Annals of Botany 100: 1475–1481, 2007
doi:10.1093/aob/mcm253, available online at www.aob.oxfordjournals.org
Pollination Drop in Juniperus communis: Response to Deposited Material
S E REN A MU GN AI NI 1 , M A S S I M O N E P I 1 , M A S S I M O GU A R NI E R I 1 , BETI PIOT TO 2 and
E T TO R E PACI N I 1 , *
1
Department of Environmental Sciences “G. Sarfatti”, University of Siena, Via P.A. Mattioli 4, 53100 Siena, Italy and
2
Department of Nature Protection, Environmental Protection and Technical Services Agency (APAT), Via Curtatone
3, 0085 Roma, Italy
Received: 22 May 2007 Returned for revision: 13 July 2007 Accepted: 20 August 2007 Published electronically: 17 October 2007
† Background and Aims The pollination drop is a liquid secretion produced by the ovule and exposed outside the
micropyle. In many gymnosperms, pollen lands on the surface of the pollination drop, rehydrates and enters the
ovule as the drop retracts. The objective of this work was to study the formation of the pollination drop in
Juniperus communis, its carbohydrate composition and the response to deposition of conspecific pollen, foreign
pollen and other particulate material, in an attempt to clarify the mechanism of pollination drop retraction.
† Methods Branches with female cones close to pollination drop secretion were collected. On the first day of pollination drop exposure, an eyelash mounted on a wooden stick with paraffin was used to collect pollen or silica gel particles, which were then deposited by contact with the drop. Volume changes in pollination drops were measured by
using a stereomicroscope with a micrometer eyepiece 3 h after deposition. The volume of non-pollinated control
drops was also recorded. On the first day of secretion, drops were also collected for sugar analysis by high-performance
liquid chromatography.
† Key Results The pollination drop persisted for about 12 d if not pollinated, and formed again after removal for up to
four consecutive days. After pollination with viable conspecific pollen, the drop retracted quickly and did not form
again. Partial withdrawal occurred after deposition of other biological and non-biological material. Fructose was the
dominant sugar; glucose was also present but at a much lower percentage.
† Conclusions Sugar analysis confirmed the general trend of fructose dominance in gymnosperm pollination drops.
Complete pollination drop withdrawal appears to be triggered by a biochemical mechanism resulting from interaction between pollen and drop constituents. The results of particle deposition suggest the existence of a nonspecific, particle-size-dependent mechanism that induces partial pollination drop withdrawal. These results
suggest that the non-specific response may decrease the probability of pollen landing on the drop, reducing pollination efficiency.
Key words: Juniperus communis, female cone, gymnosperm reproduction, pollen, pollination drop withdrawal.
IN TROD UCT IO N
The different landing sites for pollen in gymnosperms and
angiosperms led Robert Brown to distinguish these land
plant groups in the third decade of the 19th century
(Maheshwari, 1960). Vaucher (1841) subsequently recognized the pollination drop as the landing site in many gymnosperms. Pollination drops appeared very early in the
phylogeny of plants. According to Doyle (1945) and
Rothwell (1977) pteridosperms and early gymnosperms
already possessed a pollination drop and this mechanism
of pollen capture was widespread during the Carboniferous
(Taylor and Millay, 1979).
The pollination drop is a liquid secretion produced by the
ovule and exposed outside the micropyle (Doyle, 1945).
Abies, Cedrus, Larix, Pseudotsuga and Tsuga sp. (Pinaceae)
have stigmatic micropyles and no pollination drop. In
Auracaria, Agathis, Tsuga dumosa and T. heterophylla
pollen lands on sites different from the micropyle (Singh,
1978; Owens et al., 1998). In other gymnosperms, pollen
lands on the surface of the pollination drop or on a nearby
cone surface and is then picked up by the pollination drop
(Owens et al., 1998), rehydrates, and enters the ovule as the
* For correspondence. E-mail [email protected]
drop retracts. It is now known that pollination drops and
cones enhance and facilitate pollen capture (Niklas, 1985;
Greenwood, 1986; Buchmann et al., 1989; Owens et al.,
1998; Roussy and Kevan, 2000).
