全 文 ：Received 24 Mar. 2003 Accepted 20 Jun. 2003
Supported by the National Natural Science Foundation of China (39970623).
* Present address: China Flower Association, No. 18 East Street, Hepingli, Beijing, 100714, China.
** Author for correspondence. Tel: +86 (0)10 82376017 ext. 604; E-mail: .
http://www.chineseplantscience.com
Calcium Distribution During Pollen Development of
Larix principis-rupprechtii
KONG Hai-Yan* , JIA Gui-Xia**
(Beijing Forestry University, Beijing 100083, China)
Abstract: Potassium pyroantimonate precipitation method was used for ultracytochemical localization
of calcium in microsporangia of Prince Rupprecht Larch (Larix principis-rupprechtii Mayr) at different
developmental stages. During meiosis, calcium precipitates were abundant on the cell wall and intercellular
space among cells of the epidermis and middle layers of the microsporangium wall. Calcium precipitates
were only found on the cell wall of the outer tangential membrane of the tapetal cells, but few was found
in microspore mother cells. At tetrad stage, a large amount of calcium precipitates were observed on the
callose wall of each tetraspore as well as the wall surrounding the whole tetrad. The quantity of calcium
precipitates decreased at free microspore stage and then gradually increased till pollen maturation. Cal-
cium around the Ubisch bodies increased from the tetrad stage till pollen maturation. This study also
discusses the relation between the distribution of calcium precipitates on the tetrad walls and the
formation of pollen wall, as well as the possible function of epidermis, middle cells, tapetum and Ubisch
bodies in transportation of Ca2+ to the pollen.
Key words: Larix principis-rupprechtii ; microsporogenesis; pollen development; potassium pyroanti-
monate precipitation; calcium ion (Ca2+)
It is well known that calcium acts as an important ubiq-
uitous messenger in plants, which can activate and modu-
late many intracellular metabolic processes of plant growth
and development. Ca2+ has been shown to play a number
of roles in sexual reproduction of flowering plants. The
crucial roles of calcium in regulating pollen germination
and tube growth (Franklin-Tong, 1999) as well as fertiliza-
tion (Digonnet et al., 1997; Yu et al., 1998; Antonie et al.,
1999; Yu et al., 1999) have attracted much attention from
plant developmental biologists. However, studies about
calcium distribution in male reproductive system were
mainly focused on pollen germination and tube growth
(Tirlapur and Cresti, 1992; Tirlapur and Willemsee, 1992),
while far less was done on microsporegenesis and pollen
development. In recent years, some researches have been
done on calcium distribution on anthers of photoperiod-sen-
sitive cytoplasmic male-sterile rice (Tian et al., 1998) and
wheat (Meng et al., 2000). But for gymnosperms, only a few
studies were conducted on the distribution of calcium in
pollen development and fertilization, one of which was done
by Gong et al. (1995) on the need of calcium during the
process of pollen germination in Pinus yunnanensis.
In this study, the potassium pyroantimonate precipita-
tion method was used for ultracytochemical localization of
calcium in microsporangia of Larix principis-rupprechtii
Mayr at different developmental stages: from pollen mother
cell stage, through meiosis and tetrad stage, till pollen
maturation. The purpose of our study is to explore the role
of calcium during pollen development in gymnosperms
which have not yet been well studied.
1 Materials and Methods
1.1 Plant materials
The microsporangia of Larix principis-rupprechtii
Mayr at different developmental stages were collected from
the Botanical Garden of Institute of Botany, The Chinese
Academy of Sciences.
1.2 Methods
The cytochemical localization procedures for calcium
follow Wang et al.’s method (Wang et al., 1994) with some
modification. The male buds were cut into two or four parts
and submerged in a fixative of 3% glutaraldehyde/2% po-
tassium pyroantimonate buffered in 100 mmol/L potas-
sium phosphate (pH 7.6) at 4 ℃ for 6 h. Then the tissues
were washed for 2 h in 2% potassium pyroantimonate buff-
ered in 100 mmol/L potassium phosphate (pH 7.6), postfixed
in 1% osmium tetraoxide / 2% pyroantimonate (pH 7.6) at 4
℃ overnight. The postfixed tissues were washed with nor-
mal redistilled water (pH 7.0) four times and pH 10.0 redis-
tilled water twice, 0.5 h each time, followed by dehydration
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 69-76
Acta Botanica Sinica 植物学报 Vol.46 No.1 200470
in a graded ethanol series and embedded in Spurr resin.
