Download Alterations in ultrastructure and subcellular

Journal of Experimental Botany, Vol. 48, No. 311, pp. 1195-1207, June 1997
Journal of
Experimental
Botany
Alterations in ultrastructure and subcellular localization
of Ca 2+ in poplar apical bud cells during the induction of
dormancy
Ling-cheng Jian 1 3 , Paul H. Li 1 ' 4 , Long-hua Sun1'3 and Tony H.H. Chen 2
1
Laboratory of Plant Hardiness, Department of Horticultural Science and Plant Biological Sciences Program,
University of Minnesota, St Paul, MN 55108, USA
2
Department of Horticulture, and Centre for Gene Research and Biotechnology, Oregon State University,
Corvallis, OR 97331, USA
Received 8 May 1996; Accepted 14 February 1997
Abstract
In poplar (Populus deltoides Bartr. ex Marsh), bud
dormancy and freezing tolerance were concomitantly
induced by short-day (SD) photoperiods. Ultrastructural changes and the alteration in subcellular localization of calcium in apical bud cells associated with
dormancy development were investigated. During the
development of dormancy, the thickness of cell walls
increased significantly, the number of starch granules
increased, and there was a significant accumulation
of storage proteins in the vacuoles of the apical bud
cells. The most striking change was the constriction
and blockage of the plasmodesmata.
It was demonstrated that antimonate precipitation
is a reliable technique for studying subcellular localization of calcium in poplar apical bud cells. Under the
long day (LD) photoperiod, electron-dense calcium
antimonate precipitates were mainly localized in vacuoles, intercellular spaces and plastids. Some antimonate precipitates were also found in the cell walls and
at the entrance of the plasmodesmata. However, there
were few Ca 2+ deposits found in the cytosol and nucleus. After 20 d of SD exposure, when development of
bud dormancy was initiated, calcium deposits in intercellular spaces were decreased, whereas some
deposits were found in the cytosol and nuclei. From
28-49 d of SD exposure, while dormancy was developing, a large number of Ca 2+ precipitates were found
in the cytosol and nuclei. When deep dormancy was
reached after 77 d of SD exposure, Ca 2+ deposits
4
3
became fewer in both cytosol and nuclei, whereas
numerous deposits were again observed in the cell
walls and in the intercellular spaces. These results
suggest that under the influence of SD photoperiods,
there are alterations in subcellular Ca 2+ localization,
and changes in ultrastructure of apical bud cells during
the development of dormancy. The constriction and
blockage of plasmodesmata may cause the cessation
of symplastic transport, limit cellular communication
and signal transduction between adjacent cells, which
in turn may lead to events associated with growth
cessation and dormancy development in buds.
Key words: Poplar, apical bud cells, Ca2+ subcellular
localization, dormancy.
Introduction
The development of vegetative dormancy is an important
adaptive strategy for the survival and growth of temperate
perennial plants. It is generally believed that dormant
plants are better able to withstand low, potentially
damaging temperatures during winter. For most temperate perennial plants, the annual growth cycle involves an
alternation between periods of active shoot growth and
dormancy (Nooden and Weber, 1978). Cessation of shoot
growth, development of cold hardiness and initiation of
dormancy in most perennial plants are either accelerated
or initiated by short days (SD) and inhibited or delayed
by long days (LD) (Vince-Prue, 1975).
As in most perennial plants, vegetative dormancy in
To whom correspondence should be addressed. Fax: + 1 612 624 4941. E-mail: Iixxx008emaroon.tc.umn.edu
Visiting scientists from The Institute of Botany, Chinese Academy of Sciences, Beijing, China.
6 Oxford University Press 1997
1196
Jian et ai.
poplar is initiated or accelerated by SD photoperiod
(Nitsch, 1957; Pauley and Perry, 1954). Dormancy development in poplar also varies in an ecotypic manner.
Generally speaking, cessation of growth and development
of dormancy of plants from northern latitudes are initiated at longer day lengths than plants from southern
latitudes (Pauley and Perry, 1954; Howe et al., 1995).
There are distinct morphological and anatomical changes
during the development of bud dormancy. The morphology and development of terminal and axillary buds
subjected to a variety of photoperiod treatments were
studied by Richards and Larson (1981) and Goffinet and
Larson (1982). However, ultrastructural changes in
bud cells in relation to dormancy induction were not
examined.
