Plant Cell Physiol. 41(11): 1267–1271 (2000)
JSPP © 2000
Autolysis during In Vitro Tracheary Element Differentiation: Formation and
Location of the Perforation
Jin Nakashima 1, 3, Keiji Takabe 2, Minoru Fujita 2 and Hiroo Fukuda 1
1
2
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 113-0033 Japan
Department of Forest Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502 Japan
;
Tracheary elements differentiated from isolated Zinnia
mesophyll cells were observed at various times of culture
under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends
in single tracheary elements. In double tracheary elements,
which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the
junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results
suggest not only that a single tracheary element has its own
program to form a perforation at one end without being
affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.
Key words: Perforation — Programmed cell death — Tracheary element differentiation — Zinnia elegans.
Introduction
Cell walls, which are characteristic morphological structures in the plant kingdom, are laid outside the protoplasts. Cell
walls are composed of various kinds of polysaccharides such as
cellulose, hemicellulose, and pectin; cell wall proteins including arabinogalactan proteins and hydroxy-proline rich glycoproteins; and amorphous polyphenolic polymers such as lignin.
Vessels are specially built up of a longitudinal series of individual cells, termed vessel members or elements, to provide the
three-dimensional pathway for the ascent of water in plants.
For water transport, vessel elements have perforations at both
ends, and sometimes also on a side wall, and other regions of
the cell walls were reinforced by lignified secondary walls. In
general, the water moves freely through perforations in the
walls. Therefore, perforation occurs in the last stage of vessel
element differentiation as an event of programmed cell death
(Esau 1965, Esau and Charvat 1978, Meylan and Butterfield
1981, Buvat 1989). In situ vessel elements are formed successively according to cells derived from the apical meristem. A
differentiating vessel element is connected with a mature dead
vessel element and a vessel precursor cell at each end, but perforation occurs only at the end wall between the mature dead
3
vessel element and the differentiating vessel element (Esau and
Charvat 1978). Therefore, perforation should be regulated temporally and spatially in situ.
An in vitro experimental system, in which isolated Zinnia
mesophyll cells differentiate into tracheary elements (TEs), is
widely used for physiological, biochemical, and molecular biological studies to elucidate the mechanism of TE formation
(Fukuda 1997). This experimental system also offers a great
opportunity for the investigation of perforation in vitro. It has
been suggested that in intact vascular tissues, end-wall hydrolysis is a sign of the wiping away of the remnants derived from
the cytoplasm by the transpiration stream (O’Brien 1981). In
isolated Zinnia cells, however, this mechanism is unlikely to
explain the formation of perforations. Rather, perforation
seems to occur autonomously in single TEs (Burgess and Linstead 1984, Burgess and Linstead 1985, Groover et al. 1997).
Furthermore, Burgess and Linstead (1984) reported that the region of the middle lamella is specially resistant to autolysis
when it is either on the outside of an isolated cell which differentiates or between neighboring cells in contact, both of which
differentiate. These results indicate that active maintenance of
the middle lamella by the undifferentiated neighbor may be
taking place even in vitro. As a first step toward understanding
perforation mechanism during programmed cell death, therefore, we observed in detail perforation of TEs formed in vitro
using a scanning electron microscope (SEM) and demonstrated that perforation is formed temporally and spatially at only
one end even in single cells.
Materials and Methods
Mesophyll cells of the first true leaves of 14-day old seedlings of
Zinnia elegans L. cv. Canary Bird (Takii Shubyo Co., Kyoto, Japan)
were isolated as described previously (Fukuda and Komamine 1980,
Sugiyama et al. 1986), and cultured in differentiation medium. The
medium contained 0.1 mg liter–1 of naphtaleneacetic acid (NAA) and
0.2 mg liter–1 of benzyladenine (BA) as growth regulators. The population of cells was adjusted to 8.4104 cell ml–1. Cultured cells were harvested at every 6 h after the secondary wall formation had started, and
fixed with 2% (v/v) glutaraldehyde in the differentiation medium for
16 h under a cooled condition according to the procedure of Franklin
(1945) with some modifications. That is, half of the cells were sectioned at 20-m thickness with a sliding microtome equipped with a
freezing device, which enables us to investigate the cell walls from inside. All of the cells were washed thoroughly with 1/15 M Sörensen’s
phosphate buffer (pH 5.59), dehydrated in a series of ethanol, substi-
Corresponding author: E-mail, [email protected]; Fax, +81-3-5841-4462.
