Plant Physiol, January 2000, Vol. 122, pp. 11-14
Explosive Discharge of Pollen Tube Contents in
Torenia fournieri1,[w]
Tetsuya
Higashiyama,*
Haruko
Kuroiwa,
Shigeyuki
Kawano, and
Tsuneyoshi
Kuroiwa
Department of Biological Sciences, Graduate School of Science
(T.H., H.K., T.K.) and Department of Integrated Biosciences, Graduate
School of Frontier Sciences (S.K.), University of Tokyo, Hongo,
Tokyo 113-0033, Japan.
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ARTICLE |
When animals copulate, the male
organ penetrates the female. Similarly, the pollen tube of a flowering
plant (the male gametophyte) penetrates the embryo sac (the female
gametophyte) and then discharges its contents, which include male
gametes. Details of the interaction between the pollen tube and the
embryo sac are difficult to observe because of the large number of
opaque ovular cells that usually enclose the embryo sac. Although 100 years have passed since the discovery of double fertilization in
flowering plants (Nawaschin, 1898
), the discharge of male contents from
the pollen tube has never been observed. We have succeeded in observing
the discharge from pollen tubes directly in vitro by using the naked
embryo sac of Torenia fournieri, which protrudes from the
micropyle of the ovule. We were able to watch how the pollen tube,
which is only 10 µm in diameter, discharges its contents explosively
at an initial rate of about 12,000 µm3
s
1, with the resultant almost instantaneous
breakdown of one of two synergid cells adjacent to the egg cell.
We recently established an in vitro system for observing the guidance
of pollen tubes using the naked embryo sac of T. fournieri (Higashiyama et al., 1998). However, the frequency
of the discharge of male gametes by the pollen tubes was too low for us
to observe the discharge directly. We have improved our system by
adding 15% (w/v) polyethylene glycol 4000 to the culture medium (the amount of Suc is reduced from 5% to 1% [w/v] to maintain
appropriate osmotic pressure). This modification supports the higher
viability of cultures, with approximately 4-fold increases in the
frequency of guidance of pollen tubes, and allows successful
video-recording of the discharge process. In our series of
observations, 70% (23/33) of embryo sacs that had received the
contents of pollen tubes showed evidence both of early embryogenesis
and of endosperm development that resembled events in vivo (Higashiyama
et al., 1997), suggesting that fertilization proceeded normally.
After its arrival at a target embryo sac, a pollen tube enters the
embryo sac at the micropylar end, thrusting its way between two
synergid cells (Fig. 1). The pollen tube
then ruptures at its tip and begins to spout its contents explosively
(Fig. 1, 0.0 s). When only rapidly moving materials were
visualized by an image-subtraction method (Fig. 1, lower panels),
female gametophytic cells and their organelles near the pollen tube
were also seen to be shaken by the impact of the discharge. The initial
rate of discharge was estimated to be 12,000 ± 5,800 µm3 s1 (Fig.
2; n = 6). This rate
corresponded to a rate approximately 50 times higher than that of
cytoplasmic streaming in the pollen tube before discharge. The rate of
discharge decreased rapidly during the first 0.1 s. Thereafter,
however, the contents flowed into the embryo sac at an almost constant
rate (Fig. 2).
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Figure 1.
Discharge of the contents of a pollen tube into
the embryo sac. The top panels show a series of sequential
light-microscopic images. The time after the start of discharge is
indicated at the upper left of each photograph. Arrows indicate the
expanding contents of the pollen tube in the embryo sac. The bottom
panels show subtraction images that reveal only rapidly moving
materials at each time point. Subtraction was performed according to
the following formula:
where F0 refers to each frame
shown in the top line, and three frames correspond to 0.1 s.
Spectral colors correspond to the output of each pixel, with
red representing the highest level. A small arrow indicates the
rupturing plasma membrane of a synergid cell. Photoshop (Adobe Systems,
Mountain View, CA) was used for the calculation of images. Bar
represents 10 µm. ec, Egg cell; es, embryo sac; pt, pollen tube; sy,
synergid cell. A video of this process can be seen at
http://www.plantphysiol.org.
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Figure 2.
Rate of discharge of the contents of a pollen tube
into the embryo sac. The instantaneous rate (with SD) in
each video frame was calculated from the distance moved by the contents
(organelles) of the pollen tube and the diameter of the tube
(n = 6). The dotted line before time 0.0 indicates
the average rate of cytoplasmic streaming in the pollen tube. The
average diameter of pollen tubes was 10 ± 1 µm.
