Journal of Experimental Botany, Vol. 53, No. 368, pp. 483–488, March 2002
Fusiform cells in the cambium of Kalopanax pictus are
exclusively mononucleate
Peter Kitin1,2, Yuzou Sano1 and Ryo Funada1,3
1
2
Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Department of Dendrology, University of Forestry, Kliment Ohridski str. 10, Sofia 1756, Bulgaria
Received 22 June 2001; Accepted 28 September 2001
Abstract
While it is generally accepted that most plant cells
are mononucleate, it has been argued with some
vehemence that fusiform cambial cells can be multinucleate. The controversy has not been resolved
since to date, studies by conventional microscopy
and transmission electron microscopy have failed to
confirm unambiguously whether cambial cells are
mononucleate or multinucleate. In this study, semithin sections of epoxy-embedded specimens and
thick slices of cambial tissues from the hardwood
Kalopanax pictus were analysed by confocal laser
scanning microscopy. Tangential sections of cambium, regardless of the thickness of the section, are
likely to contain portions of cells in several adjacent
layers of cells and, at the lower resolution of conventional microscopy, several adjacent cells can appear
to be a single cell with more than one nucleus. The
higher resolution in the third dimension of confocal
microscopy allowed clearly adjacent layers of cells
in the cambium to be distinguished and the number
of nuclei per cell to be determined. In this tree, the
cambial cells were mononucleate in all cases.
Key words: Cambium, confocal microscopy, Kalopanax
pictus, nuclei.
Introduction
Cambial cells are cells whose specialized function is the
production of elements of the secondary phloem and
xylem. The structure and function of cambium have been
reviewed frequently and in depth (Philipson et al., 1971;
Barnett, 1981; Catesson, 1990, 1994; Iqbal, 1990; Larson,
1994; Chaffey, 1999; Lachaud et al., 1999). It has been
3
established that the dimensions and the morphology of
the nuclei in cambial cells, as well as the number of
nucleoli, vary within a single species according to both
the season and the stage of cambial activity (Barlow,
1985; Mellerowicz et al., 1989, 1990, 1992; Lloyd et al.,
1994, 1996).
It is generally accepted that each cambial cell contains
a single nucleus (Bailey, 1920; Larson, 1994). However,
for the past 100 years, some botanists have argued that
large and extremely elongated plant cells, such as cambial
cells, might contain many nuclei (for references, see
Bailey, 1920). In addition, during the past 30 years there
have been reports that the fusiform cambial cells in
some tropical species are multinucleate (Ghouse and
Khan, 1977; Iqbal and Ghouse, 1987; Venugopal and
Krishnamurthy, 1989; Iqbal, 1990; also, for references,
see Larson, 1994). Seasonal variations in the number of
nuclei have also been reported and, in some species,
cells containing five nuclei (Ghouse and Khan, 1977) or
eight to ten nuclei (Iqbal and Ghouse, 1987) have been
described. However, the existence of multinucleate
cambial cells has not been established unambiguously.
It has been suggested that a cambial cell might appear
erroneously to be multinucleate under the microscope if
two or more cells are superimposed in a tangential section
(for discussion, see Larson, 1994; Farrar and Evert, 1997).
Nevertheless, it has been noted that, if multinucleate
cambial cells do indeed exist, they represent a curious
phenomenon that necessitates detailed studies of the
division of such cells, providing new avenues of research
for cambial cytologists (Iqbal and Ghouse, 1990;
Catesson, 1994; Lachaud et al., 1999). The number of
nuclei in cambial cells has been inadequately studied and
it remains to be clearly determined whether cambial cells
are mononucleate or multinucleate.