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Perforation of TEs formed in vitro
Fig. 2 A typical SEM image of a single TE. This scanning electron
micrograph shows a overview of the typical single TE cultured for
96 h in the differentiation medium. The perforation with a central big
hole is observed on the primary wall regions at one longitudinal end.
The secondary wall thickenings in the wall are shown as strong reflecting regions which surrounded the primary walls with the mesh-like
structure. PW, primary wall; SW, secondary wall thickening.
Fig. 1 Time course of tracheary element (TE) differentiation from
Zinnia mesophyll cells cultured in the differentiation medium, which
contains 0.1 mg liter–1 of naphtaleneacetic acid (NAA) and 0.2 mg
liter–1 of benzyladenine (BA) as growth regulators. The values represent the average of two samples.
tuted in a 2-methyl-2-propanol, and lyophilized in the critical state.
The specimens were mounted on the specimen carrier stubs, coated
with gold, and observed under a SEM operated at 10 kV (JEOL JSM-T
330A).
Results and Discussion
Time course of the formation of TEs in the differentiation
medium under a light microscope is shown in Fig. 1. TEs were
formed at high frequency accounted for up to 54% among the
total cells at the end of differentiation. There was high degree
of synchrony in the formation of TEs after 48 h of culture. At
72 h, transmission electron microscopic observation revealed
that most of TEs lost their cytoplasms, indicating the death of
them (data not shown). Figure 2 shows an overview of a typical
single TE cultured for 96 h. The secondary thickenings in the
wall were shown as strong reflecting regions which surrounded the primary walls with the mesh-like structure. A big hole
(an arrow) is observed on the primary wall region at one end.
Because the thickening rim around this pore was not observed,
this pore was considered to be perforation of vessel elements.
Observation of many TEs allowed us to categorize types of
perforation into three types, that is, perforations with central
big holes (over 2 mm in diameter) surrounded by small numerous pores (Fig. 3a), with simple big holes (Fig. 3b), and with
small numerous pores (Fig. 3c). Figure 4 shows the changes in
three types of perforations when 100 single TEs were observed
at 6 h-intervals after 60 h of culture. At 72 h of culture, perforations were first observed and over 90% of them were classified
into the “small numerous pores” type (Fig. 3c, 4). When the
maturation of TEs proceeded, the proportion of the “small numerous pores” type drastically decreased, and in turn remarkably increased the proportion of the “central big holes surrounded by small numerous pores” type. At 96 h of culture, the latter
type occupied 82% of the TEs in which perforations could be
observed. The “simple big holes” type did not appear before
78 h of culture, but thereafter occupied constantly about 15%
of the TEs in which perforations could be observed. These results suggest that small numerous pores develop to a few big
holes during maturation of TEs and that this development occurs after TEs have lost cell contents. Even at mature stage,
most TEs had many small pores in addition to big pores. Such
characteristic perforations in TEs appear to differ from TEs
formed in situ, which have simple big pores. These results imply that end-wall remnants in situ may be completely swept
away by the transpiration water. The percentage of TEs with
perforations did not reach 100%, because, in the experimental
procedure for SEM, substantial numbers of TEs were stuck
onto the specimen carrier stub with the perforation sites. As
typically shown in Fig. 5, both ends could be clearly observed
in 13 TEs at 90 and 96 h of culture. All TEs had perforations at
only one end. These results suggest that the perforations are
formed only one longitudinal end on most single TEs.
In the Zinnia culture system, some single cells differentiate into two TEs after dividing into two, called double TE.
Study of this double TE may give information about perfora-
Perforation of TEs formed in vitro
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Fig. 3 Three types of perforations of TEs. These scanning electron micrographs represent the magnified view of the portion of perforations in
the single TEs cultured for 96 h, allowing us to categorize types of the perforation into three. That is, perforation with the “central big holes (over
2 mm in diameter) surrounded by small numerous pores” type (a), with the “simple big holes” type (b), and with the “small numerous pores” type
(c), respectively.
