Environmental and Experimental Botany 48 (2002) 161168

Effect of heat stress on calcium ultrastructural distribution

in pepper anther
Chun-Lan Yan, Jian-Bo Wang *, Rong-Qian Li
Key Laboratory of MOE for Plant De6elopmental Biology, College of Life Sciences, Wuhan Uni6ersity,
Wuhan 430072, Peoples Republic of China
Received 10 April 2001; received in revised form 12 March 2002; accepted 13 March 2002
Abstract
Potassium antimonate was used to locate loosely bound calcium in the pepper (Capsicum annuum) anther under
normal and heat stress environments. In the microspore mother cell, a few calcium precipitates were deposited on the
surface of the cell, a few in the cytoplasm and almost no precipitates were formed in the nucleus. After 12 h at 40 C,
antimonate calcium deposits increased in the cytoplasm and the nucleus and many emerged on the inner surface of
the vacuole membrane. After 24 h heat stress, some cells were partly deformed, numerous calcium precipitates
appeared in the cytoplasm and deposited on the surface of the vacuole membrane and in the vacuoles. Compared to
the pollen mother cell, there was a signicant increase in calcium deposit quantities on the surface and in the
cytoplasm of the tetraspore. By heat stressing for 24 h, precipitates obviously increased in the cytoplasm and nucleus
of the tetraspore in contrast to the control. In the microspore, many calcium precipitates were formed on the baculae,
the inner surface of plasma membrane, vacuole membrane, but only a few in the cytoplasm and nucleus. After 12 h
heat exposure, precipitates on plasma membrane became abundant, a few in the cytoplasm and the peripheral
nucleus, while no precipitates were seen on the vacuole membrane. As for the anthers exposed to 24-h heat stress,
precipitates increased on the inner surface of the plasma membrane, cytoplasm and nucleus. In mature pollen, there
was a layer of calcium-induced precipitates on the pollen wall, but few in the cytoplasm and plasma membrane. No
obvious calcium changes occurred on mature pollen after 12 or 24 h heat exposure. The relationship between heat
stress and calcium distribution was discussed. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Antimonate precipitation; Cytoplasm; Microspore; Pollen development; Vacuole
www.elsevier.com/locate/envexpbot
1. Introduction
Heat stress is a common environmental stress in
the development of plants and it may be the
major constraints to vegetable growth. Heat toler-
ance is becoming a desirable trait for vegetables in
heat-stressed environments, so more and more
studies are focusing on the mechanism of heat
stress and thermotolerance.
Physiological responses of plants to heat stress,
such as the damage of structure and the disorder
of physiological metabolism, have been well docu-
mented (Vierling, 1991; Blum, 1996; Wang et al.,
* Corresponding author.
E-mail address: jbwang@whu.edu.cn (J.-B. Wang).
S0098-8472/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0098- 8472( 02) 00021- 7
C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 162
1997). Although the damage and death of cells are
caused by very heavy heat stress, many plants can
survive in otherwise lethal high-temperatures
treatments if they are rst subjected to a pretreat-
ment at non-lethal high temperatures. Extensive
experiments have shown that pretreatments which
lead to acquisition of thermotolerance are condi-
tions under which heat-shock proteins (HSPs) are
synthesized (Vierling, 1991; Waters et al., 1996;
Schof et al., 1998). However, the pathways by
which heat shock signals are perceived and trans-
ducted to activate the gene expression of HSPs in
order to induce thermotolerance are unclear
(Schof et al., 1998).
As a secondary messenger in plant signaling,
calcium plays a vital role in plant growth and
development, inuences plant response and adap-
tation to various environments. Intracellular cal-
cium changed frequently with response to various
environmental stress signals, such as salinity
(Lynch et al., 1989), oxidative stress (Price et al.,
1994) and anoxia stress (Subbaiah et al., 1994).
