全 文 ：Received 6 Jun. 2003 Accepted 17 Jul. 2003
Supported by the National Natural Science Foundation of China (30070361) and Beijing Natural Science Foundation (6022009).
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Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 319-327
Glandular Characteristics of the Stigma During the Development
of Cucumis sativus Female Flowers
PENG Yi-Ben1, BAI Shu-Nong1, XU Zhi-Hong1, LI Yi-Qin2*
(1. Peking University-Yale Joint Research Center of Agricultural and Plant Molecular Biology, College of Life Sciences,
Peking University, Beijing 100871, China;
2. Protein Science Laboratory of the Ministry of Education, School of Life Sciences and Engineering,
Tsinghua University, Beijing 100084, China)
Abstract: The stigma initiation and development of Cucumis sativus L. female flower was studied using
electron microscopy. The differentiation of stigmatic cells could be recognized when the floral bud was
about 1 mm in length. After the pinnate structure of the stigma appeared, three regions indicating the
papillae, the transmitting tissue and the secretory tissue were observed. The pinnate stigma was
characterized as a partition structure for naming immature and mature stigma in the following investigation.
The ultrastructure of various tissue cells of the stigma during the development was observed using
transmission electron microscopy. Throughout the whole developmental process the cytoplasm of papillae
and secretory tissue cells was filled with many endoplasmic reticula (ER). Most of the ER was tube-like and
rough with enlarged cisterna from which many vesicles were produced. In the mature stigma, numbers of
plasmodesmata were found between the secretory and the transmitting tissue cells. The papillae and
secretory tissue cells are highly vacuolated and the plasmalemma was invaginated or exvaginated. The
nuclear envelope of secretory tissue cells was enlarged, which led to the formation of plurivalvis nucleus
during stigma development. Apparently, nuclear envelope became more strongly lamellate at mature stage.
In different tissue cells of mature stigma, ATPase activity was localized along the plasmalemma and
vacuole membrane. The PM-H+-ATPase specific activity increased during stigma development. Our
results revealed the glandular characteristics of the developing stigma of cucumber female flowers.
Key words: Cucumis sativus (cucumber); stigma development; ultrastructure; ATPase
Cucumber (Cucumis sativus) is a typical diclinous plant,
which produces male (staminate) and female (pistillate)
flowers. The ontogenesis of cucumber unisexual (female
or male) flowers is thought to be related to floral organ
development and regulated by multiple genes (Astmon and
Galun, 1960; Dellaporta, 1994; Ainsworth, 1999; Yang et al.,
2000). It is essential to understand developmental charac-
teristics of floral organs in order to elucidate genetic mecha-
nisms of the organ formation of the unisexual flowers.
The stigma plays an important role in the processes of
pollination and fertilization (Schou, 1984; Dellaporta, 1994).
Both the acceptance of compatible pollen and the rejection
of incompatible pollen are assumed to be related to the
stigmatic exudate (Martin and Telek, 1971; Cynthia et al.,
1990; Kachroo et al., 2001). Till now most of the studies
have focused on the ultrastructure of mature stigmata of
various plant species, such as Lilium longiflorum (Aspinall
and Rosell, 1978; Ghosh and Shivanna, 1980), Lycopersicum
peruvianum (Dumas et al., 1978), Phaseolus vulgaris (Lord
et al., 1979), Petunia hybrida (Herrero et al., 1979),
Nicotiana tabacum (Cresti et al., 1986), Solanum tuberosum
(Cynthia et al., 1990), Citrullus lanatus (Sedgley, 1981).
However, reports on the stigma of Cucurbitaceae plants, in
particular on the developmental feature of the stigma of
cucumber is still lacking.
The precursors of stigmatic exudates come from
photoassimilate and the stigmatic exudates are finally se-
creted from papilla cells (Dumas et al., 1978; Miki-Hirosige
et al., 1987). It is evidenced that the ATPase is associated
with the movement of photoassimilate in plant cells (Ruan
and Patrick, 1995; Patrick, 1997; Sze et al., 1999). It would
be, therefore, interesting to observe the ultrastructural fea-
tures of the developing stigma and to investigate whether
the transportation pathway of stigmatic exudates is related
to ATPase .
