Explosive Discharge of Pollen Tube Contents inTorenia fournieri

• Copyright © 2000 American Society of Plant Physiologists

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 μm³s⁻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 s⁻¹ (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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Fig. 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 F ₀ 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 athttp://www.plantphysiol.org.

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Fig. 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.

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, inT. 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.

• Received September 9, 1999.
• Accepted September 25, 1999.