This study was designed to analyse the nuclear status
of fusiform cambial cells in the hardwood Kalopanax
To whom correspondence should be addressed: Fax: q81 11 736 1791. E-mail: [email protected]
ß Society for Experimental Biology 2002
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Kitin et al.
pictus Nakai (Araliaceae). Conventional microscopic
examinations of 15 mm-thick tangential sections of
cambium revealed cambial cells that appeared to have
multiple nuclei (P Kitin and R Funada, unpublished
observations). By contrast, no multinucleate cells were
detected among isolated cambial cells in macerated tissue
(Kitin et al., 1999) or in single cells after threedimensional (3-D) reconstruction using micrographs of
serial sections of epoxy-embedded tissue (Kitin et al.,
2000). In the present study, it was attempted, by serial
optical sectioning with a confocal laser scanning microscope, to determine the number of the nuclei in individual
cells. The confocal microscope is an appropriate tool with
which to study large intact cells in thick slices of tissue
and allows detailed analysis of the nuclear status of
cambial cells (Kitin et al., 2000; Funada, 2001).
Materials and methods
Plant material
Small blocks of tissue, including cambium and the adjacent
phloem and xylem, were cut with a sharp knife and a chisel from
the stem of a single specimen of K. pictus that was growing on
the campus of Hokkaido University. Blocks were fixed in FAA
or glutaraldehyde solution, as described previously (Kitin et al.,
1999). Samples were obtained in late autumn (5 November) and
in spring (3 April, 22 April and 1 May).
Preparation of samples and microscopy
Samples of cambium were examined by conventional microscopy and by confocal laser scanning microscopy (CLSM).
The samples examined included tangential and radial sections
(15–20 mm thick) of celloidin-embedded specimens, serial semithin tangential sections (1–5 mm thick) of epoxy-embedded
specimens, and thick tangential sections (60 –80 mm thick), as
described previously (Kitin et al., 1999, 2000). All specimens
were stained with a 1% solution of safranin in 30% ethanol
for 10 min under a vacuum and then for 1 h in an incubator
at 35 8C (Kitin et al., 2000).
The 1–5 mm-thick and 15 –20 mm-thick sections were mounted
in mounting medium (Bioleit; Oken-shoji, Tokyo, Japan) for
preparation of permanent microscope slides. The slides were
observed with a confocal laser scanning microscope (LSM-310;
Carl Zeiss, Oberkochen, Germany) under transmitted visible
light or after excitation by incident light from an argon ion laser
(wavelength, 488 nm) with a band-pass (BP) filter (515–565 nm)
or from a helium neon laser (wavelength, 543 nm) with a
long-pass (LP) filter (590 nm).
The sections of 60 –80 mm thickness were treated to remove
excess stain from cells. They were dehydrated through a graded
acetone series (30%, 50%, 75%, 90%, and 100%; 15–30 min at
each concentration), with frequent changes of the respective
solutions of acetone, until no further colour was extracted from
the specimens. Then the specimens were rehydrated by passage
through decreasing concentrations of acetone and finally they
were placed in distilled water. Next, the specimens were passed
through increasing concentrations of glycerol (25%, 50%, 75%,
100% for 1 h or more per solution), with two or three changes
of each solution, and left overnight in 100% glycerol. They were
mounted on glass slides in glycerol and coverslips were placed
on the samples. Incident light from a helium neon laser (wavelength, 543 nm; LP filter, 590 nm) was used for excitation for
observations of the 60 –80 mm-thick sections of cambium by
CLSM. Consecutive confocal images of tangential sections of
cambium were obtained at intervals of 1, 2 or 3 mm. Either a
Plan Neofluar 40 3 u0.75 air or a Plan Neofluar 63 3 u1.25 oil
objective lens (both from Carl Zeiss) were used.
The digital confocal images were stored on a computer and
printed with a digital colour printer (UP-D8800; Sony, Tokyo,
Japan) as described previously (Furusawa et al., 1998). Entire
cambial cells were analysed to determine their nuclear status
on images of serial optical sections on a computer using the
software of the confocal laser scanning microscope.