Our previous report demonstrated the arrested devel-
opment of the carpel primodia in cucumber male flower
(Yang et al., 2000). In the present study, we focused on the
glandular characteristics of the developing stigma of cu-
cumber female flowers, attempting to accumulate
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004320
knowledge that is favorable to the further study on the
mechanisms of the floral organ development of diclinous
plants.
1 Materials and Methods
1. 1 Plant materials
Cucumber (Cucumis sativus L.) plants were grown in a
greenhouse at Institute of Vegetable, The Chinese Acad-
emy of Agricultural Sciences in April and May 2002. Stig-
mata of female flowers were dissected at different stages of
its development from the floral bud initiation to the anthesis.
1. 2 Light microscopy
The samples were fixed in FAA at 4 ℃ for 24 h, and then
washed twice with 50% ethanol. After staining with hema-
toxylin at room temperature for 30 min, samples were dehy-
drated in an ethanol series, followed by infiltration and
embedding in paraffin. Eight-µm sections were made with
microtome (Leitz-1512, Germany) and mounted on the slides
coated with Poly-L-lysine (Sigma, USA). The sections was
dewaxed with xylene and ethanol and then rehydrated in a
decreasing ethanol series. The samples were observed un-
der a light microscope (Olympus BH-2).
1. 3 Transmission electron microscopy
Fragments of stigma (2-3 mm in length) were treated as
follows: fixed with 4% glutaraldehyde in 0.1 mol/L phos-
phate-buffered saline (PBS, pH 7.2) at room temperature for
3 h, washed three times with PBS, postfixed with 1% os-
mium tetroxide in PBS at 4 ℃ for 2 h, rinsed in PBS, and
dehydrated in an ethanol series. Samples were then infil-
trated with Spurr’s resin (Dow Chemical Co., USA) at room
temperature for 24 h. Polymerization was conducted at 70
℃ for 8 h. The specimen was first semi-thin sectioned and
observed under the light microscope to localize the stigma
cells. Thereafter, ultrathin sections were made with micro-
tome (Leitz-1512, Germany) and collected on 100-mesh cop-
per-grids covered with 0.3% Formvar films. Samples were
stained with 2% uranyl acetate in 50% ethanol at 25 ℃ for
15 min and then with alkaline lead citrate for 10 min. All
samples were examined under a JEM-100S (Japan) trans-
mission electron microscope.
1. 4 Ultracytochemical localization of ATPase activity
Cytochemical investigation of ATPase activity was car-
ried out according to the method reported by Bentwood
and Cronshaw (1978). Stigma tissue samples were collected
and immersed in 4% formaldehyde and 3% glutaraldehyde
for 2 h, and then rinsed twice with 0.2 mol/L cacodylate-
acetate buffer (pH 7.2). After being washed in 0.05 mol/L
Tris-maleate buffer (pH 7.2) for 2 h, the samples were incu-
bated in a solution containing 2 mmol/L 5-ATP, 5 mmol/L
MgSO4, 3.5 mmol/L Pb(NO3)2 and 50 mmol/L Tris-maleate
(pH 7.2) at 22 ℃ for 2 h. Control experiments were con-
ducted by applying 10 mmol/L NaF, an inhibitor of ATPase,
instead of ATP. All samples were washed in Tris-maleate
buffer and cacodylate-acetate buffer for 1 h, followed by
post-fixation in 1% osmium tetroxide at 4 ℃ overnight. The
samples were dehydrated by acetone series and embedded
in Spurr’s resin (Dow Chemical Co., USA). Polymerization
was conducted at 70 ℃ for 8 h. Localization of ATPase
activity was observed using transmission electron micros-
copy .
1. 5 PM-H+-ATPase activity assay
The PM-H+-ATPase activity assay was conducted ac-
cording to Peng et al. (2003). Briefly, stigma samples were
collected at immature and mature stages. The samples were
triturated in liquid nitrogen in a mortar and the enzyme re-
action medium containing 30 mmol/L Tris-Mes (pH 6.5), 3
mmol/L MgSO4, 0.5 mmol/L Na2MoO4, 0.01 % (W/W) Tri-
ton X-100, and corresponding inhibitors (50 mmol/L KNO3
and 1 mmol/L NaN3). The reaction was started by adding 3
mmol/L ATP to the medium, kept at 37 ℃ for 30 min, and
then stopped by adding 250 mL 10% (W/V) sodium dodecyl
sulphate (SDS). Total inorganic phosphate (Pi) resulted from
ATP hydrolysis was determined by Ames (1966).
