SCIENTIFIC CORRESPONDENCE Explosive Discharge of Pollen Tube Contents in Torenia fournieri 1[w]

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 mm in diameter, discharges its contents explosively at an initial rate of about 12,000 mm s, 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 6 5,800 mm s (Fig. 2; n 5 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). The breakdown of the plasma membrane of one synergid cell occurred 0.6 6 0.6 s after the start of discharge (n 5 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 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 [email protected]. ac.jp; fax 81–3–3814 –1408. Plant Physiology, January 2000, Vol. 122, pp. 11–13, www.plantphysiol.org © 2000 American Society of Plant Physiologists