Tree Physiology 25, 269–275
© 2005 Heron Publishing—Victoria, Canada
Visualizing water-conduction pathways of living trees: selection of dyes
and tissue preparation methods
YUZOU SANO,1,2 YASUKO OKAMURA1 and YASUHIRO UTSUMI1,3
1
Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2
Corresponding author ([email protected])
3
Present address: Miyazaki Experimental Forest, Graduate School of Agriculture, Kyushu University, Shiiba 883-0402, Japan
Received January 29, 2004; accepted September 14, 2004; published online January 4, 2005
Summary 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.
Keywords: acid fuchsin, Populus sieboldii, ray parenchyma,
safranin, vessel, water transport, wood fiber.
Introduction
Water-conduction pathways in living trees have generally been
monitored by following the distribution of dyes introduced
into the sap stream (Greenidge 1958, Kozlowski and Winget
1963, Chaney and Kozlowski 1977, Barnett et al. 1993, Tyree
et al. 1999, Sakamoto and Sano 2000, Tyree and Zimmermann
2002). Similar methods have been adopted in studies with herbaceous plants (e.g., Canny 1990a, Schurr et al. 1996, Tang
and Boyer 2002). However, reports vary with respect to the
methods of introducing the dye. In addition, published methods
of sample preparation for microscopical visualization of dyes
introduced into the xylem are often too brief to allow evaluation. The reliability and the limitations of each reported
method have been rigorously determined in only a few cases,
e.g., the technique of freeze-substitution and anhydrous embedding and sectioning (Canny and McCully 1986) and handcut sectioning under paraffin oil (O’Dowd and Canny 1993),
that were devised to examine the apoplastic pathway of water-conduction in leaves.
The selection of dye is important for the analysis of water
flow in living trees, because the extent of dye spreading in the
water transporting system of living trees differs significantly
depending on the dye (e.g., Canny 1990a, Tyree and Zimmermann 2002). Differences between basic and acid dyes are easily seen when solutions of each are allowed to permeate filter
paper (Kishima and Hayashi 1960, Canny 1986 and references
therein, IIda et al. 1992). Basic dyes spread less extensively
than water (Figure 1A), whereas no such separation of dye and
water occurs in the case of acid dyes (Figure 1B). However, little attention has been paid to such differences, and dyes have
not been used consistently in recent studies. To develop appropriate and reproducible methods for visualizing pathways of
water flow in living trees, we compared the spread of safranin,
which is basic, with that of acid fuchsin, after aqueous solutions of each had been introduced into the water transport systems of living trees. In addition, we compared several techniques for preparing stem tissue sections for light-microscopic
evaluation of the pathways of water flow at the cellular level.
Materials and methods
Plant materials
We studied 3-year-old Populus sieboldii Miquel trees growing
in the nursery of the Sapporo Experimental Forest of Hokkaido University (Table 1). Dye solutions were introduced into
the transpiration stream on a sunny afternoon in autumn (ambient temperature was about 20 °C), when cambial activity had
already ceased.
Introduction of dye and collection of samples
Each dye solution was introduced as described by Bernholt
(1941). In brief, receptacles made from plastic funnels (21 cm
in diameter) were fitted around stems with sticky vinyl tape at
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SANO, OKAMURA AND UTSUMI
Figure 1. Distribution of water and dye in a 0.5% aqueous solution of
safranin (A) or acid fuchsin (B) recorded 30 s after a drop of the solution had been placed on a piece of filter paper (Qualitative No. 2;
Advantec, Tokyo, Japan). Bar = 1 cm. In panel A, the large arrow indicates the distribution of safranin and the small arrow indicates the distribution of water.
the base of each sample tree and at 1 m above it (Figure 2). An
aqueous solution of 0.5% (w/v) acid fuchsin (Wako Pure
Chemical Industries, Osaka, Japan; molecular weight = 585)
or safranin (Wako Pure Chemical Industries; molecular weight
= 350) was prepared. The solution of safranin was filtered
through a 0.22-µm filter (GV, Millipore, Billerica, MA) before
use to remove dye that remained undissolved after vigorous
stirring for several hours at room temperature. The aqueous
dye solution was placed in the receptacle positioned at the base
of each sample tree. A notch was cut to the depth of the pith
with a chisel below the solution surface in each funnel, and the
solution allowed to flow into the xylem through the notch for
50 min. During this time, three leaves were cut from each sample tree and water potential was measured with a pressure
chamber (Model 3000-40, Soil Moisture, Goleta, CA) (Table 1).