2 Results
2. 1 Morphology of the developing stigma
The differentiation of pistil cells of cucumber female flow-
ers could be recognized when the floral bud was about 1
mm in length (Fig.1). The pistil continued to develop while
the stamen development was stagnated (Fig.2). When the
floral bud became about 10 mm in length the pinnately struc-
ture of pistil appeared (Fig.3). For simplifying the
description, the developmental stage earlier than the ap-
pearance of the pinnately structure was named as immature
stage and the stage later than that as mature stage. In ma-
ture stigma three structural regions could be distinguished,
i.e. the papillae, the transmitting tissue which was com-
posed of phloem and parenchyma cells, and the secretory
tissue which was located among the papillae and transmit-
ting tissue cells (Fig.4).
2. 2 Ultrastructural features of the stigma cells during
its development
At the immature stage, a thin electron-dense layer ap-
peared to cover the surface of the stigma (Figs.5, 6). In
some cases, the wall was exvaginated in the common bound-
ary between the adjacent cells at the outer surface (Figs. 5,
6). The plasmalemma was invaginated or exvaginated in the
immature stigma cells (Figs.5, 7). At the mature stage, the
PENG Yi-Ben et al.: Glandular Characteristics of the Stigma During the Development of Cucumis sativus Female Flowers 321
shape of the papillae was longer than that of the stigma
parenchyma cells. The papilla cell was highly vacuolated,
and the cytoplasm was extruded to the vicinity of the cell
wall as a big vacuole was located in the center of the papilla
cell (Fig.9). The invagination and exvagination of plasmale-
mma occurred in papillae and secretory tissue cells (Figs.
8-11).
In immature stigma, the nuclear envelope of the cells
located on the surface was enlarged, which led to the for-
mation of plurivalvis nucleus (Fig.11). At mature stage more
strongly lamellate nuclei were found in cells of secretory
tissue (Fig.12). In addition, numerous plasmodesmata were
detected between the parenchyma cells at mature stage
(Fig.12).
The cytoplasm was filled with electron-dense materials
and was rich in endoplasmic reticulum (ER) in the immature
stigma cells (Fig.13). Most of the ER were tube-like and
surrounded by many vesicles. At the mature stage, numer-
ous ER, Golgi body and mitochondria appeared in the cyto-
plasm throughout the papillae developmental process (Figs.
14,15). Similar to that found at the immature stage, most ER
appeared tube-like with enlarged cisterna from which many
vesicles were produced. Some of the vesicles merged into a
central vacuole or fused with each other (Fig.15). The secre-
tory cells became highly vacuolated, and each cell con-
tained at least one large vacuole and several small ones
(Figs.10, 12). A large number of vesicles fused with vacu-
oles (Figs.16, 17). In the mature stigma the sieve elements
(SE) contained a wide sieve area (Fig.18). There were some
vesicles close to the open-sieve pore. In companion cells
(CC), many vesicles were accumulated together, forming
the multivesicular body (Fig.18). Phloem parenchyma cells
had a similar ultrastructure as described in secretory tissue
cells (not shown).
2. 3 Localization of ATPase activity and PM-H+-ATPase
activity assay
Figures 19-22 demonstrate the localization of ATPase
activity detected by using cytochemical technique. In the
stigma cells at the immature stage, ATPase activity was low
as little lead phosphate precipitates were found (Fig.19).
However, at the mature stage, ATPase activity was detected
along the membrane system. Figure 20 shows that ATPase
activity was dominantly localized in the plasmalemma of
the SE-CC complex. ATPase activity was also observed in
the plasmalemma and vacuole membrane of secretory tis-
sue cells (Fig.21). In papilla cells, ATPase activity was lo-
calized preferentially in the plasmalemma (Fig.22). Follow-
ing the development of the stigmata PM-H+-ATPase spe-
cific activity quantitatively increased, from 1.43 mg Pi·mg-1
protein·min-1 detected in immature stigmata to 3.41 mg
Pi·mg-1 protein·min-1 detected in mature stigmata (Fig. 23).