Metabolic changes associated with the seasonal cycle
of dormancy have been studied extensively in poplar.
Changes in carbohydrates (Fege and Brown, 1984; Nelson
and Dickson, 1981), amino acids (Cote et al., 1989;
Sagisaka, 1974a), and enzyme activities (Sagisaka, 1974ft)
all demonstrated that metabolic changes are closely associated with the annual growth cycle of poplar. In addition,
a 32 kDa poplar bark storage protein was accumulated
during dormancy induction by short days (Coleman et al.,
1991); and a full-length cDNA and a gene encoding for
poplar 32 kDa bark storage protein have been isolated
and sequenced (Coleman et al., 1992; Coleman and
Chen, 1993). Although physiological changes that occur
throughout the dormancy cycle have been wellcharacterized, little is known about the process of SD
signal transduction and ultrastructural changes associated
with the development of bud dormancy in poplar plants.
It is well-known that calcium plays an important role
in plant growth and development. The alteration in
cellular Ca2+ level regulates an astonishing variety of
cellular processes from control of ion transport to gene
expression (Hepler and Wayne, 1985; Bush, 1995). It is
also known that environmental stimuli, such as temperature and light, etc., can alter cellular Ca 2+ distribution
(Hepler and Wayne, 1985; Bush, 1995). It was investigated whether there is a relationship between the alteration in cellular Ca 2+ localization and ultrastructural
changes and the development of dormancy in apical bud
cells of poplar and the results suggest that SD-induced
alteration in subcellular Ca 2+ localization, and ultrastructural changes in plasmodesmata and cell walls are closely
associated with the development of dormancy in poplar
buds.
Materials and methods
Plant material
Poplar plants (Populus deltoides Bartr. ex Marsh.) were
established from greenwood stem cuttings that were rooted and
grown in the greenhouse in individual 1.5 1 pots containing a
mix of 2 peat moss:l soil:l prolite (by vol.) under a photoperiod
of 16 h light and 8h darkness, and temperatures of 25/21 °C
day/night. When the plants grew to about 40-50 cm tall, they
were transferred into a SD growth chamber with photoperiod
of 8 h light and 16 h darkness and temperatures of 25/21°C
day/night for up to 77 d. Plants which had been grown for
different time periods in SD were used as experimental materials.
Measurement of dormancy status
At each sampling date (0, 10, 20, 28, 35, 49, and 77 d of SD
exposure), one set of six poplar plants were transferred from
SD growth chamber to LD greenhouse conditions at 25/21 °C
day/night to determine dormancy status. Plants were manually
defoliated and the shoot tips (about 2.5 cm) were removed.
Dormancy status was expressed as the number of days to first
bud break (the first visible shoot growth) in the decapitated
and defoliated plants.
Measurements of freezing tolerance
The shoots were cut into short stem segments, each with two
nodes. Five stem segments were placed in each of the test tubes,
and there were three replicates (tubes) per sample. Tubes with
samples were placed vertically in a low temperature liquid bath
and then cooled down at a rate of 4-5 °Ch"'. When the sample
temperature reached — 2°C, ice pieces were added to the tubes
to inoculate the stem segments to prevent supercooling. Samples
were then lowered to each of —6, —8, —10, —12, —14, —16,
and — 18°C for 3 h. The tubes were removed from the liquid
bath at each temperature interval and the stem segments were
removed from the tuber and immediately placed on moist filter
papers in Petri dishes which were held in an ice-filled box. After
slowly thawing overnight in a cold room (2-4 °C), stem
segments were incubated in a growth chamber at 25/21 °C
day/night temperature and 14 h photoperiod. After 5 d, viability
of the bark of the stems and buds was examined by visual
observation of tissue browning. When bark and bud tissues
became brown, death was recorded. Temperatures which caused
50% death of the total samples were referred to as killing
temperature (LTi0).