Ultrasections were obtained with an LKB2088 ultramicro-
tome equipped with glass knife. After post-stained with
uranyl acetate, the sections were viewed and photographed
under a Hitachi H-600A transmission electron microscope.
For control experiments, grids with sections were floated
on a solution of 1 mmol/L EGTA (pH 8.0) at 60 ℃ for 0.5 h
to remove the calcium pyroantimonate precipitates from
the sections.
2 Results
2.1 Calcium distribution in microspores and pollen
According to previous research (Jia et al., 1994), the
microspore mother cells of Larix principis-rupprechtii are
formed in September. At that time, the cells are of diamond
shape and in close contact with each other. This state will
last to the second half of the next February. Before meiosis,
the microspore mother cells separate from each other and
their shapes become oval. A large nucleus occupies most
of the space of the cell, and abundant plastids, mitochon-
dria and dictyosomes are seen in the cytoplasm. Most of
the plastids contain a big starch grain and concentrate
around the nucleus. At this stage, calcium is seldom found
in different parts of the cell, except a small amount along
the cell wall. Sporopollenin is found in the microsporangium
locule (Fig.1).
During meiosis, the number of plastids, mitochondria
and dictyosomes in the microspore mother cells is still
increasing. Rough endoplasmic reticulum (RER) is observed
in the cytoplasm (Fig.2). Lipid droplets are found located
along the cell membrane (Fig.3). At this stage, the chromo-
somes are mainly located in the equatorial area. The amount
of calcium on the cell wall is more than that at the previous
stage (Fig.4). A large amount of calcium is observed within
the plastids (Fig.5), while fewer in the cytoplasm and the
nucleus.
At tetrad stage (Fig.6), there are still a large amount of
plastids and mitochondria, which are mainly located at the
proximal end, while the nucleus is located at the distal end.
At this stage, the pollen ectexine and endexine start to form.
Calcium precipitates are abundant in the callose wall and
the intercellular space among the microspore cells (Figs.7,
8). The amount of calcium in the cytoplasm is more than
before. A few abnormal cells are observed, which are se-
verely contracted, with more calcium precipitates along the
membrane and vacuoles than that in the normal cells (Fig.
9).
With degradation of the callose wall, the microspores
are released from the tetrad. Organelles are abundant in the
free microspores and the lipid droplets are more than before.
A lot of sporopollenin are seen on the ectexine, and the
endexine consists of four to five lamellae with a few of
calcium precipitates on it (Fig.10). Almost no calcium is
observed in the cytoplasm and the nucleus.
The mature pollen consists of five cells (Fig.11), includ-
ing two degraded prothallial cells, one body cell, one stalk
cell and one tube cell (Jia et al., 1994). Abundant lipid drop-
lets and starch grains are observed in the cytoplasm. Be-
sides ectexine and endexine, calcium precipitates are also
localized in the connection part between exine and intine.
But those on the ectexine are larger in volume and less in
density than that on the endexine (Fig.12).
2.2 Calcium distribution on the microsporangium wall
The microsporangium wall of Larix principis-
rupprechtii consists of an epidermis, one to three layers of
middle cells and a tapetum.
During meiosis, starch grains are rich in the cells of the
epidermis and middle cells of the microsporangium wall.
Calcium precipitates are abundant in the cell wall, intercel-
lular space and tonoplast (Fig.13). The plasmalemma of the
tapetal cells becomes wavy and some irregular invagina-
tions appear. A few of pro-Ubisch bodies are seen attach-
ing to the tapetal cells or in the microsporangium locule
(Fig.14). In the tapetal cells, organelles and rough endo-
plasmic reticulum are abundant (Fig.15), and more calcium
precipitates are localized on the cell membrane, tonoplasts
and mitochondria. Sporopollenin bodies are observed in
the microsporangium locule (Fig.16), which might play an
important role in the formation of pollen exine.
