Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

Summary

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

Abstract

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN₂). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (< ca. −0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN₂ for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Introduction

Availability of water resources (i.e., precipitation, soil water content) strictly determines the mortality and geographic distribution of plant species, since they need to absorb water from the soil and transport it to the leaves for photosynthetic production. Plants must maintain their water transport system under fluctuating water supplies. In particular, woody plants generate high tensions in their conduits along the transpiration streams as, in some cases, they need to hold their crown more than ~100 m above ground. To maintain water columns under such high negative pressure, xylem conduits consist of a continuum of tubular cells with rigid and hydrophobic-lignif....

Protocol

NOTE: A schematic chart of this protocol is shown in Figure 1.

1. Sampling: Tension Relaxation within Water Column of Xylem Conduits

NOTE: The following tension relaxation treatment is recommended before the LN₂ application to avoid both freezing and tension–induced artifacts in the xylem water distribution.

1. Enclose a branch and leaves for sampling with a black plastic bag to equilibrate the water potential between xylem and leaves more than two hours before sampling.
2. Determine the water potential of at least two leaves from the sample using a p....

Results

Representative images of transverse-cut surfaces of coniferous and broadleaved tree xylem, observed by cryo-SEM, are shown in Figure 2. At low magnification, the black area in the images indicates the cavities from which water entirely or partly disappears, and the gray area indicates xylem cell walls, cytoplasm, and water (Figure 2A). At high magnification, it is apparent that the water is not entirely lost from.......

Discussion

The cryo-SEM observation methods introduced in this paper are practical for clearly visualizing water distribution on a cellular scale. Through this method, exploring the changes in the distribution of water within xylem can potentially help clarify the mechanism of tree species tolerance to abiotic stress (water shortage or freezing) or biotic stress (tree disease).

The most crucial step in this method is preserving the water distribution characteristic of the native water status during sampl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
coating material	JOEL Ltd., Japan	Gold wire, 0.50 × 1000 mm, 99.99 %, Parts No. 125000499
cryo scanning electron microscope	JOEL Ltd., Japan	JSM-6510 installed with MP-Z09085T / MP-51020ALS
cryostat	Thermo Scientific	CryoStar NX70
microtome blade	Thermo Scientific	HP35 ULTRA Disposable Microtome Blades, 3153735
tissue freezing embedding medium	Thermo Scientific	Shandon Cryomatrix embedding resin, 6769006