xylem-water-distribution-woody-plants-visualized-with-cryo-scanning

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 ...

Hokkaido University

To visualize water-conduction pathways in living trees, we introduced aqueous solutions of safranin and acid fuchsin into stems of Populus sieboldii Miquel. To examine the spread of each dye in the trees, we compared several techniques for preparing tissue for light microscopy. Acid fuchsin was distributed more rapidly and more widely than safranin, reflecting differences between the dye molecules in state of ionization. We prepared some sections without allowing the dye to redissolve after it had been stabilized by freeze-drying. In these sections, the dye was observed in vessels and in some of the adjacent ray parenchyma cells. Other sections were prepared without stabilizing the dye. In these sections, acid fuchsin in the sap stream left cell walls unstained, whereas safranin stained wood fibers in the vicinity of vessels, as well as the vessels themselves, provided that the sections were mounted in glycerin, which dissolves safranin. Although stained with safranin, the wood fibers contained no water. The results indicate that stabilization of the introduced dye and subsequent preparation of tissues under conditions that avoid dye resolublilization allow accurate visualization of water-conduction pathways at the cellular level.

Visualizing water-conduction pathways of living trees: selection of dyes and tissue preparation methods.

The structure of pit membranes in angiosperms has not been fully examined and our understanding about the structure is incomplete. Therefore, this study aims to illustrate the micromorphology of pit membranes in fibres and tracheids of woody species from various families.

Perforated pit membranes in imperforate tracheary elements of some angiosperms.

Imperforate tracheary elements (ITEs) in wood of vessel-bearing angiosperms may or may not transport water. Despite the significance of hydraulic transport for defining ITE types, the combination of cell structure with water transport visualization in planta has received little attention. This study provides a quantitative analysis of structural features associated with the conductive vs. non-conductive nature of ITEs.

Anatomical features associated with water transport in imperforate tracheary elements of vessel-bearing angiosperms.

Elucidation of the mechanism of adsorption of particles suspended in the gas-phase (aerosol) to the outer surfaces of leaves provides useful information for understanding the mechanisms of the effect of aerosol particles on the growth and physiological functions of trees. In the present study, we examined the localization of artificially deposited sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica, a typical Japanese coniferous tree species, by field-emission scanning electron microscopy. The clusters (aggregates) of carbon-based particles were deposited on the needle surface regions where epicuticular wax crystals were sparsely distributed. By contrast, no clusters of the particles were found on the needle surface regions with dense distribution of epicuticular wax crystals. Number of clusters of carbon-based particles per unit area showed statistically significant differences between regions with sparse epicuticular wax crystals and those with dense epicuticular wax crystals. These results suggest that epicuticular wax crystals affect distribution of carbon-based particles on needles. Therefore, densely distributed epicuticular wax crystals might prevent the deposition of sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica to retain the function of stomata. 

Effect of epicuticular wax crystals on the localization of artificially deposited sub-micron carbon-based aerosols on needles of Cryptomeria japonica.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Article	Year
Visualizing water-conduction pathways of living trees: selection of dyes and tissue preparation methods. Tree physiology\| PubMed ID: 15631975	2005
Perforated pit membranes in imperforate tracheary elements of some angiosperms. Annals of botany\| PubMed ID: 16520339	2006
Anatomical features associated with water transport in imperforate tracheary elements of vessel-bearing angiosperms. Annals of botany\| PubMed ID: 21385773	2011
Effect of epicuticular wax crystals on the localization of artificially deposited sub-micron carbon-based aerosols on needles of Cryptomeria japonica. Journal of plant research\| PubMed ID: 27294967	2016
Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells. Tree physiology\| PubMed ID: 30806711	2019

Yuzou Sano

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