The micropylar secretion contains dissolved substances,
the composition of which was unclear at the beginning of
the last century (Fujii, 1903; Tison, 1911). Subsequent
studies found carbohydrates, amino acids and proteins.
McWilliam (1958) found glucose, fructose and sucrose.
Ziegler (1959) found several amino acids, peptides and
organic acids. Sugars, and especially sucrose, glucose and
fructose, are the more frequent substances identified in
the pollination drop in more recent studies (Owens et al.,
1987; Séridi-Benkaddour and Chesnoy, 1988; Gelbart and
von Aderkas, 2002) that also revealed differences in the
relative proportions of these substances in various groups
of gymnosperms sensu latu. Ephedra, for instance, has a
high proportion of sucrose and Welwitschia has a high percentage of fructose (Carafa et al., 1992). Acid phosphatase
activity has been demonstrated in pollination drops of
Welwitschia (Carafa et al., 1992). Proteins have been
found in the micropylar drop of Taxus and Douglas fir
and have been linked to pollen selection and development
(O’Leary et al., 2004; Poulis et al., 2005).
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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1476
Mugnaini et al. — Pollination Drop in Juniper
Pollination drop secretion has been studied in several
species of gymnosperms (Owens et al., 1987; Takaso and
Owens, 1995a, b; Takaso and Owens, 1996). Ultrastructural
modifications of nucellar apex cells during pollination drop
secretion were reviewed by Takaso and Owens (1995a) and
demonstrate that these cells are responsible for pollination
drop secretion.
There is more uncertainty regarding the mechanism of
pollination drop withdrawal. According to Tomlinson
et al. (1997), pollination drop withdrawal is not determined
by evaporation or mechanical stimulation but pollen is the
sole stimulus for drop withdrawal. Biochemical and ecological aspects of pollination drops are largely unknown,
as observed by Chesnoy (1993) in her review of the subject.
The aim of the present experimental study was to obtain
information about pollination drop composition, production
and withdrawal in Juniperus communis (Cupressaceae), and
to determine if physical contact of non-pollen substances
can influence pollination drop withdrawal. This work is
part of a research programme on the reproductive biology
of Mediterranean junipers (Mugnaini et al., 2004) that are
of ecological importance because of their capacity to colonize highly stressed environments (Falinski, 1980; Piotto
and Di Noi, 2003).
M AT E R I A L S A N D M E T H O D S
Plants of Juniperus communis L. used for this research grew
close to Greve in Chianti (Florence province), 438370 6800 N
118170 6200 E at an altitude of 184 m. Material was collected
from 20 plants of approximately the same age and height
(about 2 m).
Pollination drop volume and pollination experiments
Branches with cones just about to secrete the pollination
drop were collected in the study area the evening before
the experiments and kept in the laboratory. When drops
appeared, sprigs 3 – 5 cm long with one/three female
cones were inserted in vials containing water, and maintained overnight at 15 + 1 8C and 52 + 5 % relative humidity. Next morning sprigs bearing female cones without
pollination drop were discarded. Female cones were only
used for experiments on the first day of pollination drop
exposure.
A human eyelash mounted on a wooden stick with paraffin was used to collect pollen and deposit it by contacting
the pollination drop. Contact with the human eyelash
alone did not stimulate withdrawal of the pollination drop.
The following were used for the pollination trials:
(1) viable conspecific pollen collected soon after pollen
sac opening; (2) dead conspecific pollen, killed by heat
(105 8C for 12 h); (3) particles of silica gel in two size
ranges (10 – 15, and 63– 200 mm); (4) pollen of Pyrus
communis; and (5) pollen of Pinus canariensis. The last
two treatments were chosen to test if completely different
kinds of pollen have a similar effect. Only one treatment
was applied to the cones of each sprig. Viability of
juniper pollen for treatments (1) and (2) was evaluated by
means of fluorochromatic reaction (FCR) as indicated by
Nepi et al. (2005).
A stereomicroscope with micrometer eyepiece was used
to measure volume changes in ‘undisturbed’ pollination
drops for a period of 48 h. Volume changes 3 h after pollination trials were observed via the same method.
Pollination drop diameter, assumed spherical, was
measured and its volume calculated with the formula
4/3pr 3 where r was the radius of the pollination drop.
Only that part of the pollination drop protruding from the
micropyle was observed and measured.