Results and discussion
Cambial cells that appeared to have several nuclei were
detected in the 15–20 mm-thick tangential sections in all
samples of dormant (5 November) and active (3 April,
22 April, 1 May) cambia of K. pictus (Fig. 1A, B). However, multinucleate fusiform cambial cells on tangential
sections must be examined carefully to determine whether
the cells are, indeed, single cells or actually represent
superimposed cells in adjacent layers of cells. Under the
conventional light microscope, it was not easy to
distinguish adjacent layers of cells in tangential sections
of more than 10 mm in thickness because the tangential
walls of adjacent cells were visible only with difficulty
(Fig. 1A). Observations of sections of 1–5 mm in thickness
did, however, distinguish cells in adjacent tangential
layers (Kitin et al., 2000). The tangential walls of cambial
cells could be observed with relative ease when the
sections were slightly oblique (note, for example, the
obliquely cut double walls between cells 1, 2 and 3 in
Fig. 2). In contrast to the observations of 15–20 mm-thick
sections, observations of 1–5 mm-thick sections failed to
reveal any multinucleate cells. When nuclei of adjacent
cells were visualized in the same focal plane, it was
apparent that such nuclei were positioned on both sides of
obliquely cut cell walls (Fig. 2). Similarly, in studies by
transmission electron microscopy (TEM), no multinucleate cambial cells were found in Robinia pseudoacacia
(Farrar and Evert, 1997) or in Aesculus hippocastanum
(Chaffey et al., 1997). However, it is difficult to analyse
entire cells on single sections of 1–5 mm in thickness or by
TEM using ultra-thin sections and a clear definition of
the nuclear status of cambial cells cannot be derived in
either case.
Entire cambial cells can be analysed in detail on serial
sections of epoxy-embedded tissue. However, the procedure is very time-consuming (Kitin et al., 2000). In the
present study, the nuclear status of cambial cells was
analysed on serial optical sections of thick slices of tissue
(60 –80 mm thick) by CLSM. Multinucleate cambial cells
were visualized in projection images by CLSM (Fig. 3A).
Three-dimensional (3-D) analysis of the same slices of
tissue did not, however, show any cambial cells with
Fusiform cambial cells are mononucleate
485
Fig. 2. A confocal image of a tangential section (5 mm thick) of epoxyembedded tissue. The sample was excited by the argon laser (488 nm)
with a BP filter (515–565 nm). The section includes three adjacent layers
of cells. The cells indicated by numbers 1, 2 and 3 are adjacent cells in a
radial file of cambial cells. The thin tangential walls between these cells,
which have been cut obliquely, are visible as dark strips. Arrows point to
nucleoli, which are seen on both sides of the double wall between cells 2
and 3. Scale bar ¼ 20 mm.
Fig. 1. A tangential section of cambium in celloidin-embedded tissue
(20 mm thick). The sample was obtained at cambial reactivation (3 April).
(A) Transmitted-light image. Several nuclei (arrows) are apparent within
the contours of some cells. (B) The same section visualized by CLSM
after staining with safranin and excitation by the helium neon laser
(543 nm; LP filter, 590 nm). A projection image of 10 optical sections,
obtained at 1 mm steps, is shown. Within a depth of 9 mm along the ‘z’
axis of the specimen, three nuclei appear within the contours of one cell
(arrows on the left) and four nuclei appear within the contours of
another cell (arrows on the right). Scale bar ¼ 50 mm.
more than one nucleus. The higher resolution of the
confocal images in the third dimension (‘z’-resolution)
revealed the tangential walls between adjacent cells and
showed that each nucleus did, in fact, belong to a
different cell (Fig. 3B, C, D). The bright strip between
cells 1 and 2 in Fig. 3B, C and D is due to fluorescence
from the obliquely cut tangential walls between these
two cells. The nucleus of cell 1 is seen in panels B and C in
Fig. 3 and the nucleus of cell 2 is seen in panel D in the
same figure.