Sample preparation for cytochemical localization of Ca2 + and
ultrastructural observations
Cytochemical localization of calcium was performed following
the method of Slocum and Roux (1982) and adapted by Wang
and Jian (1994). The samples were collected from the plants
after either 10, 20, 28, 35, 49, and 77 d of SD exposure or LD
plants (0 d in SD). To minimize the possible effects of circadian
rhythm on intracellular Ca2+ fluctuation induced by environmental stimulus, such as photoperiod, all sample collections
were conducted at 11.00 a.m., about 3h after the beginning
of light illumination. The apical buds were excised, cut into
0.5 x 0.5 x 0.5 mm slices, and immediately immersed in a fixative
solution containing 4% glutaraldehyde and 2% potassium
antimonate (K2 H2 Sb207.4H20) in 0.1 M potassium phosphate
buffer (pH 7.6) for 4 h at 4°C. After fixation, the samples were
washed three times, 20 min each, with 0.1 M potassium
phosphate buffer (pH 7.6) containing 2% potassium antimonate,
and then post-fixed in 1% osmium tetroxide (OsO4) and 2%
potassium animonate in 0.1 M potassium phosphate buffer
(pH 7.6) at 4°C overnight. After post-fixation, the samples
were washed twice in phosphate buffer containing 2% potassium
antimonate, and followed by twice with pH 10 distilled water
adjusted with KOH. Thereafter, the samples were dehydrated
in an ethanol series, and embedded in EMbed 812 (EMS, New
Jersey, USA). The embedded samples were then sectioned with
Poplar apical bud cells during dormancy
1197
glass knives and a RMC (Tucson, Arizona, USA) MT4000
ultramicrotome. The sections were stained with uranyl acetate,
and then observed and photographed under a Philips (Mahwah,
New Jersey, USA) CM 12 TEM operated at 60 kV. At the same
time, half of the samples were fixed in a fixative solution
containing no antimonate. These tissue sections were used as
the control for cytochemical localization of Ca2 + , as well as
used for the observation of ultrastructural changes.
In order to verify the location of calcium, additional treatment
involving the chelation of calcium ion with EGTA was
performed. Grids mounted with tissue sections previously
examined by TEM were immersed in the solution of 100 mM
EGTA (pH 8.0), and incubated at 60 °C for 1 h. After treatment,
the grids were rinsed briefly in distilled water and stained with
uranyl acetate again, and then observed under EM.
20
28
35
49
77
Days in SD
Results
Changes in dormancy status and freezing tolerance
Under 8 h SD photoperiod and warm temperature, the
growth rate of poplar plants decreased immediately upon
transfer to SD from LD, and completely stopped by 20 d
of SD treatment (data not shown). However, there was
neither significant change in dormancy status, expressed
as the days to bud break (Fig. 1), nor in freezing tolerance
(Fig. 2) for plants exposed to SD for up to 20 d. There
was a gradual increase in the degree of dormancy as
evidenced by the delayed bud break which increased from
14-29 d after 28 d and 49 d of SD exposure, respectively.
After 77 d of SD exposure, it took more than 3 months
before any sign of regrowth became visible, indicating
that a high degree of dormancy had been reached. The
degree of dormancy of the terminal buds was similar to
that of lateral buds of the same plants (Jian et al,
Fig. 2. The induction of freezing tolerance in poplar buds and stem
bark under growth chamber conditions of warm temperature and short
day photopenod. See Materials and methods section for detail. Vertical
bar = LSD (P< 0.05).
unpublished results). During the SD treatment, there was
also a gradual increase in freezing tolerance in both the
stem and buds (Fig. 2). In plants that had been maintained under LD and warm conditions, the killing temperature was about — 6 °C and was constant throughout the
experimental period of 77 d. Under SD, plants developed
a higher degree of freezing tolerance when exposed to SD
for more than 28 d. Plants given 77 d of SD exposure
were able to tolerate about 12°C lower temperature than
LD plants. Thus, the SD photoperiod clearly induced
development of dormancy and cold hardiness simultaneously and in a similar manner to that observed for other
temperate perennial plants (Weiser, 1970).
Ultrastructural changes under SD conditions
©
1
10
20
28
35
49
77
Days in SD
Fig. 1. Changes in the dormancy status of poplar buds under growth
chamber conditons of warm temperature and short-day photoperiod.
Dormancy status was expressed by the number of days needed when
50% of SD-exposed plants showed bud break after transferring to LD.
See Materials and methods section for detail.