After the introduction of dye, liquid nitrogen was placed in
another receptacle positioned 1 m above the site of dye introduction (Figure 2). After 5 min, two discs (2 cm thick) were
cut from the frozen part of each sample tree and stored in liquid nitrogen. One disc from each sample tree was used for light
microscopy and the other for cryo-scanning electron microscopy (cryo-SEM).
After collection of the frozen specimens, cylindrical pieces
of unfrozen stems (5 cm in length) were cut about 20 cm below
the frozen part. Both cut ends were coated with petroleum jelly
and wrapped in aluminum foil to prevent dehydration. The
cylinders were taken to a laboratory and used immediately for
Figure 2. Schematic representation of the methods used for introducing dye and for freezing samples for light microscopy and cryo-scanning electron microscopy.
tissue preparation, i.e., without dye stabilization. Cylindrical
pieces of unfrozen stem (5 cm in length) were also cut at 10- to
20-cm intervals above the dye-introduction site for macroscopic observations of the dye distribution. These samples
were stored in liquid nitrogen until analyzed.
Macroscopic observations of dye distribution
The cylindrical specimens were freeze-dried. Discs (1 cm
thick) were cut from the central part of each specimen with a
disc saw. The distribution of dye was examined macroscopically on the transverse surface of each disc.
Light microscopy without stabilization of dye
Slides were prepared as soon as possible after the collection of
samples in the field. The procedures are summarized in Figure 3A. Transverse sections (15–20 µm thick) were cut on a
sliding microtome with disposable steel blades. Some sections
were mounted in glycerin, in which both of the dyes are
readily soluble (Figures 4A and 4B), without prior rinsing in
other solutions. Other sections were rapidly dehydrated in absolute ethanol and rinsed in xylene before mounting in Bioleit
(Ohkenshoji, Tokyo, Japan; main component is styrol), in
Table 1. Height, diameter at breast height (DBH), leaf water potential of sample trees and maximum ascent of sap determined by safranin and acid
fuchsin in sample trees. Each water potential is the mean (SD) for three leaves per tree. Maximum ascent of dye refers to the distance from the site
of dye injection to the top of the colored zone.
Sample tree
Height (m)
DBH (cm)
Dye
Water potential (MPa)
Maximum ascent of dye (m)
1
2
3
4
3.0
3.9
3.4
3.6
2.0
3.5
2.5
2.5
Safranin
Safranin
Acid fuchsin
Acid fuchsin
–1.1 (0.03)
–1.3 (0.07)
–0.9 (0.18)
–1.2 (0.18)
1.5
1.4
2.0
3.6
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VISUALIZATION OF WATER-CONDUCTION PATHWAYS
271
Figure 3. Flow chart of methods used
for preparing tissue for light microscopy.
which neither dye dissolves (Figures 4A and 4B). Sections
were observed by standard light microscopy.
Results
Macroscopic distribution of dye
Light microscopy after stabilization of dye
To stabilize the dye where it was present at the time of sample
collection, the frozen stem discs were freeze-dried. We then
cut 2-mm 3 cubes from the stained region of each disc. We
rinsed half of the cubes in xylene overnight and then infiltrated
them with Bioleit and hardened them at 60 °C. We immersed
the remaining cubes in n-butyl glycidyl ether overnight, which
was then replaced with an epoxy resin that contained triglycidyl ether of glycerol (Epon 812; TAAB, Berkshire,
U.K.), methyl nadic anhydride (Wako Pure Chemical Industries), dodecenylsuccinic anhydride (Wako Pure Chemical Industries) and tri(dimethylaminomethyl)phenol (Ohkenshoji)
(45:30:24:1 (v/v)). The resin was polymerized at 60 °C for
72 h. Acid fuchsin was insoluble in all of the solutions used for
both embedding procedures (Figure 4B). Therefore, the distribution of acid fuchsin was unchanged once the tissue had been
freeze-dried. In contrast, the distribution of safranin may have
been altered by the embedding process, because safranin is
soluble in both n-butyl glycidyl ether and in the resin components (Figure 4A).