3 Discussion
3.1 Ontogenesis of stigma in cucumber female flower
In cucumber floral bud when the pistil primordia and
stamen primordia appeared at the same level the develop-
ment of stamen was stagnated (Figs.1-3). Along with the
continuous development of the pistil the differentiated cells
were obviously observed and the stigma became a pinnate
structure. Under electron microscope three types of cells,
i.e. papillae, secretory and transmitting cells, could be ob-
served on the longitudinal section of a cucumber stigma. It
Figs. 1-4. 1. Early stage of the development, stigma (S) and
stamen (St) appeared at the same level. 2. Late stage of the
development, the stigma continued to develop while the stamen
was arrested. 3. Mature stigma, apparent pinnate structure and
emerged nectary (Ne). 4. Longitudinal section of the upper part
of a mature stigma of Cucumis sativus. Three structural regions
represent papillae (P), secretory tissue (ST) and transmitting tis-
sue (TT).
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004322
resembled the stigmata of bisexual flowers in the plant spe-
cies studied, such as Lycopersicum peruvianum, Nicoti-
ana tabacum, Petunia hybrida, Solanum tuberosum
(Dumas et al., 1978; Herrero et al., 1979; Cresti et al., 1986;
Cynthia et al., 1990).
3.2 Ultrastructural alteration along with the develop-
ment displayed the glandular characteristics of the stigma
Sedgley (1981) reported that in watermelon stigma the
Figs. 5-12. 5. Immature stigma, a thin, electron-dense layer appeared on the surface of the stigma (arrow), ×8 000. 6. The cell wall
was exvaginated at the boundary between the adjacent cells, ×12 000. 7. Immture stigma cells, the plasmalemma appeared invaginated
(arrow), ×10 000. 8. Mature stigma, in secretory tissue cell, the plasmalemma was invaginated (arrow), ×10 000. 9. Mature stigma,
The papilla was highly vacuolated, ×10 000. 10. Mature stigma, secretory tissue cells were also highly vacuolated, ×10 000. 11.
Immature stigma, the nuclear envelope was enlarged which led to forming lamella nucleus, and paramural body was found (arrow),
×12 000. 12. Mature stigma, the nucleus in secretory tissue cell was even more lamellate, ×10 000. Abbreviations: CW, cell wall; N,
nucleus; V, vesicle.
PENG Yi-Ben et al.: Glandular Characteristics of the Stigma During the Development of Cucumis sativus Female Flowers 323
papilla cells had simple wall thickening, which were associ-
ated with dictyosomes and secretory vesicles. We did not
find the wall thickening in cucumber papilla cells, but found
cell wall exvagination and a electron-dense layer appeared
on the surface of the stigma at immature stage (Figs.5, 6).
Following the cucumber stigma development, the plas-
malemma appeared invaginated or exvaginated as shown
in Figs.7-9. The invagination of plasmalemma could bring
Figs. 13-18. 13. Immature stigma, cytoplasm was filled with ER and ER-produced vesicles. Many plasmodesmate were found
between the cells (arrow), ×25 000. 14. Mature stigma, numerous Golgi bodies and mitochondria appeared in the cytoplasm of papilla,
×20 000. 15. Mature stigma, cytoplasm of papilla was filled with tube-like ER. The ER vesicles were fused to each other, ×30 000.
16. In secretory cells many vesicles (arrow) were produced from ER and the vesicles merged into each other, ×20 000. 17. In secretory
cells many vesicles (arrow) were produced from Golgi body, ×35 000. 18. SE-CC complex of the transmitting tissue with a wide sieve
area, open sieve pores and multivesicular body in the companion cell, ×20 000. Abbreviations: CC, companion cell; ER, endoplasmic
reticula; G, Golgi body; M, mitochondria; MB, multivesicular body; SE, sieve element; v, vesicle.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004324
about paramural bodies, which might be associated with
the transport of the exudate via the apoplast (Harris, 1981).
The fact that numerous plasmodesmata were found be-
tween parenchyma cells in the mature stigma is very
important, as the plasmodesmata may play a decisive role
in the transportation of substances through the symplast
(Oparka, 1990). Furthermore, during the development in
parenchyma cells the nuclei became more and more lamellar.
The plurivalvis nucleus was considered to be related to the
nucleus/cytoplasm operation of the parenchyma cell (Gerace
et al., 1984). It is consistent with the finding in the transfer
cells of maize caryopses (Davis et al., 1990). These results
may also indicate that cucumber stigmatic parenchyma cells
possess the principle function of the transfer cell.