Klein and Ferguson (1987) found that the uptake
of calcium in pear cells was signicantly enhanced
under heat stress and Biyaseheva et al. (1993)
showed that heat shock resulted in increased in-
tracellular calcium in pea mesophyll protoplasts.
Several authors have suggested that pretreatment,
such as Ca
2+
, Ca
2+
chelator EGTA, plasma
membrane Ca
2+
channel blockers La
3+
or vera-
pamil, could change the intrinsic heat tolerance of
various plants (Gong et al., 1997; Zhang et al.,
2000).
In the studies of heat stress, the responses of
plant leaves to heat shock/stress have been widely
investigated (Gong et al., 1997; Zhang et al.,
2000). In contrast, little is known about how heat
stresses affect the development of anthers. Some
cytological data suggested that heat stress would
lead to a decrease of pollens viability (Han et al.,
1996); some studies indicated that calcium was
involved in pollen germination and pollen tube
elongation (Tirlapur and Cresti, 1992; Tirlapur
and Willemse, 1992; Gong and Cao, 1995), but
the interaction of heat stress and anthers develop-
ment is not clear.
Presently, several approaches have been applied
to study the calcium localization and alteration in
plant cells. The cytochemical method of anti-
monate precipitation is widely used for subcellular
calcium localization (Jian et al., 1999; Meng et al.,
2000). The present study was designed to locate
the loosely bound calcium in pepper anthers by
using antimonate precipitates. In this study, it was
hypothesized that: (i) there would be interaction
not only between calcium and the normal anther
development, but also between calcium and the
effect of heat stress in the anther development. As
secondary messenger, calcium in the anther cells
may increase under heat stress, while a prolonged
exposure to increasing Ca
2+
may be toxic to the
cells and be correlated with the decrease of pol-
lens viability; (ii) the appropriate restoration of
Ca
2+
homeostasis would be necessary to prevent
heat injury. To test the hypothesis, pepper anthers
were treated under different levels of heat stress
environments. The study was focused on the ef-
fects of heat stress on calcium distribution in
anther cells.
2. Materials and methods
Pepper (Capsicum annuum L., cv. XiangYan
No. 1) was grown in a green house with the
temperature ranged from 25 to 28 C. When the
plants were in the blossom period, they were
transferred to a 40 C growth chamber without
light. The anthers of different lengths were col-
lected after being stressed for 0 h (control, before
exposure), 12 and 24 h, respectively.
Subcellular calcium localization was analyzed
according to the method of Slocum and Roux
(1982) with minor modication. Anthers were im-
mediately immersed in a xative containing 2.5%
gluteraldehyde and 2% potassium antimonate
(K
2
H
2
Sb
2
O
7
4H
2
O) in 0.2 mol/l potassium phos-
phate buffer (pH 7.8) for 4 h at 4 C. Then the
anthers were washed four times, 30 min each, with
0.2 mol/l potassium phosphate buffer (pH 7.8)
containing 2% potassium antimonate. Then the
samples were xed in potassium phosphate buffer
(pH 7.8) containing 1% osmium tetroxide (OsO
4
)
and 2% potassium antimonate at 4 C overnight.
After the second xation, the samples were bathed
four times in 0.1 mol/l phosphate buffer, 30 min
C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 163
each. Thereafter, the samples were dehydrated in
a graded acetone series and embedded in Spurr
resin. The embedded samples were sectioned with
a glass knife using a LKB8800 ultramicrotome.
The sections were stained with uranyl acetate or
not, then observed and photographed with JEM-
100/CXII transmission electron microscopy
(TEM) operated at 60 kV.
In order to conrm that the deposits contain
Ca
2+
, chelation of calcium ions with EGTA (eth-
ylene glycol-bisN,N,N%N%-tetraacetic acid) was
performed. The grids mounted with tissue sections
that had been examined by TEM, were immersed
in 100 mmol/l EGTA (pH 8.0) and incubated at
37 C for 1.5 h. After incubation, the grids were
rinsed briey with distilled water, stained with
uranyl acetate again and examined under TEM.