Plate 1A-C shows the ultrastructural features of the meristematic cells of poplar apical buds from plants which
were grown under LD photoperiod. These cells contained
relatively large nuclei which were localized in the centre
of the cells, and which were characterized by their dense
cytoplasm and thin walls. Many mitochondria, plastids,
endoplasmic reticuli, and ribosomes could be identified
in the cytoplasm, and many plasmodesmata traversed the
walls of neighbouring cells. After 77 d of SD exposure,
many starch grains accumulated in the apical bud cells;
the heterochromatin in the nuclei of these cells decreased
or disappeared; the walls of these cells were markedly
thickened; and the plasmodesmata in the cell walls were
not visible (Plate ID). This is in contrast to that observed
in the samples collected from LD plants, in which many
plasmodesmata were observed in the walls between two
neighbouring cells (arrows, Plate 1A, B).
Plate 2A-D shows the high magnification view of
plasmodesmata electron micrographs. Plate 2A is the cells
of apical buds grown in LD; many plasmodesmata in the
1198 Jian etal.
Plate 1. Ultrastructural comparison of apical bud cells between LD and 77 d SD-grown plants. (A-C) The meristematic cells of apical buds in
plants grown under LD condition. Note the relatively large-sized nucleus (N), dense cytoplasm, many mitochondria (M), plastids (P), endoplasmic
reticulum (ER), small vacuoles (V) and thin cell wall (W); and many plasmodesmata (PD) traverse the cell walls of neighbouring cells (arrows)
(A) x4500; (B) x 7000; (C) x 18000. (D) After 77 d of SD exposure, the hetrochromatin in nucleus decreased or disappeared; the cell walls were
thickened markedly; the plasmodesmata are not visible in the cell walls; x 6000. S: starch granule. Bar: 1 fun.
walls of the neighbouring cells could be observed and
their pores were generally larger in diameter. Plate 2B
shows the cells from plants which had been exposed to
SD photoperiod for 35 d. The walls of these cells were
thickened and the pore diameter of plasmodesmata was
reduced. After 77 d of SD exposure when the apical buds
had reached a high degree of dormancy (Plate 2C, D),
the plasmodesmata of these cells appeared to undergo a
further alteration, i.e. the plasmodesmata were no longer
distinguishable from the cell walls. Both ends of the
plasmodesmata seemed to be blocked and the plasma
membranes at the entrance of the plasmodesmata
appeared to be fused with each other and formed a
continuous membrane which was parallel with the cell
wall. A number of electron-dense grains, which may be
the remains of the original plasmodesmata, could still be
identified at the plasmalemma (arrows, Plate 2C, D).
Plate 3A-D shows the ultrastructure of bud cortical
cells from plants of 0, 28, 35, and 77 d of SD exposure,
respectively, at the same magnification (x4500). It was
clearly observed that during the induction of dormancy
the cell walls were thickened; the precipitates in vacuoles
increased; the central vacuoles in the cells from plants
exposed to SD for 77 d were segmented to form many
Poplar apical bud cells during dormancy
1199
Plate 2. Plasmodesmata in apical bud cells of poplar plants which had been exposed to SD tor various penods ot time. (A) Cells of LD-grown
plants. After 35 d (B) and 77 d of SD exposure (C, D). (A) x 9000; (B) x 5000; (C) x 24000; (D) x 24000. PM: plasmamembrane. Bar: 1 ^m.
smaller vacuoles; andfinallythe number of starch granules
in the cells was markedly increased. The plasmodesmata
in the samples collected from plants that had been exposed
to SD for 28 d were constricted (arrows, Plate 3B). In the
cells of the samples collected from plants after 35 d of
SD exposure, only a few small plasmodesmata could be
observed in the walls of neighbouring cells (arrows,
Plate 3C). After 77 d of SD exposure (Plate 3D), almost
no distinct plasmodesmata could be found in these
cell walls, the plasma membrane lost its intercellular
connection, and thus the intercellular transport and
communication of symplast might be discontinued.
These results indicate that during the development of
dormancy induced by short days, there were a number of
ultrastructural changes in both apical meristematic cells
and differentiated cells directly under the apical meristematic cells. The changes could be summarized as follows:
(1) the heterochromatin in nuclei decreased
or disappeared; (2) starch granules increased; (3) a
large number of precipitates, possibly storage proteins
(Coleman et cil., 1991; Wetzel et ai, 1989), were
accumulated in vacuoles; (4) the cell walls were thickened;
and (5) the plasmodesmata were constricted, and their
pores were blocked.