Pollination drop volume at time zero (i.e. just before
deposition of pollen or particulate) was compared with
that 3 h later by the Wilcoxon test for paired data.
Although the time required for complete withdrawal of
the droplet after pollination with conspecific pollen was
1 –2 h, pollination drop volume was monitored for longer
to be certain of any volume fluctuations or new secretion.
Three hours after deposition of material on the pollination
drop, it was possible to distinguish three different
responses: complete withdrawal when pollination drop
volume dropped to 0; partial withdrawal when pollination
drop volume decreased significantly but did not drop to
zero; and no withdrawal when there was not a statistically
significant variation in pollination drop volume or a
volume increase. The statistical significance of different
behaviour of the pollination drop in response to the different treatments was evaluated by the chi square test applied
to a 7 3 (treatments response categories) contingency
table.
Pollination drop collection and analysis
For chemical analysis, droplets were removed from
sprigs, stored as described above, with a microcapillary
tube (5 mL) on the first day of secretion. Samples were
stored at 280 8C. The content of six capillaries, each containing 40 – 300 pollination drops, was analysed for sugar
content by high-performance liquid chromatography
(HPLC) as reported by Nepi et al. (2003). Before analysis,
samples were thawed to ambient temperature and diluted
1 : 50 with distilled water. A 20-mL volume of sample
and standard solution was injected into a Waters LC
module 1 apparatus. Mobile phase was water (MilliQ,
pH7). The flow rate was set at 0.5 mL min21 and column
temperature at 85– 90 8C. Sugars were separated in a
Waters Sugar-Pack I (6.5 – 300 mm) column and identified
with a refractive index detector (Waters 2410).
R E S U LT S
Pollination drop formation
The pollination period lasts about 1 month (Table 1). The
three upper scales of the female cone open 1 – 2 d before
pollination drop production and micropylar apertures are
revealed. Pollination drop production starts in a few cones
of each plant, reaching a peak when almost all cones of
all plants in a study area have pollination drops.
Pollination drop production in a single cone is not always
a synchronous phenomenon.
Mugnaini et al. — Pollination Drop in Juniper
TA B L E 1. Reproductive features of Juniperus communis
Pollination period ¼
February–March
Pollination drop volume (mean; mm3)
Pollen diameter (mm)
Dry pollen
Fully hydrated in water
Pollination drop persistence*
Sugar content of pollination drop (mg mL21)
Glucose
Fructose
Mannitol
Sucrose
0.02 + 0.01 (n ¼ 30)
20.53 + 2.51 (n ¼ 50)
28.72 + 1.7 (n ¼ 50)
Approx. 12 d
8.30 + 0.35 (n ¼ 6)
45.49 + 0.59 (n ¼ 6)
0.60 + 0.33 (n ¼ 6)
Not detected
All measurements are mean + s.d.
* Laboratory conditions.
Under laboratory conditions, ‘undisturbed’ pollination
drops persisted for up to 12 d (Table 1). Average pollination
drop diameter was 0.31 mm (Fig. 1), about 4 –5 times larger
than that of the micropyle (0.07 mm). Mean volume on the
first day of emission was 0.02 mm3 (range 0.002–
0.122 mm3). The volume of unpollinated pollination drops
followed for 48 h fluctuated slightly in time under
uniform laboratory conditions.
Because female cones have three ovules and the pollination drops are very close to each other, they sometimes
merge spontaneously or in response to handling. When pollination drops were removed with a microcapillary tube,
they formed again the next day or the day after. New pollination drops formed again up to four times; after that the
ability to produce the drop was lost.
1477
Pollination drop composition
Glucose, fructose and mannitol were detected. Fructose
concentration was much higher (45.49 + 0.59 mg mL21)
than that of glucose (8.30 + 0.35 mg mL21; Table 1).
Mannitol was found in small amounts. Sucrose was not
detected.
Response of pollination drop to pollen or other powdery
material
When the pollination drop was touched with an eyelash
free of pollen it did not retract or change in volume.
Withdrawal occurred only when powdery material
adhered to its surface (Fig. 2A – F). When pollen of
Juniperus communis was applied, some grains went directly
into the pollination drop but others remained on the surface,
moving to the lowest point of the pollination drop along the
surface. Pollen deposition caused evident pollination drop
withdrawal in about 30 min.