3. Results
Calcium distribution was determined from the
microspore mother cell stage to mature pollen
stage in both normal and heat stressed anthers.
We focused on four stages of development: (i)
microspore mother cell; (ii) tetrad; (iii) mi-
crospore; and (iv) mature pollen.
3.1. Calcium distribution differences between
normal and treated microspore mother cell
(MMC)
MMCs in normal anthers were distinct from
the tapetal tissue and the anther wall. They were
angular in outline and each cell had the features
of a relatively undifferentiated cell bounded by a
simple wall. In the MMC, a number of small
vacuoles were seen in the cytoplasm. A few cal-
cium-induced precipitates occurred on the surface
of the cell, few in the cytoplasm and nearly no
precipitates in the nucleus (Fig. 1A).
After 12 h heat stress at 40 C, calcium de-
posits (4059 ppt mm
2
), which can be observed
on the surface of MMC, were more abundant
than those in the control (2039 ppt mm
2
). The
number of deposits increased continuously in
both cytoplasm and the nuclei (Fig. 1B). At the
same time, many vacuoles formed in the MMC,
many calcium precipitates deposited on the inner
surface of vacuole membrane and numerous cal-
cium-induced precipitates occurred on the surface
of MMC opposite to the tapetum.
As for the anthers treated with 24 h heat stress,
numerous calcium precipitates emerged in the cy-
toplasm and also occurred on the surface of vac-
uole membrane and in the vacuoles (Fig. 1C).
3.2. Calcium distribution differences between
normal and stressed tetrad
When anthers developed to the tetrad stage,
tetrads of microspores, in a tetrahedral arrange-
ment, were encased in callose each that was a light
Fig. 1. Electron micrographs of cells in MMC stage exposed to
normal environment (A: bar=2 mm), 12 h heat stress (B:
bar=3 mm), 24 h heat stress (C: bar=3 mm) and tetrad stage
in normal environment (D: bar=3 mm), 24 h heat stress (E:
bar=4 mm). (F) The calcium-free control section incubated by
EGTA (bar=2 mm). The difference in calcium precipitates
distribution after different durations of heat treatment was
evident. Arrows indicate calcium precipitates. MMC, mi-
crospore mother cell; N, nucleus; P, pollen; Sp, microspore; V,
vacuole.
C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 164
layer of electron-lucent and nearly no precipitates
formed in it. Moreover, the number of calcium
precipitates on the surface of the tetraspore was
increasing and the volume became bigger. Cal-
cium precipitates also increased in the cytoplasm
and distributed regularly. Some calcium deposits
formed on the surface of vacuole (Fig. 1D).
After exposure to 40 C for 24 h compared
with the control, precipitates signicantly in-
creased and accumulated in the cytoplasm and
nucleus of the tetraspores (Fig. 1E).
3.3. Calcium distribution differences between
normal and stressed microspore
Released from the callose tetrad, the mi-
crospores had irregular shape and dense cyto-
plasm. Exine deposition on the wall of the
microspores, which just released from the tetrad,
was well developed. At the early stage of mi-
crospore development, baculae were irregularly
spaced between a spongy tectum and a dense foot
layer that was complete around the microspore.
Many big volume precipitates were on the baculae
discontinuously. There were many calcium precip-
itates in the plasma membrane where the future
colporal regions were formed (data not shown).
The later uninucleate microspores contained a
large central vacuole and a peripheral nucleus.
Abundant calcium precipitates were deposited not
only on the surface of the baculae, but also on the
inner surface of the plasma membrane and vac-
uole membrane, with only a few precipitates in
the cytoplasm and nucleus (Fig. 2A).
In those microspores that endured 12 h heat
stress, a few calcium deposits were found on the
baculae in the later uninucleate microspores, but
precipitates on the plasma membrane became
abundant. In addition, a few precipitates occurred
in the cytoplasm and the peripheral nucleus.