Ultrastructural investigations have primarily dealt with
how stigmatic exudates are secreted from the papillae to
the stigma surface. Most of the previous studies reported
that the secretion of stigmatic exudates from papillae to the
stigma surface is related to the ER vesicles. But a study on
Citrullus lanatus has shown that Golgi vesicles were in-
volved in the transportation of stigmatic exudates (Sedgley
and Blesing, 1983). In Lycopersicum peruvianum, some
secretion droplets merged into Golgi vesicles or fused with
each other (Dumas et al., 1984; Knox, 1984). However,
Cynthia et al. (1990) declared that he did not find any struc-
ture that could be interpreted as a fusion between Golgi
vesicles and secretion droplets in Solanum tuberosum stig-
matic tissue. The present study clearly showed that the
cytoplasm of the papilla cells and secretory tissue cells
was filled with numerous endoplasmic reticula (ER) through-
out the whole developmental process. It was also found
that Golgi bodies secreted some vesicles in papilla cells
and secretive cells. However, the amounts of vesicles se-
creted from Golgi bodies were less than those from ER in
papilla cells and secretive cells (Figs. 14-17). It could be
speculated that the Golgi bodies were associated with trans-
portation of stigmatic exudates, but might be less active
than ER in stigmatic tissue of cucumber female flower.
Figs. 19-22. 19. In immature stigma cells, ATPase activity was low, ×12 000. 20. Mature stage. In the sieve element/companion cell
complex of the transmitting tissue ATPase activity was located along the plasmalemma, × 8 000. 21. In the secretory cells of mature
stigma, ATPase activity was observed in the plasmalemma and vacuole membrane (arrow), × 20 000. 22. In the papilla cell, ATPase
activity was located in the vicinity of plasmalemma (arrow), × 20 000. Abbreviations: CC, companion cell; CW, cell wall; SE, sieve
element; Va, vacuole.
PENG Yi-Ben et al.: Glandular Characteristics of the Stigma During the Development of Cucumis sativus Female Flowers 325
3. 3 ATPase activity in stigma cells is responsible to its
secretory function
The enzyme to catalyze ATP hydrolysis mostly con-
tains P-type ATPase and V-type ATPase in plant cells
(Serrano, 1989; Sze et al., 1999; Palmgren, 2001). P-type
ATPase includes H+-ATPase and Ca2+-ATPase, the former
is usually localized in the plasmalemma and the latter as in
intracellular membranes, i.e. the plasmalemma, vacuole mem-
brane and endoplasm reticulum (Sze et al., 1999; Palmgren,
2001). The PM-H+-ATPase was suggested to be involved
in the photoassimilate unloading and transportation of
metabolic products across the plasmalemma among plant
cells (Cronshaw, 1980; DeWitt and Harper, 1991; DeWitt
and Sussman, 1995; Langhans et al., 2001). By the use of
cytochemical technique we find that ATPase activity was
low in immature stigma cells of the cucumber female flower.
However, during the development of the stigma, an
ATPase activity was localized on the plasmalemma and
vacuole membrane in different stigmatic tissue cell types.
This is in accordance with the cytochemical localization of
ATPase in the phloem of Pisum sativum (Bentwood and
Cronshaw, 1978) and the investigation of H+-ATPase
immunolocalization in the stem of Ricinus communis
(Langhans et al., 2001). We found that lead particles were
densely accumulated along the plasmalemma of SE-CC (Fig.
20). It could imply that the transportation of the stigmatic-
secrete precursors from SE-CC to phloem parenchyma cells
was strongly ATPase-dependent. ATPase activity was also
localized in the plasmalemma of the papillae and secretive
cells, as well as in the vacuole membrane of the secretive
cells. To convince the cytochemical results we carried out
quantitative measurements of PM-H+-ATPase specific
activity. It was found that PM-H+-ATPase specific activity
increased by 138% in mature in comparison with that of the
immature stigmata. It is proposed that the ATPase is in-
volved in stigmatic secretion from the secretory tissue ones
to papilla cells and then to the surface.
Acknowledgements: We thank Prof. Hu Shi-Yi, College of
Life Sciences of Peking University, for her inspiring sug-
gestions on this research, Mrs. Liu Hai-Hong and Jia Jun-
Zheng, China Agricultural University, for providing TEM
facility and Prof. He Chao-Xing, Chinese Academy of Agri-
cultural Sciences, for his help in cucumber plant cultivation.
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