At tetrad stage, the tapetal cells contract severely and
Figs.1-7. 1. A microspore mother cell, showing abundant cell organelles in cytoplasm and a few calcium precipitates on the cell wall.
Some sporopollenin bodies enter the microsporangium locule, ×2 914. 2-5. Meiosis stage. 2. Rough endoplasmic reticulum (RER) and
some small calcium precipitates appear in the cytoplasm, ×17 484. 3. Lipid droplets along the cell membrane, ×29 140. 4. Calcium
distribution at the inner tangential side of the tapetal cell and the cell wall of microspore mother cell, ×17 484. 5. Calcium precipitates
in the plastids, ×21 855. 6. Calcium distribution at tetrad stage, ×3 643. 7. Calcium precipitates on the callose wall of the tetrad
microspore, ×21 855. Abbreviations: BC, body cell; C, callose wall; Ca, calcium precipitate; D, dictyosome; ECT, ectexine; END,
endexine; L, lipid droplet; M, mitochondrion; PC, prothallial cell; PU, pro-Ubisch body; RER, rough endoplasmic reticulum; S, starch
grain; SB, sporopollenin body; SC, stalk cell; T, tapetal cell; TC, tube cell; U, Ubisch body; V, vacuole.
→
KONG Hai-Yan et al.: Calcium Distribution During Pollen Development of Larix principis-rupprechtii 71
Acta Botanica Sinica 植物学报 Vol.46 No.1 200472
KONG Hai-Yan et al.: Calcium Distribution During Pollen Development of Larix principis-rupprechtii 73
Acta Botanica Sinica 植物学报 Vol.46 No.1 200474
begin to degenerate. A lot of mitochondria and vacuoles
are observed in the cytoplasm. Calcium precipitates are
abundant on the cell membrane, tonoplasts and
mitochondria. Interestingly, calcium precipitates are also
found around the Ubisch bodies and in the microspo-
rangium locule (Fig.17).
During free microspore stage, the amount of calcium
precipitates increases in the mitochondria of the tapetal
cells (Fig.18). Furthermore, a lot of Ubisch bodies change
to global, claval and other irregular shapes, quite different
from those in Pinus bungeana (Chen et al., 1987) and
Cunninghamia lanceolata (Jia, 1996). Calcium precipitates
are obviously seen around the Ubisch bodies (Fig.19).
At mature pollen stage, the epidermis and middle cells
of the microsporangia begin to vacuolate. The tapetal cells
have almost degenerated, with a lot of calcium precipitates
located on the cell membrane and some in the cytoplasm
(Fig.20). The Ubisch bodies are still of irregular shapes,
with large amount of sporopollenin and calcium around
them (Fig.21).
In grids of control sections treated with EGTA, the pre-
cipitates become transparent, which proves that they are
really calcium precipitates (Fig.22).
3 Discussion
The present paper gives evidence in Larix principis-
rupprechtii that the amount of calcium gradually increases
on the callose wall from microsporocyte stage to tetrad
stage. After that, it begins to decrease at the free microspore
stage, but increases again at mature pollen stage, espe-
cially on the endexine and the intine. Previous studies on
pollen development of gymnosperms showed that pollen
wall begins to form from the tetrad stage (Jia et al., 1998).
Gong et al. (1990) consider that cell wall is one of the larg-
est Ca2+ store in plant cells, and pollen wall holds the high-
est calcium level in pollen grains (Stanley and Linskens,
1974). Furthermore, Gong et al. (1995) report that there is a
significant Ca2+ release from pine and tobacco pollen dur-
ing the hydration and early germination phase. We sug-
gest that the accumulation of calcium in the callose wall of
the tetrad might be correlated with the formation of pollen
wall, and the accumulation of calcium in the mature pollen
wall might be a storage of calcium for pollen germination.
In our study, calcium distribution on different layers of
the microsporangia and the Ubisch bodies suggests a
gradual transportation of calcium from microsporangia wall
to pollen during pollen development. Similar results have
been reported by Tian et al. （1998） in photoperiod-
sensitive cytoplasmic male-sterile rice and by Meng et al.
