-SIMS spectrum obtained from the transverse surface of the freeze-fixed stem of M. kobus is shown in (d). Standard chemicals were dissolved at 100 mM concentration in 100 mM aqueous solution of KCl and frozen for the measurements. All spectra were obtained in bunched mode and are displayed as relative intensity to total ion counts.

Figure 2

Radial distribution of salicifoline and syringin evaluated by HPLC using serial tangential sections of sample blocks (thickness, 10 mm; circular sector of radius, 5 mm; and central angle, π/16). Measurements were performed in triplicate using three different sample blocks cut from the same disk.

Figure 3

Transverse surface images of freeze-fixed M. kobus visualized by cryo-TOF-SIMS/SEM. (a) Cryo-SEM image taken after cryo-TOF-SIMS measurement and appropriate freeze etching. Cryo-TOF-SIMS positive ion images of the (b) total ion, (c) potassium, (d) phosphatidylcholine, and (e) salicifoline. (f) Optical microscopic image of a freeze-fixed stem of M. kobus in a sample holder showing the measurement area (approximately 2.4 × 0.4 mm). Scale bars are 100 μm for a, b, c, d, and e and 1.0 mm for f. Arrows above and below the sides of the images of a, b, c, d, and e indicate the cambial zone. Grey scaled tetragons at both sides of the images a, b, c, d, and e indicate the cell wall formation stages of wood fibres.

Figure 4

Images obtained by (a) cryo-SEM and (b) overlay of cryo-TOF-SIMS m/z 210.15 ion (red) on the cryo-SEM image, illustrating the distribution of salicifoline in the freeze-fixed stem of M. kobus. Nearly the same region was observed by (c) optical microscopy with toluidine-blue staining, and the assigned tissues were separately coloured to distinguish them (d). Letters in d show the positions of cortex (Co), phloem fibre (PF), sclereid (Sc), secondary phloem (SP), and ray cells (R). Scale bar is 100 μm.