Sections (4–8 µm thick) were cut from the embedded specimens on an ultramicrotome with a glass knife. We routinely
handle such sections with a wire loop after floating them on
water (e.g., Hayat 1989). However, in this study, sections were
cut under dry conditions to keep the dye in place. The sections
were picked up with fine forceps, mounted in Bioleit and observed by light microscopy (Figure 3B).
Cryo-SEM
To monitor the distribution of water at the time of sample collection, we performed cryo-SEM with the freeze-planing technique that has been described previously (e.g., Sano et al.
1995, Utsumi et al. 1996, 2003). In brief, the samples were
trimmed and planed on a sliding microtome with disposable
steel blades in a low temperature room at –23 °C. The samples
were slightly etched, coated with platinum and carbon in the
cryo-SEM system (JSM840-a equipped with CRU-7000;
JEOL, Tokyo, Japan) and observed at 3 to 5 kV.
Acid fuchsin ascended stems to greater heights than safranin.
In addition, acid fuchsin was distributed more widely in the
tangential direction than safranin when compared at the same
distance above the site of dye introduction (Table 1, Figures 4C and 4D).
Light microscopy
Specimens prepared without dye stabilization The dye distribution in sections prepared from trees that had taken up
safranin differed depending on the method of preparation. Vessels, and the wood fibers and ray parenchyma surrounding
them, were stained in samples mounted in glycerin immediately after sectioning (Figure 4E). The extent of the stained
area increased with time (Figure 4F). In contrast, sections
mounted in Bioleit after dehydration showed no obvious staining of wood fibers, even though vessels and some of the adjacent ray parenchyma were stained (Figure 4G).
In sections cut from trees that had taken up acid fuchsin and
which were immediately mounted in glycerin, no staining of
cell walls was visible (Figure 4H). Moreover, no cell walls
were stained in similar sections mounted in Bioleit.
Specimens prepared after dye stabilization In specimens with
safranin, the Bioleit and Epon 812 methods of embedding resulted in similar dye distributions. In both cases, the cell walls
of vessel elements and some of the adjacent ray parenchyma
were stained (Figures 4I and 4J). Safranin was observed to
spread occasionally from stained ray parenchyma to adjacent
wood fibers (arrows in Figures 4I and 4J). There were some
slight differences between the results obtained with the two
embedding methods, but these differences were not clearly visible in the light micrographs (Figures 4I and 4J). Embedding
medium present in the lumina of the vessel elements was
slightly stained in sections that had been prepared from Epon812-embedded specimens, probably because the safranin had
redissolved and relocated there during rinsing in n-butyl
glycidyl ether and embedding in Epon 812. In contrast, the lu-
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272
SANO, OKAMURA AND UTSUMI
Figure 4. (A, B) Dissolution of safranin (A) and acid fuchsin (B) in solutions 12 h after 10 mg of the dyes had been placed and shaken in each solution used for the preparation of tissue. Abbreviations: Gly = glycerin; Bio = Bioleit (mainly styrol); Epo = Epon 812; Eth = ethanol; Xyl = xylene;
and n-b = n-butyl glycidyl ether. (C, D) Discs of dye-injected trees (C, Tree 2; D, Tree 3; see Table 1). The number beside each disc indicates distance (m) from the site of introduction of the dye. The upper side of each disc corresponds to the side of the stem into which dye was introduced.
Bars = 1 cm. (E–G) Transverse sections prepared from safranin-stained xylem without dye stabilization. Transverse sections in E and F show a
single sample photographed immediately and 24 h after mounting in glycerin, respectively. The transverse section in G shows a sample mounted in
Bioleit after dehydration. Bars = 50 µm. (H) Transverse section of acid-fuchsin-stained xylem without dye stabilization and mounted in glycerin.