Localization of Ca2 + in poplar apical bud cells grown in LD
For apical bud sections fixed with potassium antimonate,
the calcium antimonate precipitates, which are indicative
of calcium localization, appeared as electron-dense particles when observed under transmission electron microscope. The electron-dense calcium precipitates in the
apical bud cells of LD plants were mainly localized in
vacuoles (Plate 4A, B), implying that the vacuole is a
main pool of Ca2+ in cells of actively growing plants.
There were many large electron-dense grains in intercellular spaces and plastids (Plate 4A, B). In addition,
some antimonate precipitates were observed in the cell
walls and at both ends of plasmodesmata (Plate 4A, B).
On the other hand, few visible Ca 2+ deposits were
observed in cytosol and nucleus (Plate 4A, B).
To demonstrate that the electron-dense grains were
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Jian et al.
D
Plate 3. Ultrastructural changes in the differentiated cortical cells of poplar apical buds during the induction of dormancy by short days.
Ultrastnictures of cells from LD (A), and from 28 d (B), 35 d (C), and 77 d (D) of SD-grown plants. Note the changes in plasmodesmata (arrows).
x4500. Bar: 1 /un.
indeed calcium deposits and not osmiophilic globules
(lipid body), the tissue sections fixed in solutions with
or without potassium antimonate were compared, and
between sections which were fixed in solutions containing
potassium antimonate but then treated with EGTA, a
treatment that is known to remove calcium. The cells
fixed without potassium antimonate did not show any
electron-dense precipitates in plastids, intercellular spaces
and vacuoles (Plate 4C). Furthermore, in the tissue sections that were treated with EGTA, there were small
transparent holes in plastids, intercellular spaces and
vacuoles, corresponding to where the electron-dense
grains were localized before EGTA treatment (Plate 4D).
Taken together, these results suggested that the location
of electron-dense precipitates is a reliable indicator of
cellular calcium localization.
Changes of calcium distribution in poplar apical buds during
dormancy induction by short day
After 10 d of exposure to SD, the distribution of calcium
in apical buds appeared to be similar to that of LD
photoperiod, and no significant change was observed.
Again, a large number of calcium antimonate precipitates
were observed in intercellular spaces, vacuoles and
plastids; and there were few visible Ca 2+ deposits in
cytosol (Plate 5A).
After plants were subjected to SD photoperiod for
20 d, some marked changes occurred: the antimonate
precipitates in intercellular spaces were decreased markedly, some calcium deposits were found to be localized
in cell walls and on the external side of plasma membrane,
and a few antimonate precipitates appeared in cytosol
and nucleus (arrows, Plate 5B).
Poplar apical bud cells dunng dormancy
1201
N
I
s>
w
w
IS
O
Plate 4. Subcellular localization of Ca 2 + antimonate deposits in the apical bud cells of poplar grown under 16 h LD photopenod. (A, B) The
electron-dense calcium antimonate deposits (indicated by arrows) were mainly localized in vacuoles (V), intercellular spaces (IS) and plastids (P).
Some were also observed at the entrances of the plasmodesmata (arrows) and in the cell wall (W). Few Ca 2 + deposits were found in cytosol and
nucleus. (A) x8500; (B) x9000. (C) Samples were fixed without potassium antimonate, x 5000. (D) Tissue sections were treated with EGTA. As
indicated by the arrows, there were transparent holes in plastids, intercellular spaces and vacuoles, where the electron-dense grains were located
before EGTA treatment, x 5000. Bar- 1 /xm.
After 28 d of exposure to SD, few Ca 2+ deposits were
observed in intercellular spaces. On the contrary, many
antimonate precipitates distributed between the cell walls
and the plasmalemma (arrows, Plate 5C), and many
small-sized calcium deposits showed up in cytosol and
nucleus. In addition, there were still some calcium antimonate precipitates observed in vacuoles and plastids
(Plate 5C, D).