Compared with the control, no calcium deposits
granules were seen on the vacuole membrane (Fig.
2B).
In microspores treated for 24 h, besides a layer
of abundant calcium precipitates was present on
the baculae, precipitates also occurred on the
inner surface of the plasma membrane and the
precipitates in the cytoplasm and nucleus were
Fig. 2. Electron micrographs of cells in microspore stage
exposed to normal environment (A); 12 h heat stress (B); 24 h
heat stress (C); mature pollen stage in normal environment
(D); 12 h heat stress (E); and 24 h heat stress (F). Arrows
indicate calcium precipitates. L, lipid body; N, nucleus; P,
pollen; PM, cytoplasm membrane; S, starch grain; V, vacuole;
(bar=3 mm).
abundant. In the nucleus, the volume of precipi-
tates was bigger than in the cytoplasm (Fig. 2C).
3.4. Calcium distribution differences between
normal and treated mature pollen
In mature pollen, cytoplasm became dense
again and storage materials, such as starch and
lipids, accumulated inside the grains. The pollen
wall was composed of a lightly sculptured tectum
and a fully developed intine. On the pollen wall,
there was a layer of calcium-induced precipitates
on baculae, but few in the cytoplasm and plasma
membrane (Fig. 2D).
The quantities and the distribution of calcium-
induced precipitates in mature pollen after 12 and
24 h heat stress were similar to that in the control
(Fig. 2E, F).
C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 165
Calcium accumulation trends are summarized
in Table 1.
4. Discussion
As an electron microscopic cytochemical tech-
nique for in situ localization of exchangeable cel-
lular Ca
2+
-calcium that may be readily converted
to free calcium upon environmental modication,
potassium antimonate precipitation has been
widely used in the relevant studies. Although the
mechanism of precipitate formation was not clear
(Wick and Hepler, 1982), recent studies indicate
that the precipitates are calcium antimonate: (i)
the specic chelator EGTA was capable of chelat-
ing the Ca
2+
precipitates (Meng et al., 2000); and
(ii) energy-dispersive X-ray microanalysis (EDX)
indicated that a ratio of Sb to Ca of nearly 2:1 is
expected (Slocum and Roux, 1982; Jian et al.,
1999). Free cytoplasmic calcium is usually below
the limit of detection (10
6
mol/l Ca
2+
), whereas
less soluble compound (e.g. calcium phosphates
or carbonates) did not appear to release calcium
for binding with antimonate. The localization of
calcium antimonate seems useful in identifying
loosely bound calcium.
4.1. Calcium distribution during normal anther
de6elopment
Calcium is known to inuence cell functions
through spatial and temporal changes induced by
stimuli (Bush, 1995). Calcium levels in developing
tissues of anther appear to be dynamic, pre-
sumably allowing transfers of Ca
2+
between free
cytoplasmic pools, loosely bound pools and
tightly bound pools. Antimonate preferentially la-
bels the loosely bound pools.
In microspore mother cell, calcium-induced pre-
cipitates were mainly found on plasma membrane.
However in tetrad, calcium precipitates emerged
equably in the cytoplasm. In microspore, calcium
precipitates accumulated on the inner surface of
plasma membrane and the sites opposite to the
future colporal region and some calcium deposits
also occurred in the nucleus, while few calcium
precipitates accumulated in the cytoplasm of ma-
ture pollen. The differentiation of calcium concen-
tration and distribution in anther development
had also been reported in Casteria 6errucosa and
wheat (Tirlapur and Willemse, 1992; Meng et al.,
2000).
We also observed calcium-induced precipitates
gradually accumulated on the microspore surface
of tetrad, evidently increased on the surface of the
microspore and deposited a frequent calcium
layer on the mature pollen exine. The same cal-
cium deposits changes in the pollen wall forma-
tion also appear in the rice and wheat (Tian et al.,
1998; Meng et al., 2000). All these results indi-
cated that strategically located concentrations of
calcium were related to normal anther
development.