All treatments were associated with a significant decrease
in pollination drop volume except in the case of silica gel
particles of 63– 200 mm, which were associated with an
increasing trend in pollination drop volume (Fig. 2D) and
in the case of control pollination drops that did not alter
in volume (Fig. 2G). The treatment that gave the most
homogeneous response was that with viable pollen
(variance ¼ 20.26); variability of response was greater
with non-viable pollen (variance ¼ 944.56). The greatest
variation in response was observed with silica gel,
especially that with large grain size (variance ¼ 22^
381.92; Fig. 2D).
There was a statistically significant relationship between
certain treatments and pollination drop response (x2 ¼
78.06, d.f. ¼ 12, P , 0.001). Complete pollination drop
withdrawal was recorded in a significantly greater number
of drops pollinated with viable conspecific pollen of
J. communis (Fig. 3A). Partial withdrawal was characteristic of drops pollinated with non-viable pollen (Fig. 3B).
Silica gel of 10– 15 mm, Pinus and Pyrus pollen caused
partial or total pollination drop withdrawal (Fig. 3C, E, F).
A significantly higher number of non-withdrawals was
recorded only in drops treated with silica gel of
60– 200 mm and in control drops (Fig. 3D, G).
DISCUSSION
Pollination drop production
F I G . 1. A female cone of Juniperus communis with three micropiles, each
one with a pollination drop. Scale bar ¼ 0.3 mm.
Gymnosperms show variations in the interval during which
the pollination drop remains on the micropyle. Indeed, in
genera such as Juniperus the pollination drop may persist
for several weeks (Table 1); in other species it seems to
be a cyclical phenomenon, i.e. secretion at night or in the
early morning, when the atmosphere has high relative
humidity, and evaporation or withdrawal during the day,
with the cycle repeating the next day (Singh, 1978;
Owens et al., 1980). The cycles may be one or many,
according to species. For instance, Cupressus sempervirens
has five cycles, irrespective of whether or not pollination
occurs (our unpublished data). Most authors (McWilliam,
1478
Mugnaini et al. — Pollination Drop in Juniper
F I G . 2. Effects of deposition of different powdery material on volume of pollination drop of Juniperus communis: (A) viable pollen; (B) non-viable
pollen; (C) silica gel, 10–15 mm; (D) silica gel, 63– 200 mm; (E) Pinus pollen; (F) Pyrus pollen; (G) control. Closed symbols indicate pollination
drop volume before treatment, open symbols volume 3 h after treatment. Each point on the x-axis corresponds to a pollination drop. The table summarizes
the statistical significance of each treatment according to the Wilcoxon test for paired data: n, number of trials; Z, statistics of the test; P, probability.
1958; Barner and Christiansen, 1960; Owens et al., 1980)
have related these cycles to mechanisms that indicate a
tree’s water status as the driving force behind drop production. By contrast, O’Leary and von Aderkas (2006)
found that post-pollination drop production of hybrid
larch is not regulated by a tree’s overall water status, but
is under the control of localized structures such as the
cone or ovule.
Unpollinated drops remain exposed for up to 2 weeks in
Taxus brevifolia (Anderson and Owens, 2000) with the
maximum volume observed in the early morning.
Tomlinson et al. (1997) found that unpollinated drops of
Phyllocladus (Phyllocladaceae) in field conditions evaporated slowly and could persist for over 1 h, but they persisted
for several days in closed containers, becoming increasingly
viscous, as suggested by stretching their surface with a
Mugnaini et al. — Pollination Drop in Juniper
1479
needle. This phenomenon was not observed in the present
laboratory experiment with persistent unpollinated drops
of J. communis that were exposed for almost 12 d. It therefore seems that in J. communis, an equilibrium establishes
after some time between secretion and evaporation of the
pollination drop. The lack of increase in viscosity suggests
the existence of an osmotic mechanism of regulation affecting drop concentration. It is worth remembering that the
laboratory conditions under which these observations
were made may favour water availability, thus lowering
solute concentrations of the pollination drop.
In Juniperus communis, when the pollination drop was
removed, up to four cycles of re-secretion were possible,
after which the pollination drop was not produced again.