After plants were exposed to SD for 35 and 49 d, a
large number of calcium deposits were localized between
cell wall and plasmalemma. The number of Ca 2+ deposits
in cytosol and nucleus significantly increased. There were
also many antimonate precipitates in plastids, mitochondria and small vacuoles (arrows, Plate 6A-D). In addition,
Ca2+ deposits were detected in some intercellular spaces
of the cells in plants which had been exposed to SD
photoperiod for 49 d (Plate 6D).
After 77 d of SD exposure, the distribution of Ca 2+ in
apical buds changed again. Calcium deposits in cytosol
and nucleus appeared to be decreased (Plate 7A, B). In
some cells, although many Ca2+ deposits were still visible
in cytosol, their distribution seemed to be concentrated
in some regions but not uniformly distributed (arrows,
Plate 7A, B). The calcium deposits distributed between
the cell wall and the plasma membrane were also reduced.
It is worth noting that a large number of Ca 2+ deposits
occurred again in intercellular spaces and in the cell walls
(Plate 7A-D). The deposits were not observed in the
1202
Jian et al.
w
V *
P
• '
PM
PM
»
N
W
' W %PM
*#
IS
Plate 5. Subcellular localization of Ca1+ antimonate deposits in apical bud cells of poplar under an 8 h SD photoperiod. (A) After 10 d of SD
exposure, the pattern of Ca2+ deposit distribution was similar to that of LD (see Plate 4A, B), x 11 000. (B) After 20 d of SD exposure, changes
in Ca2+ deposit localization occurred: deposits were markedly reduced in intercellular spaces; some calcium deposits were located on the outer face
of plasma membrane (arrows); and some deposits were observed in cytosol and nuclei (arrows), x 13 000 (C, D) After 28 d of SD exposure,
essentially no deposits were found in intercellular spaces, but a large number of antimonate precipitates distributed between the cell wall and the
plasmalemma (arrowheads); many small calcium deposits were found in cytosol and nucleus; and there were still calcium antimonate precipitates
in vacuoles and plastids. (C) x 11 000; (D) x 8500. Ban 1 ^m.
intercellular spaces of the cells of the samples collected
from plants that had been exposed to SD for 77 d, but
were fixed without potassium antimonate (Plate 7E).
Again, this demonstrated that the observed calcium antimonate precipitates are a true and reliable indicator
of calcium localization, and not an artefact. In these
cells, some lipid-protein bodies were also observed
(Plate 7A-E).
Discussion
Changes in cellular calcium distribution during the induction
of dormancy
There are several histo- and cytochemical methods that
have been used to determine calcium localization in plant
cells. Two of the most commonly used methods are
X-ray microanalysis and the chlorotetracycline technique
Poplar apical bud cells during dormancy
:PW
1203
w
N
: •> \
M
M
P.
•^ A
-PM
w
w
N
M
v
ill
D
Plate 6. Subcellular localization of Ca 2 + antimonate deposits in the apical bud cells of poplar plants grown in SD condition for either 35 d (A, B)
or 49 d (C, D). A large number of electron-dense calcium precipitates were widely distributed among cell wall, plasmalemma, plastids, cytosol and
nucleus. Ca 2 + deposits were also found in some intercellular spaces of the cells exposed to SD photopenod for 49 d (D). (A) x 4800; (B) x 10000;
(C) x8000; (D) x7000. Bar: 1 ^m.
(Tretyn and Kopcewicz, 1988). Recently,fluorescentdyes
(e.g. Quin 2, Fura-2, Fluo-3, Indo-1) have also been
widely used to measure the changes in cytoplasmic Ca 2+
(Williams et al., 1990; Gilroy et ai, 1991; McAinsh et ai,
1992; Subbaiah et al., 1994). However, all of these
methods have some limitations. For example, X-ray
microanalysis can only show the average calcium concentration in a particular area, whereas the chlorotetracycline
method can only reveal so-called membrane-associated
Ca2+ (Tretyn and Kopcewicz, 1988). When using fluorescent probes (dyes) to quantify the level of cytoplasmic
free Ca 2+ , it is not possible to determine the ultrastructural localization of the calcium ion, even by means of
laser-scanning confocal microscopy. In the present study,
the antimonate precipitation cytochemical-technique was
used. This procedure has been widely used for localization
of Ca2+ in animal cells (Weakley, 1979; Reith and Boyde,
1985). Wick and Hepler (1982), as well as Slocum and
Roux (1982) have demonstrated the usefulness of this
technique in localizing Ca2+ in plant cells. Since then,
many researchers have successfully applied this technique
to determine the cellular localization of Ca 2+ in plant
cells (Dauwalder et al., 1985; Lazzaro and Thomson,
1992; Tretyn et al., 1992; Wang and Jian, 1994; Hilaire
et al., 1995). As mentioned above, the experimental
results allowed the visualization of the dynamic changes
1204
Jian et al.