Bar = 50 µm. (I, J) Transverse sections prepared from safranin-stained xylem after dye stabilization. Panels I and J show samples from specimens
embedded in Epon 812 and Bioleit, respectively. Arrows indicate partial staining of walls of wood fibers by safranin. Bars = 50 µm. (K) Transverse
section prepared from acid-fuchsin-stained xylem after dye stabilization. This section was cut from a specimen embedded in Epon 812. Bar = 50 µm.
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VISUALIZATION OF WATER-CONDUCTION PATHWAYS
mina of vessel elements were unstained in sections of Bioleitembedded tissues.
In acid-fuchsin-stained specimens, there were no differences in the distribution of dye between the two methods of
embedding samples. Acid fuchsin was concentrated on the inner surface of vessel walls and in the lumina of some ray parenchyma cells that were in contact with the stained vessels
(Figure 4K). Unlike safranin, acid fuchsin did not spread from
ray parenchyma that contained the dye into adjacent wood fibers (Figure 4K).
The embedding medium influenced the quality of sections.
For example, cell walls were undulating in sections prepared
from Bioleit-embedded specimens (Figure 4J), whereas no
similar cell-wall deformation was visible in sections cut from
Epon-812-embedded specimens (Figures 4I and 4K).
Cryo-SEM
Most vessels were filled with water in the dye-stained regions
of the stem xylem, irrespective of dye type (Figure 5A). In
contrast, wood fibers were rarely water-filled, even in the vicinity of vessels, although occasionally water droplets were
observed in some wood fibers (Figure 5B).
Discussion
Differences in the macroscopic distribution of dye-stained xylem between stems into which acid fuchsin or safranin had
been introduced were mainly a result of the difference in ionization state between the dyes in solution (Canny and McCully
1986). The concentration of safranin probably decreased gradually with the movement of the dye solution because safranin
273
is positively charged and therefore easily adheres to negatively
charged cell wall components. As a result, it was impossible to
detect dye-staining near the flow front of the safranin solution
at some distance from the site of its introduction into stems. By
contrast, no similar dilution of acid fuchsin dye occurred after
its introduction because this negatively charged dye rarely adheres to cell wall components. Thus, based on the rate and the
extent of water flow in trees measured by dye tracers, staining
by acid fuchsin ought to reveal the pathways of the aqueous solution more faithfully than staining by safranin, as previously
noted (e.g., Canny and McCully 1986).
Early studies in which acid fuchsin was used as a tracer to
examine the ascent of water in trees did not include microscopic examinations (Greenidge 1958, Rudinsky and Vité
1959, Kozlowski and Winget 1963), presumably because acid
fuchsin rarely adheres to cell wall components and is easily
washed out during tissue preparation. Schurr et al. (1996) used
a light microscope to examine the distribution of acid fuchsin
introduced into the xylem of the herbaceous plant Ricinus
communis L., in which tumors had been artificially induced by
Agrobacterium. However, their observations were limited to
longitudinal hand-cut sections at low magnification. Our microscopic observations of acid fuchsin in the xylem seem to indicate that the distribution of this dye in embedded sections accurately reflected its distribution at the time of sample collection, because the acid fuchsin was stabilized by freeze-drying
and did not redissolve during tissue preparation. The distribution of safranin in the Bioleit-embedded sections also accurately reflected the distribution of the dye at the time of sample
collection. Thus, we conclude that these methods are as reliable for the visualization of water-conduction pathways in
trees as the technique of freeze-substitution and anhydrous
Figure 5. Cryo-scanning electron micrographs of stained xylem of a tree that had taken up safranin (No. 2). (A) Transverse surface of the middle
layer of the outermost annual ring. Bar = 100 µm. (B) An enlarged view of a region similar to that in A. Bar = 50 µm.
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SANO, OKAMURA AND UTSUMI
embedding and sectioning that was devised by Canny and
McCully (1986). In particular, Epon-812-embedding of acid
fuchsin-treated samples was most useful because it caused no
deformation of cell walls.