4.2. The relationship between heat stress and
Ca
2+
distribution
Calcium has been found to be involved in the
regulation of responses of plants to environmental
stresses (Bush, 1995; Braam et al., 1996; Webb et
al., 1996). Intracellular calcium levels in plant cells
often signicantly increase under various stresses,
such as salinity (Lynch et al., 1989), touch, wind
stimulation and cold shock (Jian et al., 1999),
anoxia (Subbaiah et al., 1994) and oxidative stress
(Price et al., 1994) and there is increasing evidence
that the same affairs may happen in heat shock/
stress (Biyaseheva et al., 1993; Gong et al., 1997;
Torrecilla et al., 2000). In this study, we also
found those calcium precipitates increased by heat
stress.
Several studies have revealed that there are
genotypic differences in the sensitivity of maize
pollen germination to high temperature (Herrero
and Johnson, 1980; Lyakh et al., 1991). Frova et
al. (1989) demonstrated that mature maize pollen
was unable to mount a heat-shock response when
it was exposed to supraoptimal temperatures. The
same phenomenon had not been demonstrated
from cytochemical studies. In our study, we ob-
served that calcium precipitates changed greatly in
stressed microspore mother cell and tetraspore,
compared with the control, respectively, while no
obvious calcium distribution changes occurred in
mature pollen. Given the potential role of HSPs
C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 166
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C.-L. Yan et al. / En6ironmental and Experimental Botany 48 (2002) 161168 167
in protecting against heat stress (Vierling, 1991),
one hypothesis to explain pollen thermosensitivity
may be that the transition from uninucleate to
binucleate pollen marks the onset of the progres-
sive decrease in the pollens ability to synthesize
HSPs, eventually leading to the complete inhibi-
tion of HSPs synthesis in mature pollen
(Gagliardi et al., 1995; Magnard et al., 1996).
Whether this is in part a result of the pollens
viability decrease under heat stress is speculative.
That the increasing calcium levels restore to the
low resting levels (i.e. the restoration of Ca
2+
homeostasis) is essential not only to permit the
next signal to occur, but also to protect the cell
from the toxic effects of high Ca
2+
concentra-
tions. It is generally known that increased levels
of calcium are necessary for proper plant cell
functions, but a prolonged high cytoplasmic Ca
2+
concentration is toxic to the cells (Minorsky,
1985; Biyaseheva et al., 1993; David, 1995; Jian et
al., 1999). In our study, cytotoxicity occurred
after 24 h heat stress, as evidenced by the damage
of the cell when high levels of Ca
2+
still remained
in the cytoplasm and the nucleus. After 12 h heat
stress, we did not see the ne structural damage in
cells, although the elevation of calcium deposits
formed in cytoplasm and nucleus. Our ndings
suggest that the transient Ca
2+
elevation plays an
important role for percepting heat stress; how-
ever, the appropriate restoration of Ca
2+
homeostasis is necessary to prevent heat injury.
The restoration of Ca
2+
homeostasis in pollen
probably depends on the activity of plasma mem-
brane (PM) and endoplasmic reticulum (ER)
Ca
2+
-ATPase (Evans et al., 1991; Thomson et al.,
1993; Askerlund and Sommarin, 1996). Further-
more, those plants which Ca
2+
homeostasis re-
store at an appropriate pace during a prolonged
stress can increase their tolerance to the same
stress (Gong et al., 1997; Jian et al., 1999; Tor-
recilla et al., 2000). In further studies, we antici-
pate the focus on the activity of PM
Ca
2+
-ATPase during heat stress, which is impor-
tant for cells to restore and maintain Ca
2+
homeostasis through which cytotoxicity caused by
heat-induced levels of intracellular Ca
2+
could be
prevented.
Acknowledgements
This research was supported by the Natural
Science Foundation of Hubei Province of the
Peoples Republic of China.
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