The number of nuclei in cambial cells has been studied,
for the most part, in tangential sections by conventional
light microscopy. Iqbal and Ghouse reported as many as
10 nuclei per cell in their analysis of 10–12 mm-thick
longitudinal tangential sections of the cambium of Acacia
nilotica (Iqbal and Ghouse, 1987, 1990). These authors
argued that a 10 –12 mm-thick tangential section would
contain, at most, two or three layers of cambial cells
since the radial diameter of fusiform cambial cells was
only 4 –10 mm. Therefore, the detection of, apparently,
10 nuclei per cell in 10 –12 mm-thick sections was taken
as proof of the existence of multinucleate cells.
In the present study, cambial cells that were apparently multinucleate were also observed in relatively thin
sections by conventional microscopy or in projection
images obtained by CLSM. For example, Fig. 1B is a
projection image that was constructed from ten optical
sections obtained, at steps of 1 mm, by CLSM. The tissue
scanned by CLSM corresponded, in this case, to a
histological section of 9 mm in thickness. The image in
Fig. 1B clearly shows the fluorescence from several nuclei
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Kitin et al.
Fig. 3. A confocal image of a thick tangential section of cambium (thickness, approximately 60 mm) that was sampled at cambial reactivation
(3 April). The section was stained with safranin and excited by the helium neon laser (543 nm) with an LP filter (590 nm). The images show the
bright fluorescence from cell walls and nucleoli. (A) A projection image of eight optical sections, obtained in 2 mm steps. Two nuclei appear
within the contours of some of the fusiform cells (arrows). However, an analysis of confocal images shows that each of the nuclei belongs to a different
cell. (B–D) Sequential optical sections in 2 mm steps of the sample in (A). Cells designated 1 and 2 in (B–D) are adjacent cells in a radial file of cambial
fusiform cells. The bright strip between cells 1 and 2 is a portion of the tangential wall between these two cells, which was cut obliquely. The nucleus
of cell 1 is seen in panels (B) and (C) and the nucleus of cell 2 is seen in panel (D). Scale bar ¼ 50 mm.
per cell, which are included within the 9 mm-thick optical
slice of cambial tissue. Since the radial width of fusiform cambial cells in the stem of K. pictus is 6–12 mm,
a tangential section of 9 mm would normally contain
portions of at most one or two layers of cells. Therefore,
the visualization of four nuclei within a tangential section
of 9 mm in thickness suggests the presence of cells with
more than one nucleus or, at least, of two superimposed
cells that are in the process of mitosis, provided that the
section is oriented exactly along the cell axes. However,
the analysis by CLSM revealed that, in every case, the
tangential sections of cambium were slightly oblique.
Even thin sections, such as the section shown in Fig. 2,
included portions of several adjacent cambial cells.
Figure 4 shows that it is extremely difficult and may even
be impossible to cut longitudinal sections precisely along
the axes of cambial cells. Tangential sections for
observations of cambium are commonly large (side AB
in Fig. 4). Thus, a very small shift in the plane of
sectioning from the axial direction (the angle a8 in Fig. 4)
would result in non-negligible obliqueness of the section.
Since the length of the fusiform cambial cells in K. pictus
can be as much as 600 mm (Kitin et al., 1999), the
longitudinal extent of any observation on a microscopic
Fusiform cambial cells are mononucleate
487
section, appearing to be a single cell with several nuclei,
because of the large depth of focus and low resolution in
the third dimension of the conventional light microscope.
By contrast, analysis by CLSM of serial optical sections
of thick slices of tissue clearly revealed that each cambial
cell of K. pictus contains a single nucleus.
Acknowledgements
The authors thank Dr J Ohtani and Dr S Fujikawa for their
valuable comments. This work was supported in part by a
Grant-in-Aid for Scientific Research from the Japan Society for
the Promotion of Science (No. JSPS-RFTF 96L00605).
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Fig. 4. A radial section of dormant cambium and a diagram showing
possible directions of longitudinal–tangential sectioning. The sides AB
and BC of the triangle ABC represent oblique and exactly oriented
tangential sections, respectively. The angle between AB and BC (a8)
represents the shift from the axial orientation of the plane of sectioning.
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