Similar observations were reported by Tomlinson et al.
(1997) for Phyllocladus and by von Aderkas and Leary
(1999) for Douglas fir and larches. It can be concluded
that a limited number of secretion processes of the pollination drop can occur.
The chemical composition of the pollination drop
The chemical composition of the droplet and its concentration may be involved in the development of appropriate
(conspecific) pollen. Sugars, amino acids and other substances and their relative abundance may create environments
more or less favourable for pollen germination and tube
growth. Thus, the pollination drop may exercise direct
female selection of conspecific pollen and therefore have a
prezygotic selective role (Gelbart and von Aderkas, 2002;
Poulis et al., 2005).
Pollination drop sugar composition does not appear to be
very different among the species studied thus far. Although
sucrose is the preferred compound for carbon transfer in
conifers (Gelbart and von Aderkas, 2002), fructose is the
dominant sugar in Pinus nigra, Pinus engelmannii
(McWilliam, 1958), Cephalotaxus drupacea, Thuya orientalis, Taxus baccata (Séridi-Benkaddour and Chesnoy,
1988) and Welwitschia mirabilis (Carafa et al., 1992).
The present study also confirmed this trend in Juniperus.
Only two exceptions seem to exist: Picea engelmannii
has glucose as the dominant sugar in the pollination drop
(Owens et al., 1998) and Ephedra helvetica has sucrose
(Ziegler, 1959). The absence of sucrose or its subordination
to glucose and fructose may be the effect of invertase
activity in the pollination drop, as demonstrated in
Douglas fir, in which two of the most abundant proteins
in ovule secretions are invertases (Poulis et al., 2005).
Pinus mugo pollen cultivated in vitro shows preferential
uptake of fructose with respect to other sugars (Nygaard,
R
F I G . 3. Frequency histograms of the response of Juniperus communis pollination drop to deposition of different material (treatments): 1, complete
withdrawal (pollination drop volume dropped to 0); 2, partial withdrawal
(pollination drop volume decreased significantly but did not drop to
zero); 3, no withdrawal (there was no significant variation in pollination
drop volume or a volume increase). (A) Viable pollen; (B) non-viable
pollen; (C) silica gel, 10– 15 mm; (D) silica gel, 63–200 mm; (E) Pinus
pollen; (F) Pyrus pollen; (G) control.
1480
Mugnaini et al. — Pollination Drop in Juniper
1977). Fructose is presumably the ‘preferred’ sugar for
development once pollen reaches the pollination drop.
Pollination drop withdrawal
The process of withdrawal has rarely been studied. It is
evidently a quick process but it is not easy to discern
what is responsible for this withdrawal and which cells
collect/store the liquid. The first observations of withdrawal
of the pollination drop after pollen deposition were made
with cones of Pinus (Doyle and O’Leary, 1935) and subsequently with several other species such as Callitris,
Chamaecyparis, Cryptomeria and Thuja (Tomlinson
et al., 1997, and references therein).
According to Singh (1978) and references therein, pollination drop withdrawal takes from 10 min, as in Pinus, to
much longer, as in Abrotaxis. These data cannot be taken
alone: to understand the process it is necessary to know
other features, such as pollination drop composition/viscosity, ovule morphology and climatic conditions at pollination. Pollination drop withdrawal seems hindered by
high relative humidity, as demonstrated by the experiments
of Lill and Sweet (1977) and Tomlinson (1994).
Different species of gymnosperms respond differently to
pollen deposition: for some of them, pollen arrival is the
stimulus for pollination drop withdrawal; for others pollination drop reduction simply occurs by evaporation, as for
example in Podocarpaceae (Tomlinson et al., 1991, 1997).
The present results with J. communis suggest that withdrawal of the pollination drop occurs in two phases that
may occur simultaneously or sequentially.
A first phase involves partial withdrawal and occurs
whenever particles are deposited on the pollination drop
surface, irrespective of their biological or non-biological
nature. In every case there is a significant variation in
drop volume, suggesting that at least at first, the mechanism
of withdrawal is linked to a non-specific physical stimulus.