（2000） in wheat. Our result suggests that in pollen de-
velopment of Larix principis-rupprechtii, calcium in the
pollen wall is mainly provided by the tapetum, and Ubisch
bodies might play an important role in the transportation of
calcium. Similar results have been reported by Tirlapur et
al. （1992）and Meng et al. (1999) in tobacco and wheat,
respectively.
As for the over-accumulation of calcium on cell mem-
brane and even in cytoplasm of abnormal tetrads, we sup-
pose that it might be the cause of the abnormal develop-
ment of tetrads. Previous studies have shown that in
tobacco, wheat and rice, abnormal distribution of calcium
in the anther wall and pollen might lead to the abortion of
pollen (Tian et al., 1998; Meng et al., 2000; Li et al., 2001).
An interesting point is that calcium is also found abun-
dant in the plastids of microspore mother cells during
meiosis. Some studies on tobacco egg cell, maize root cell
and pea root tip showed that calmodulin (CaM) has been
found in the amyloplasts (Dauwalder et al., 1986; Li et al.,
1993; Fu et al., 1998). Yang et al. (2002) report that the amy-
loplasts in the embryo sac are rich in CaM before and after
fertilization. They suggest that CaM might promote starch
hydrolysis and provide nutrition for the alteration from egg
Figs.8-22. 8. Abundant calcium precipitates on the tetrad wall, ×29 140. 9. Severely contracted tetrad-microspore and its calcium
distribution, ×7 285. 10. Microspore structure and calcium distribution at free microspore stage, showing the relationship among
microspore, tapetal cell, Ubisch bodies and calcium precipitation, ×11 656. 11. The structure of mature pollen grain, ×2 186. 12.
Distribution of calcium precipitates, starch grain and lipid droplet in mature pollen grain, ×5 099. 13,14. Microsporangia at meiosis
stage, showing the structure and calcium distribution. 13. Calcium distribution on the outer and medium layer of microsporangium wall,
×5 828. 14. The tapetal cell, pro-Ubisch bodies and Ubisch bodies, ×17 484. 15. RER with plenty of ribosomes in the tapetal cells at
meiosis stage, ×14 570. 16. Sporopollenin bodies in the microsporangium locule at meiosis stage, ×29 140. 17. Tapetal cell, pro-Ubisch
bodies and Ubisch bodies at tetrad stage, ×17 484. 18,19. Tapetum at free microspore stage. 18. Calcium precipitates on the mitochondria,
×36 425. 19. Ubisch bodies of irregular form at the inner tangential side of the tapetum and calcium distribution around them, ×11 656.
20, 21. Microsporangium wall at mature pollen stage, showing degenerated tapetal cells and calcium distribution. 20. Calcium distribution
(hollow arrow pointing the calcium precipitate) in degenerated tapetal cells, ×2 914. 21. Calcium distribution (arrow) around the
irregular-formed Ubisch bodies, ×14 570. 22. Calcium-free control section incubated in a solution of EGTA, calcium precipitates change
into small holes (arrow), ×11 656. The abbreviations are the same as in Figs.1-7.
　←
KONG Hai-Yan et al.: Calcium Distribution During Pollen Development of Larix principis-rupprechtii 75
cell to zygote. As it is well known that CaM is a target
protein of calcium signaling, we suggest that calcium in the
plastids might also be related to starch hydrolysis and pro-
vide nutrition for the meiosis of the microspore mother cell.
References:
Antonie A F, Faure J E, Cordeiro S, Dumas C, Rougier M, Feijo J
A. 1999. A calcium influx is triggered and propagates in the
zygote as a wavefront during in vitro fertilization of flowering
plants. Proc Natl Acad Sci USA, 97:10643-10648.
Chen Z-K , Wang F-X , Zhou F . 1987. Investigation on the
ultrastructure of the tapetum in Pinus bungeana. Acta Bot Sin
, 29:486-491. (in Chinese with English abstract)
Dauwalder M, Roux S J, Hardison L. 1986. Distribution of
calmodulin in pea seedlings: immunocytochemical localiza-
tion in plumules and root apices. Planta, 168:461-470.
Digonnet C, Aldon D, Leduc N, Dumas C, Rougier M. 1997. First
evidence of a calcium transient in flowering plants at
fertilization. Development, 124:2867-2874.