Plate 7. Localization of calaum antimonate deposits in apical bud cells of poplar plants after 77 d of SD exposure. (A) Antimonate precipitates in
cytosol and nucleus seemed to decrease in some cells, although many Ca J + deposits were still observed in the cytosol. However, in these cells,
cytosolic deposits tended to be regionally aggregated as shown (arrows), x 7000. (B) A large number of Ca 2 + deposits were observed on the
outside surface of the cell wall (in intercellular spaces), x 7000, and in the cell walls (C x 12 000 and D x 15 000). (E) Cells fixed without potassium
antimonate, x 6500. No electron-dense precipitates were seen. Some lipid-protein bodies were also observed in the cells (arrows, A-E)
LP: Lipid-protein body. Bar: 1 ^.m.
of Ca 2+ localization in the cells of poplar apical buds
during the development of dormancy induced by SD
photoperiod.
Changes in subcellular Ca i + distribution in bud cells
seemed to be closely associated with the development of
dormancy in buds under the growth chamber conditions
of warm temperature and SD photoperiod. The dormancy
status, expressed as days to bud break (Fig. 1), indicates
that there was a low level of dormancy (< 10 d to bud
break) in buds from plants which had been exposed to
SD for up to 20 d. After 28 d of SD exposure, buds
started to develop a higher level of dormancy (> 10 d to
bud break). From 28-49 d of SD exposure, the degree of
dormancy further increased. By 77 d of SD exposure, a
high degree of dormancy was detected (>90d to bud
break). With the development of dormancy, there was
also an increase in freezing tolerance in both buds and
stem bark under SD photoperiod. The development of
Poplar apical bud cells during dormancy
1205
freezing tolerance started after 28 d of exposure to SD
(Fig. 2). By the end of 77 days of SD treatment, freezing
tolerance in buds and stem bark was increased to — 18 °C
from — 6°C, a level of freezing tolerance observed in
LD poplar.
Changes in calcium distribution in bud cells had already
occurred prior to the initiation of dormancy, i.e. up to
20 d of SD exposure calcium deposits were decreased in
intercellular spaces and increased in cytosol and nucleus,
respectively. From 28-49 d of SD exposure, when the
dormancy and freezing tolerance were developing, more
calcium deposits accumulated in the cytosol and nuclei.
When a high degree of dormancy was reached after 77 d
of SD exposure, the levels of Ca 2 + in cytosol and nuclei
were decreased, while accumulation of calcium in intercellular spaces and in cell walls was again observed. These
results suggest that there is a dynamic change in Ca 2 +
distribution in apical bud cells during the development
of dormancy. It is not known whether the antimonate
precipitates derived from free Ca 2 + , bound Ca 2 + or both.
Neither is it known what roles calcium may have in
dormancy development, but the changes in subcellular
calcium distribution, as detected by the antimonate cytochemical technique, do suggest the involvement of calcium
in the initiation and development of bud dormancy
induced by SD.
the plasmodesmata between cells were blocked by
glycoproteins or disconnected due to the contraction of
protoplast (Jian and Sun, 1992). Such a change in plasmodesmata was considered to be an important mechanism
for plant survival in a cold winter by preventing a rapid
de-acclimation when the ambient temperature rose rapidly
(Jian and Sun, 1992). In the present study, it was observed
that the changes in plasmodesmata were closely related
to the development of dormancy under SD photoperiod.
During SD exposure and the development of dormancy,
the frequency of plasmodesmata in the cell walls between
neighbouring cells in apical buds decreased and the diameter of pores reduced. This was especially obvious in
plants which had developed a high level of dormancy
after 77 d of SD exposure. The number of plasmodesmata
observed in the cell walls was reduced and those visible
appeared to be disconnected, so that the symplastic
connection between cells seemed to be discontinued. It is
possible that these dramatic changes in plasmodesmata
result in reduction or blockage of the intercellular transport of materials and of intercellular communication.