Fig. 5 A TE showing both longitudinal ends. This scanning electron
micrograph shows a overview of the typical single TE cultured for
96 h in the differentiation medium. Both ends can be clearly observed
from this electron micrograph, which suggests that perforations are
formed at only one end (arrow).
Fig. 4 Changes in three types of perforations of TEs during culture.
Hundred single TEs were observed at 6 h-intervals after 60 h of culture. Perforations with the “central big holes (over 2 mm in diameter)
surrounded by small numerous pores” type are represented in gray columns, perforations with the “simple big holes” type in open columns,
and perforations with the “small numerous pores” type in black columns, respectively.
tions in planta. When the cells were sectioned with the microtome, some TEs were accidentally fractured and exhibited the
cell walls between two TEs of the double TE. Figures 6a and b
are two examples of such TEs showing that, after a cell divided
almost parallel to the longitudinal axis, the perforation was
formed on the primary wall between the two TEs. The perforations on cell walls between the two TEs were always the “simple big holes” type bounded by the secondary wall thickenings. Observation of 13 double TEs showed that all TEs had
perforations (at most four) between their two TEs. These
perforations are remarkably similar to the perforations observed on the lateral primary walls between two vessel ele-
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Perforation of TEs formed in vitro
Fig. 7 Perforations of double TEs that developed after a cell divided
almost perpendicularly to the longitudinal axis. The perforations are
formed at the two positions, that is, at the junction of the two TEs
(arrow a) and at a longitudinal end of one of the two TEs (arrow b).
Inlet panel indicates the illustration of the fractured position.
Fig. 6 Perforations of double TEs that developed after a cell divided
almost parallel to the longitudinal axes. The perforations are formed
on the primary walls between the two TEs. They were always the
“simple big holes” type bounded by the secondary wall thickenings
(arrows). Inlet panels indicate the illustration of the fractured position.
ments in roots and rhizomes of ferns (Carlquist and Schneider
1999, Carlquist and Schneider 2000) and in the xylem of
woody plants (Meylan and Butterfield 1981). When the two
successive TEs developed after a cell divided almost perpendicularly to the longitudinal axis, perforations occurred at the
two positions, that is, at the junction of the two TEs and at the
end of only one of the two TEs (Fig. 7). Similar but non-fractured double TEs had perforations at only one longitudinal end.
Therefore, it is strongly suggested that a TE perforates only at
one end even in divided TEs. Thus, TEs formed in cultured
Zinnia cells have the morphological feature of the vessel elements, and most perforations are formed near the longitudinal
end of TEs.
These results indicate that single TEs formed in vitro have
their own program to form perforations at a given end, which
must be one of the features in programmed cell death at the cellular level. These results also suggest the presence of cell-po-
larity even in a single TE and of well organized cell-cell communication in double TEs. Indeed, an antibody revealed
polarized localization of a cell wall component in differentiating single TEs in culture (Shinohara et al. 2000). The fact that
auxin transport inhibitors prevent TE formation in cultured
Zinnia cells (Burgess and Linstead 1984) also supports the idea
that polarity in the cell is involved in in vitro TE formation.
When the successive differentiation of vascular networks actively progresses, perforations are formed at the side of mature
dead TEs, but not of immature living TEs, which is another important characteristic for programmed cell death at the cellular
level. Such organized perforations should be carried out by various wall-degradating enzymes. Recently, we found that wallbound polygalacturonase activity and mRNAs for two polygalacturonase genes are upregulated in association with TE formation (Y. Ohdaira, K. Iwamoto, M. Sugiyama, and H. Fukuda,
in preparation). These wall-bound polygalacturonases might be
some of the key enzymes which spatially and temporally control the formation of perforations. Efforts are under way to elucidate the involvement of these polygalacturonases in the formation of perforations during programmed cell death.
Acknowledgements
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (nos.
10304063, 10219201, and 10182101 to H.F.), and from the Japan Society for the Promotion of Science (JSPS-RF096749 to H.F.), and by
Japan Society for the Promotion of Science Research Fellowships for
Young Scientists (to J.N.).
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(Received July 11, 2000; Accepted August 23, 2000)