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The breakdown of the plasma membrane of one synergid cell occurred
0.6 ± 0.6 s after the start of discharge (n = 6; Fig. 1, 1.8 s: note that female gametophytic cells and their
organelles are shaken again). It is impossible to monitor such an
exceedingly rapid sequence of events using present methods for the
fixation of ovules. In general, in flowering plants, the contents of a pollen tube are received by the selectively degenerated synergid cell
for further transport of non-motile male gametes (Russell, 1993). The
receptive synergid cell begins to degenerate before arrival of the
pollen tube in many plant species. However, the exact timing of the
breakdown of the synergid cell is still a matter of considerable debate
(Jensen, 1973; Russell, 1996). Our observations suggest that, in
T. fournieri, the discharge from the pollen tube triggers
the breakdown of the receptive synergid cell instantaneously. It is
possible that the rapid accumulation of the discharged contents of the
pollen tube in the embryo sac increases the physical pressure within
the sac and ruptures the somewhat degenerating synergid cell
selectively. The rate of discharge decreased gradually and the
discharge ceased in approximately 1 min; the other synergid cell
remained persistent at least for a few days.
The two synergid cells have been implicated in pollen tube guidance.
The synergid cell is morphologically active in secretory functions
(Huang and Russell, 1992) and contains a relatively high
concentration of calcium (Chaubal and Reger, 1992), which can
potentially control the direction of pollen tube growth (Malhó, 1998). When one of two synergid cells is broken during cultivation of
ovules of T. fournieri, pollen tubes are rarely guided to
such an ovule (Higashiyama et al., 1998). In our observations, two synergid cells remained intact at the start of discharge by the pollen
tube. It seems possible that both synergid cells actively secrete
chemoattractants of pollen tubes until a tube reaches the synergid
cells. Once the pollen tube begins to discharge, the receptive synergid
cell might immolate itself for the succeeding transport of male gametes
to their target female gametes.
We are now able to watch the discharge of pollen tube contents directly
in vitro. For further investigations in our system, it will be useful
to be able to observe the behavior of two sperm cells. It is unknown
how non-motile sperm cells of flowering plants can fertilize with their
target female gametes
in the embryo sac, in spite of the great interest in this issue.
It has been proposed that the two sperm cells are transported along
actin bundles in the embryo sac by actomyosin interactions (Russell,
1996; Zhang and Russell, 1999). We are now trying to establish a
staining method of sperm cells in living pollen tubes to directly
observe how two sperm cells are discharged into the embryo sac and
fertilize with their respective target female gametes.
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FOOTNOTES |
Received September 9, 1999; accepted September 25, 1999.
1
This work was supported by a research fellowship
to T.H. (no. 4770) from the Japan Society for the Promotion of Science
for Young Scientists and by grants both for Specially Promoted Research (project no. 06101002 to T.K.) and for Scientific Research in Priority
Areas (no. 11163206 to T.K.) from the Ministry of Education, Science
and Culture of Japan.
[w]
The online version of this article contains Web-only
data for Figure 1. This version is available at
www.plantphysiol.org.
*
Corresponding author; e-mail higashi{at}biol.s.u-tokyo.ac.jp; fax
81-3-3814-1408.
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LITERATURE CITED |
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Chaubal R, Reger BJ
(1992)
Sex Plant Reprod
5: 34-46
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Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T
(1997)
Planta
203: 101-110
[CrossRef][ISI]
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Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T
(1998)
Plant Cell
10: 2019-2031
[Abstract/Free Full Text]
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Huang BQ, Russell SD
(1992)
Int Rev Cytol
140: 233-293
[ISI]
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Jensen WA
(1973)
BioScience
23: 21-27
[CrossRef][ISI]
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Malhó R
(1998)
Sex Plant Reprod
11: 242-244
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Nawaschin S
(1898)
Bull Acad Imp Sci St Pétersbourg
9: 377-382
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Russell SD
(1993)
Plant Cell
5: 1349-1359
[Free Full Text]
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Russell SD
(1996)
Sex Plant Reprod
9: 337-342
[CrossRef]
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Zhang Z, Russell SD
(1999)
Planta
208: 539-544
[CrossRef]
© 2000 American Society of Plant Physiologists
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LITERATURE CITED
