Name: xylem water distribution in woody plants visualized with a cryo-scanning electron microscope
Uploaded: 2019-06-20T12:30:00
Description: Watch this Scientific Journal Video about Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope at JoVE.com

This work was supported by JSPS KAKENHI (No. 20120009, 20120010, 19780129, 25292110, 23780190, 23248022, 15H02450, 16H04936, 16H04948, 17H03825, 18H02258)

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&#8211;induced artifacts in the xylem water distribution.
<ol>
	<li>Enclose a branch and leaves for sampling with a black plastic bag to equilibrate the water potential ...

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...

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-lignified cell walls1. The vulnerability to xylem dysfunction of xylem conduits in each species is a good determinant of the species survival under fluctuating water supply2. In addition, studying the water status of xylem conduits is important for the evaluation of the health condition of individual trees subjected to abiotic or biotic stresses. Measuring sap flow or water potential can provide estimates of a woody plant&#39;s water status due to the integrated hydraulic function of xylem conduits. Furthermore, visualizing the distribution of water in xylem cells can clarify the condition of individual components of the xylem hydraulic system.
Several techniques for visualizing the water status of xylem conduits exist3. The classical and useful methods for observing water pathways in woody tissue involve staining the water column by immersing the ends of cut branches into a dye or by injecting a dye into standing tree stems4. Soft X-ray photography also allows the visualization of water distribution of sliced wood disks due to the differential X-ray absorption intensity of the moisture in xylem5,6. These methods, however, only provide tracks of water movement or demonstrate macroscopic distributions of water. Recently, non-destructive observation techniques, such as micro focus X-ray computed tomography (&#181;CT)7,8,9,10and magnetic resonance imaging (MRI)11,12, have been significantly improved to allow observation of water in xylem conduits within intact saplings. These non-destructive methods have great advantages in that we can observe the xylem&#39;s water status without artificial cutting effects, and we can track water flow dynamics by sequential imaging or introducing a contrast agent10. However, we need to use a customized MRI for plant imaging or a specialized facility for synchrotron-based &#181;CT in order to obtain the images which can identify cellular level water content. In addition, although the synchrotron-based &#181;CT system enabled to obtain fine images with high spatial resolution, which is comparable to light microscopy7,8,9, living cells can be injured by the radiation of high energy X-ray13,14. Employing a scanning electron microscope in which cryo-units are installed (cryo-SEM) is a very useful method for precisely locating the water in xylem at a cellular level, although this requires destructively harvesting the sample for observation. To fix the water in xylem conduits, a portion of the stems (i.e., twigs, branches or stems) are frozen in situ by liquid nitrogen (LN2). Observations of the surface of trimmed, frozen specimens by cryo-SEM provide highly-magnified images of the xylem structure from which we can identify the water in xylem conduits as ice. A significant limitation of this method is that sequential observation of water movability within the same sample is impossible. However, the application of &#181;CT or MRI for sequential observation of trees that live in a field is extremely challenging because these instruments are not portable. In contrast, cryo-SEM has a potential for using this technique on big trees in field experiments to clearly visualize water contents at not only the cellular level but also at a finer structure level, e.g., water in intervascular pits15, water in intercellular spaces16, or bubbles in water column17.
Many studies observing xylem water by cryo-SEM have been reported 5,12,18,19,20,21,23. Utsumi et al. (1996) initially established the protocol for observation of xylem in situ&#160;by freeze-fixation of a living trunk via filling LN2&#160;into a container set onto the stem21. The temperature of the sample was maintained below -20 &#176;C during sample collection and during cryo-SEM preparation in order to avoid melting the ice within xylem conduits. This method has been used to observe the water in xylem in order to clarify water distribution under changing water regime11,12,24,25,26,27,28, the seasonal variation of water distribution21,29,30, the effect of freeze-thaw cycles17,31,32, the distribution of water in wet wood5, changes in the water distribution during the transition from sapwood to heartwood20, seasonal time course of cambial activity and differentiation of vessels33, and cavitation induced by certain biotic stresses23,34. Hydraulic conductivity and conduits vulnerability to cavitation have also been verified using cryo-SEM35,36. Cryo-SEM equipped with energy dispersive X-ray spectrometry (EDX or EDS) has been used to study element distribution over the surface of a specimen containing water37.
Freeze-fixation of a living trunk which contains conduits under high hydraulic tension sometimes causes artificial cavitations which are observed by cryo-SEM as fractured ice crystals in the lumen of conduits38,39. In particular, broadleaved species with longer and wider conduits are vulnerable to tension-induced artifacts, such as cavitation caused by sample cutting, even if conducted under water3,40. Cavitation artifacts become conspicuous after sampling of a transpiring tree (i.e., sampling during the day time) or under severe drought conditions and they can mislead to an overestimation of cavitation occurrence3,38,39. Therefore, the tension working in the conduits has to be released in order to avoid the artifactual cavitation3,12,39.
The freeze-fracture technique using a knife installed in a specimen chamber is often employed to expose specimen surface for cryo-SEM observation. However, freeze-fractured planes of woody plant tissues, especially transverse sections of secondary xylem, are too rough to clearly observe the anatomical features and water in the tissue6. The application of a cryostat for trimming a specimen allows rapid and high-quality preparation of sample surfaces20,23. The overall goal of this method is providing evidence with electron microscopy resolution of the water distribution in various kinds of xylem cells in situ without the occurrence of sampling artifacts. We introduce our updated procedure, which has been steadily improved since we first adopted it, regarding the sampling, trimming and cleaning the specimen surface for obtaining high-quality electron micrographs of cryo-fixed samples of xylem.

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			<td height="41" style="height:41px;width:249px;">coating material</td>
			<td style="width:179px;">JOEL Ltd., Japan</td>
			<td style="width:284px;">Gold wire, 0.50 &#215; 1000 mm, 99.99 %, Parts No. 125000499&#160;</td>
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			<td height="58" style="height:59px;width:249px;">cryo scanning electron microscope</td>
			<td style="width:179px;">JOEL Ltd., Japan</td>
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			<td height="47" style="height:48px;width:249px;">cryostat</td>
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			<td height="38" style="height:38px;width:249px;">microtome blade</td>
			<td style="width:179px;">Thermo Scientific</td>
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			<td height="38" style="height:38px;width:249px;">tissue freezing embedding medium</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:284px;">Shandon Cryomatrix embedding resin, 6769006</td>
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xylem water distribution in woody plants visualized with a cryo-scanning electron microscope

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&#160;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 (&#60; ca. &#8722;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&#160;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.

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...

Watch this Scientific Journal Video about Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope at JoVE.com

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

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&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

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