It was difficult to interpret some features of the subcellular
distribution of the dye in embedded sections. For example, in
the embedded sections of trees, acid fuchsin accumulated
along the surface of vessel walls (Figure 4K). Redistribution of
dye during freeze-drying may have been responsible for this
accumulation. Freeze-substitution, which was devised for this
kind of study by Canny and McCully (1986), might minimize
this artifact. Another phenomenon that is difficult to explain is
the partial staining of the walls of wood fibers that were in contact with ray parenchyma that stained strongly with safranin
(Figures 4I and 4J). Cryo-SEM demonstrated that the corresponding wood fibers were not filled with water. The spread of
dye from ray parenchyma to wood fibers was not observed in
acid-fuchsin-stained sections. Thus, it is unlikely that the dye
solution penetrated the lumina of wood fibers from the ray parenchyma. It is also unlikely that such staining was caused by
differential stainability within the tissue, because such partial
staining was not found in sections of safranin-stained xylem
(not shown). Safranin may have reached these wood fibers as a
result of diffusion through cell walls (Canny 1990b).
In sections that were prepared from Epon-812-embedded
specimens, contamination caused by diffusion of safranin may
have occurred because safranin was soluble in the embedding
media. Similar contamination may also have occurred during
sectioning and subsequent processing of sections cut from
safranin-stained xylem without stabilization of the dye, although the distribution of the introduced safranin in these sections was similar to that in sections prepared from Bioleit-embedded specimens. In Epon-812-embedded specimens, secondary changes in the distribution of the stabilized dye may
have been too small to detect because of the low solubility of
safranin in the embedding media (Figure 4A). In Bioleitmounted sections without dye stabilization, excess safranin
may have been removed before it could adhere to cell wall
components because sectioning and dehydration of tissues
were carried out quickly. Thus, the two methods of embedding
appear to be acceptable for practical applications despite the
potential for contamination during tissue preparation.
The solubility of dye in the mounting medium had a considerable effect on the accuracy of detection of water-conduction
pathways. Changes in the distribution of the tracer dye were
apparent after mounting sections in glycerin. In sections that
had been prepared from safranin-stained xylem without dye
stabilization, the cell walls of wood fibers in the vicinity of
vessels were stained with the dye (Figure 4E). It is unlikely
that such wood fibers conducted water because they were not
filled with water. The staining of these wood fibers was therefore probably a result of the diffusion of safranin in glycerin
after mounting, indicating the importance of using a mounting
medium in which the dye is insoluble.
It has been shown by dye introduction that the active transport of water occurs in the radial direction through ray paren-
chyma (e.g,. Barnett et al. 1993, Tyree et al. 1999, Tyree and
Zimmermann 2002). However, we found no evidence of radial
transport through radial files of ray parenchyma. The dye appeared only in some ray parenchyma cells adjacent to vessel
elements, and it did not penetrate further into adjacent ray
cells, irrespective of the nature of the dye (Figures 2I–2K).
This negative result may be associated with differences in
function between the two kinds of ray parenchyma cells in
Populus species. Ray parenchyma cells are classified as contact cells or isolation cells based on the presence or absence of
pit pairs with vessel elements (Braun 1967). The two types of
ray cell differ in fine structure, function and the process
whereby they differentiate (e.g., Sauter and Kloth 1986, Murakami et al. 1999). It appears that the contents of contact cells
move into adjacent isolation cells only with difficulty when
the flow of water occurs in adjacent vessels. The application of
our optimized methods of dye injection and of dye visualization by light microscopy should improve understanding of the
radial transport of water through the ray parenchyma in trees.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 12760112). The authors thank Dr. Takayuki
Shiraiwa and Dr. Keita Arakawa for use of a low-temperature room at
the Institute of Low Temperature Science, Hokkaido University.
References
Barnett, J.R., P. Cooper and L.J. Bonner. 1993. The protective layer as
an extension of the apoplast. IAWA Bull. New Ser. 14:163–172.
Bernholt, W.M. 1941. Distribution by the sap stream of spores of three
fungi that induce vascular wilt diseases of elm. J. Agric. Res. 62:
637–681.
Braun, H.J. 1967. Development and structure of wood rays in view of
contact–isolation–differentiation to hydrosystem. I. The principle
of contact–isolation–differentiation. Holzforschung 21:33–37.
Canny, M.J. 1986. Water pathways in wheat leaves. III. The passage
of mestome sheath and the function of the suberised lamellae.
Physiol. Plant. 66:637–647.