It also seems that in this first phase the diameter of particles
is important. Inorganic particles 10– 15 mm in diameter
induce partial withdrawal of the drop whereas larger ones
(63 – 200 mm) have no effect. As the diameter of the micropylar canal of J. communis is about 70 mm, the absence of
the effect of the larger particles (63 – 200 mm) may be due
to their inability to enter the micropylar canal. On the basis
of the current experiments, the conclusion of Tomlinson
et al. (1997) that inorganic material did not stimulate pollination drop withdrawal may have depended on the large
size of the particles used in their experiments (glass
Balloti spheres, diameter about 75 mm).
A statistically significant increasing trend of pollination
drop volume after deposition of silica gel in the size
range 63 – 200 mm emerges from the present observations.
However, it is necessary to consider that initial pollination
drop volumes in this treatment were much smaller than
average, suggesting that these cones were in the early
stages of secretion and would naturally tend to increase in
volume. Differences in the stage of secretion may explain
the high variability of pollination drop volume observed
at any moment.
The fact that stimulation of the drop by simple temporary
contact (such as touching with an eyelash or a dissecting
needle) does not cause withdrawal had already been
observed by Tomlinson et al. (1997) and could indicate
the need for prolonged mechanical stimulation to determine
withdrawal.
In the case of pollination with conspecific ( juniper pollen)
or heterospecific pollen (Pyrus and Pinus pollen), partial
withdrawal may be caused by pollen rehydration. This is particularly true for juniper pollen, which has very thick hygroscopic intine (Pacini et al., 1999; Nepi et al., 2005), and for
pine pollen, which, although not having thick intine relative
to pollen size (Pacini et al., 1999), increases greatly in
volume on hydration. In the case of silica gel, water molecules adhere to the surface but there is no ‘sponge effect’
with increase in particle volume. This phase therefore
seems to be independent of the hygroscopic capacity of the
particles that fall on the pollination drop.
The second phase determines total withdrawal and is
only completed if viable conspecific pollen lands on the
drop; this suggests the existence of a biochemical–molecular
mechanism that can be triggered only by biological particles, such as pollen, and that does not respond to the
stimulus of non-viable pollen, heterospecific pollen or abiological particles. Only the interaction between living conspecific pollen and the complex biochemical environment
of the pollination drop leads to total drop withdrawal and
subsequent pollen development. For some angiosperm
species several types of metabolites (heterogeneous proteins – most of which are enzymes – sugars, amino acids,
phenolics, polyamnines) are known to be released by
pollen and to interact with the stigma, style and ovary
tissues, favouring pollen germination and tube growth
(Shivanna, 2003, and references therein). What kind of
metabolites are released by gymnosperm pollen and how
they can interact with the pollination drop is not known.
CON C L U S IO NS
The present results show that the pollination drop of
Juniperus communis responds differently to stimuli determined by deposition of different particles. The ability to
recognize viable and non-viable pollen is striking.
Besides clarifying the mechanisms of pollination in
J. communis, the results also show a certain non-specificity
in the pollination biology of this species. The micropylar
pollination of gymnosperms, especially J. communis, is
less selective than the stigma pollination of angiosperms.
The latter have a series of tissues and organs (stigma,
style, ovary) that function as a ‘filter’ for pollen selection
(Wilhelmi and Preuss, 1999; Shivanna, 2003). We have
demonstrated that non-viable conspecific pollen, heterospecific pollen and also abiotic particles of an appropriate
size induce partial withdrawal of the pollination drop of
J. communis. Partial withdrawal of the pollination drop
leads to a decrease in the surface exposed for pollen
capture, reducing the probability of successful pollination.
Thus, deposition of non-viable pollen or inorganic particles
may reduce pollination efficiency in Juniperus by preventing viable pollen from reaching the surface of the nucellus
Mugnaini et al. — Pollination Drop in Juniper
and may have major ecological implications. We intend to
test this hypothesis on natural populations of Juniperus
growing near sources of airborne dust, such as quarries,
cement plants and roads. According to the laboratory experiments, the reproductive efficiency (i.e. the number and
quality of seeds produced) of these populations should be
lower than that of control populations growing far from
sources of dust.
AC KN OW L E DG E M E N T S
We are grateful to Dr Francesco Pezzo for his suggestions
on statistical analysis. We thank the Environmental
Protection and Technical Services Agency (APAT, Rome,
Italy) for funding.
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