Franklin-Tong V E. 1999. Signaling and modulation of pollen tube
growth. Plant Cell, 11:727-738.
Fu Y, Chen Y-F , Liang S-P, Zhou C . 1998. Immunolocalization
of calmodulin in unfertilized and fertilized embryo sacs in
Nicotiana tabacum var. macrophylla. Acta Bot Sin , 40:683-
687. (in Chinese with English abstract)
Gong M , Yang Z-H , Tsao T-H . 1995. Initiating effect of calcium
on pollen germination and its regulatory role in pollen tube
growth. Acta Scentiarum Naturalium Universitatis Pekinensis
, 31:238-249. (in Chinese with English abstract)
Gong M , Li Y , Tsao T-H. 1990. Calcium messenger system in
plants. Chin Bull Bot, 7(3):19-29. (in Chinese)
Jia G-X , Li F-L ,Shen X-H. 1994. The formation of gameto-
phytes and embryo development in Larix principis-rupprechtii.
J Beijing Forestry Univ , 16(2):10-13. (in Chinese with En-
glish abstract)
Jia G-X . 1996. The reproductive biology of Cunninghamia
lanceolata. PhD Thesis. Beijing: Institute of Botany, The
Chinese Academy of Sciences. (in Chinese with English
abstract)
Jia G-X, Chen Z-K, Shen X-H . 1998. Pollen ontogeny and its
wall formation in Cunninghamia lanceolata. Grana, 37:343-
351.
Li J X, Liu J W, Sun D Y. 1993. Immunoelectron microscopic
localization of calmodulin in corn root cells. Cell Res, 3:11-
19.
Li R-Q , Zhu Y-G , Meng X-H , Wang J-B . 2001. The distribution
of calcium in the pollen and connective tissue of Honglian-
Yuetai cytoplasmic male sterile rice. Acta Agron Sin , 27:230-
233. (in Chinese with English abstract)
Meng X-H, Wang J-B, Li R-Q . 1999. The role of tapetal Ubisch
bodies in the transportation of Ca2+. Chin Bull Sci , 44:2640-
2643. (in Chinese)
Meng X-H, Wang J-B , Li R-Q. 2000. Effect of photoperiod on
calcium distribution in photoperiod-sensitive cytoplasmic
male-sterile wheat during anther development. Acta Bot Sin ,
42:15-22. (in Chinese with English abstract)
Stanley G, Linskens H F. 1974. Pollen. Berlin: Springer-Verlag.
127-128.
Tian H Q, Kuang A, Musgrave M E, Russell S D. 1998. Calcium
distribution in fertile and sterile anthers of photoperiod-sen-
sitive genic male-steril rice. Planta, 204:183-192.
Tirlapur U K, Cresti M. 1992. Computer-assisted video image
analysis of spatial variation in membrane associated Ca2+ and
calmodulin during pollen hydration, germination and tip growth
in Nicotiana tabacum L. Ann Bot, 69:503-508.
Tirlapur U K, Willemsee M T M. 1992. Changes in calcium and
calmodulin levels during microsporogenesis, pollen develop-
ment and germination in Gasteria verrucosa. Sex Plant Reprod,
5:214-223.
Wang H , Jian L-C , Zhang J-R. 1994. Changes of the level of Ca2+
in cells of rice seedlings under low temperature stress. Acta
Bot Sin, 36:587-591. (in Chinese with English abstract)
Yang J , Zhao J , Liang S-P , Yang H-Y . 2002. Changes of calmodulin
distribution in the embryo sac of Oryza sativa before and
after fertilization: an immunogold electron microscope study.
Acta Bot Sin , 44:264-272.
Yu F-L , Liang S-P , Yang H-Y, Wang Y . 1998. Ultracytochemical
localization of calcium in micropyle and embryo sac of Bras-
sica napus before and after pollination. Acta Bot Sin , 40:591-
597. (in Chinese with English abstract)
Yu F-L , Zhao J , Liang S-P , Yang H-Y. 1999. Ultracytochemical
localization of calcium in the gynoecium and embryo sac of
rice. Acta Bot Sin , 41:125-129. (in Chinese with English
abstract)
(Managing editor: WANG Wei)