Distribution of coniferin in freeze-fixed stem of Ginkgo biloba L. by cryo-TOF-SIMS/SEM

Abstract

To clarify the role of coniferin in planta, semi-quantitative cellular distribution of coniferin in quick-frozen Ginkgo biloba L. (ginkgo) was visualized by cryo time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. The amount and rough distribution of coniferin were confirmed through quantitative chromatography measurement using serial tangential sections of the freeze-fixed ginkgo stem. The lignification stage of the sample was estimated using microscopic observations. Coniferin distribution visualized at the transverse and radial surfaces of freeze-fixed ginkgo stem suggested that coniferin is stored in the vacuoles, and showed good agreement with the assimilation timing of coniferin to lignin in differentiating xylem. Consequently, it is suggested that coniferin is stored in the tracheid cells of differentiating xylem and is a lignin precursor.

Figure 1

Cryo-TOF-SIMS spectra and chemical structures of (a)coniferin, (b) sucrose, and (c)glucose. All chemicals were dissolved at 1 mM concentration in 10 mM KCl aqueous solution and frozen for measurement.

Figure 2. Transverse surface images of freeze-fixed ginkgo stem 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 (b) total ion, (c) K⁺ at m/z 39, and (d) coniferin at m/z 180. (e) Schematic illustration of sample preparation and the resultant optical microscopic image of transverse surface of freeze-fixed ginkgo stem on a cryo-TOF-SIMS sample holder showing the measurement area (ca. 2.3 × 0.4 mm). Scale bars are 500 μm for (a–d) and 2 mm for (e). Arrows at both sides of images suggest the line of the cambial zone. Cryo-SEM images before and after freeze-etching are displayed in Supplementary Fig. 6. Cryo-TOF-SIMS images of mono-/di- saccharides are shown in Supplementary Fig. 7.

Figure 3

The radial distribution of coniferin evaluated by (b) HPLC and (c) cryo-TOF-SIMS. Preparation procedure of tangential sections for HPLC measurements is shown in (a). In (b), serial tangential sections of 100-μm thickness were used and the position of cambial zone corresponding to the section numbers 9 and 10 was determined by the dry weight of the sections as shown in Supplementary Fig. 9. The means and standard deviations for each section in (b) were obtained from three sets of measurements using the different sample blocks cut from the same disk. In (c), the m/z 180 ion count from bark to xylem was used and arrows at the distance 1100 μm indicate the position of the cambial zone.

Figure 4. Radial surface images of freeze-fixed ginkgo stem by cryo-TOF-SIMS/SEM.

(a) Cryo-SEM image after cryo-TOF-SIMS measurement and freeze-etching. Cryo-TOF-SIMS positive ion images of (b) total ion and (d) m/z 180 ion. (c) The overlay image of cryo-TOF-SIMS m/z 180 ion on the cryo-SEM image. Scale bar is 100 μm.

Figure 5

(a) The enlarged total ion image of cryo-TOF-SIMS demonstrating the generation of ROIs (n = 4 using the lines A, B, C, and D). Resultant relative ion intensities are summarized in (b). Scale bar is 100 μm.

Figure 6

Comparative visualization of (a) the overlay image of cryo-TOF-SIMS m/z 180 ion (red) on the cryo-SEM illustrating endogenous coniferin distribution in the freeze-fixed ginkgo stem and (b) ¹⁴C microautoradiography showing ¹⁴C-lignin introduced by ¹⁴C-coniferin administration to ginkgo (rearranged from the previous research by Fukushima and Terashima11). Arrows show the positions of (X) cambial zone, (Y) start of CML lignification, and (Z) start of secondary cell wall lignification. Scale bar is 100 μm.