Whether this has any connection to the lowered metabolic
activity in the cells of dormant buds remains to be proven.
Further studies are needed to explore the role of symplastic connections in the initiation of growth cessation and
development of dormancy in poplar.
Role of plasmodesmata in dormancy development
Subcellular calcium localization may be involved in the
regulation of the dynamic changes in plasmodesmata and
cell walls
In plant cells, functional plasmodesmata form a continuous network of connected protoplast, termed symplast.
The transport of organic materials and the transduction
of signals between cells in the symplast depend upon the
frequency and conductance of plasmodesmata. Therefore,
plasmodesmata play an important role in plant growth
and development, as well as in response and adaptation
to environmental factors (Robards and Lucas, 1990;
Lucas et al., 1993). The plasmodesmal channel is a
dynamic structure, and its frequency can be variable
(Botha, 1992; Lucas et al., 1993). When viral particles
move through plasmodesmata or when plants are under
anaerobic or osmotic stresses, the diameter of plasmodesmal pores become widened (Wolf and Lucas, 1994;
Cleland et al., 1994; Schulz, 1995). Wille and Lucas
(1984) reported that plasmodesmata existed in the cell
walls between guard mother cell and neighbouring epidermal cells, as well as in the cell walls between developing
young guard cells and neighbouring epidermal cells.
During the development of the mature guard cell walls,
however, the plasmodesmata were sealed. Thus, within
the mature stomatal complex, there were no plasmodesmata connecting guard cells to sister guard cells or to
adjacent epidermal cells. A similar case was also observed
during the development of the embryo sac in angiosperms
(Hawker et al., 1991). In overwintering wheat seedlings,
The roles of Ca 2 + in the regulation of growth and
development of plants have been studied extensively
(Hepler and Wayne, 1985; Bush, 1995). Erwee and
Goodwin (1983) proposed that cytoplasmic Ca 2 + concentration may directly regulate the permeability of plasmodesmata in the plant symplast. Micro-injection of
buffered-carboxyfluorescein containing IP 3 or with Ca 2 + BAPTA into cells, revealed that elevated cytoplasmic
Ca 2 + content inhibits cell-to-cell diffusion of carboxyfluorescent dye via plasmodesmata in staminal hairs of
Setcreasea purpurea, suggesting that calcium may regulate
the opening and closing of plasmodesmal pores by modifying the conformation of the structural proteins surrounding the plasmodesmal pores (Tucker, 1988, 1990).
Shepherd and Goodwin (1992) studied cell-to-cell communication in vegetative lateral branches of Chara
corallina during winter and spring. Their experimental
results suggested that in winter Chara cells undergo a
period of vegetative dormancy characterized by relatively
low plasmalemma potential differences, restricted cell-tocell communication, inactive branch dactyls, and
restricted growth. Spring branch internodes were characterized by high plasmalemma potential differences, extensive intercellular communication and active growing
1206 Jian etal.
branch dactyl. Artificially increasing Ca2+ ion concentration in spring cells by application of ionophore A23187
or direct micro-injection of Ca 2+ significantly restricted
intercellular communication to a level similar to that
found in winter cells. Eklund and Eliasson (1990) and
Eklund (1991) reported that Ca 2+ ion concentration
influenced the synthesis of cell wall constituents. At a low
concentration of cytosolic Ca2 + , cell wall deposition was
reduced, mainly as a result of the reduction in lignin and
non-cellulosic polysaccharide deposition. High concentrations of Ca 2+ stimulated non-cellulosic polysaccharide
and lignin deposition. The results revealed that following
the increase of intracellular Ca 2+ concentration in apical
bud cells under the influence of SD photoperiod, cell
walls were thickened and stained densely. At the same
time, plasmodesmata were constricted, sealed, blocked,
and/or even disappeared. The elevation in cytosolic Ca 2+
may, therefore, be triggering the synthesis of noncellulosic polysaccharide (such as callose) and lignin
deposition in cell walls, leading to thickened cell walls
and changes in plasmodesmata, which in turn may lead
to events associated with growth cessation and dormancy
development in the buds of poplar.
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