Canny, M.J. 1990a. What becomes of the transpiration stream? New
Phytol. 114:341–368.
Canny, M.J. 1990b. Rates of apoplastic diffusion in wheat leaves.
New Phytol. 116:263–268.
Canny, M.J. and M.E. McCully. 1986. Locating water-soluble vital
stains in plant tissues by freeze-substitution and resin-embedding.
J. Microsc. 142:63–70.
Chaney, W.R. and T.T. Kozlowski. 1977. Patterns of water movement
in intact and excised stems of Fraxinus americana and Acer saccharum seedlings. Ann. Bot. 41:1093–1100.
Hayat, M.A. 1989. Principles and techniques of electron microscopy:
biological applications. 3rd Edn. Macmillan Press, Houndmills,
U.K., 469 p.
IIda, I., G.J. Zhao, W.C. Shi and W.T. Wang. 1992. Dyeing of xylem
utilizing the sap flow of living trees. (3) Investigation of permeable
dyes for wood. Bull. Kyoto Prefect. Univ. For. 36:29–36.
Greenidge, K.M.H. 1958. Rates and patterns of moisture movement
in trees. In The Physiology of Forest Trees. Ed. K.V. Thimann.
Ronald Press, New York, pp 19– 41.
Kishima, T. and S. Hayashi. 1960. Microscopic observation of the
courses of water penetration into wood. Wood Res. 24:33– 45.
TREE PHYSIOLOGY VOLUME 25, 2005
VISUALIZATION OF WATER-CONDUCTION PATHWAYS
Kozlowski, T.T. and C.H. Winget. 1963. Patterns of water movement
in forest trees. Bot. Gaz. 124:301–311.
Murakami, Y., R. Funada, Y. Sano and J. Ohtani. 1999. The differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. Ann. Bot. 84:429– 435.
O’Dowd, N.A. and M.J. Canny. 1993. A simple method for locating
the start of symplastic water flow (flumes) in leaves. New Phytol.
125:743–748.
Rudinsky, J.A. and J.P. Vité. 1959. Certain ecological and phylogenetic aspects of the pattern of water conduction in conifers. For. Sci.
5:259–266.
Sakamoto, Y. and Y. Sano. 2000. Inhibition of water conductivity
caused by watermark disease in Salix sachalinensis. IAWA J. 21:
49–60.
Sano, Y., S. Fujikawa and K. Fukazawa. 1995. Detection and features
of wetwood in Quercus mongolica var. grosseserrata. Trees 9:
261–268.
Sauter, J.J. and S. Kloth. 1986. Plasmodesmatal frequency and radial
translocation rates in ray cells of poplar (Populus × canadensis
Moench ‘robusta’). Planta 168:377–380.
275
Schurr, U., B. Schuberth, R. Aloni, K.S. Pradel, D. Schmundt,
B. Jähne and C.I. Ullrich. 1996. Structural and functional evidence
for xylem-mediated water transport and high transpiration in Agrobacterium tumefaciens-induced tumors of Ricinus communis. Bot.
Acta 109:405– 411.
Tang, A.-C. and J.S. Boyer. 2002. Growth-induced water potentials
and the growth of maize leaves. J. Exp. Bot. 53:489–503.
Tyree, M.T. and M.H. Zimmermann. 2002. Xylem structure and the
ascent of sap. 2nd Edn. Springer-Verlag, New York, 283 p.
Tyree, M.T., S. Salleo, A. Nardini, M.A. LoGullo and R. Mosca.
1999. Refilling of embolized vessels in young stems of laurel: do
we need a new paradigm? Plant Physiol. 120:11–21.
Utsumi, Y., Y. Sano, S. Fujikawa and J. Ohtani. 1996. Seasonal
changes in the distribution of water in the outer growth rings of
Fraxinus mandshurica var. japonica, a study by cryo-scanning
electron microscopy. IAWA J. 17:113–124.
Utsumi, Y., Y. Sano, R. Funada, J. Ohtani and S. Fujikawa. 2003. Seasonal and perennial changes in the distribution of water in the sapwood of conifers in a sub-frigid zone. Plant Physiol. 131:
1826–1833.
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