Home > Press Releases
JST Press Release

August 22, 2014
Japan Science and Technology Agency (JST)
5-3, Yonbancho, Chiyoda-ku, Tokyo 102-8666
URL http://www.jst.go.jp/EN/index.html

Calcium dynamics during fertilization was caught in the deep tissue
-Study on flowering plant fertilization moves forward-

Calcium is well known as a signal transduction molecule. In animals, membrane fusion process between egg cell and sperm cell, in other words, fertilization causes calcium spike and its oscillation in the egg cell. On the other hand, calcium dynamics during more complex fertilization process which involves not only egg cell but central cell fertilization in flowering plants were not investigated well. We succeeded in imaging cytosolic calcium in these two cells, and in the two synergid cells that are important for pollen tube attraction. A calcium sensor protein called YC3.60 expressed in each cell was used to observe calcium dynamics during semi-in vivo fertilization (Figure 1) under high-sensitive laser microscopes. Following pollen tube discharge and membrane fusion the egg and central cells showed transient calcium spikes, but not oscillations. Only the second calcium spike in the egg cell correlated with the membrane fusion (Figures 2 and 3). By contrast, the synergid cells displayed calcium oscillations upon pollen tube arrival (Figure 4). These calcium dynamics in the fertilization responsible cells seem to represent highly specific signatures that coordinate successful fertilization in the flowering plants (Figure 5).

Figures

Figure 1 Diagram of Arabidopsis flower, fertilization process and semi-in vivo fertilization assay

(a) A pollen grain containing the two sperm cells. It produces a pollen tube, which arrives at the ovule. After the arrival, one of the synergid cells degenerates and the pollen tube bursts to discharge its contents. One sperm cell fuses with the egg cell to form the embryo, and the other fuses with the central cell producing the endosperm. (b) Fertilization process is observed by semi-in vivo fertilization assay, wherein pollen tubes growing through a pistil are attracted by excised ovules.

Figure 2 Calcium dynamics in egg cells during fertilization

(a) Upper panels show representative calcium concentration. The square indicates the region shown in the lower panels. Lower panels show sperm cell nuclei and egg cell plasma membrane in Red, egg cell cytosol in yellow. Arrowheads indicate sperm cell nuclei. Asterisk shows the timing of egg cell fertilization. Scale bar, 10 m. (b) Time course of the calcium concentration change at the egg cell shown in (a).

Figure 3 Relationship between second calcium spike in egg cell and sperm cell fusion to the egg cell

(a) Upper panels show representative calcium concentration. The square indicates the region shown in the lower panels. Lower panels show sperm cell nuclei and egg cell plasma membrane in Red, egg cell cytosol in yellow. Arrowheads indicate sperm cell nuclei. Asterisk shows the timing of egg cell fertilization. Scale bar, 10 m. (b) Time course of the calcium concentration change at the egg cell shown in (a).

Figure 4 Calcium dynamics in two synergid cells during fertilization

Time course of the calcium concentration change in the two synergid cells.

Figure 5 Diagram of calcium dynamics in four cells responsible for double fertilization

The egg cell indicates two distinct calcium spikes during fertilization process. The first spike correlated to pollen tube discharge and the second spike corresponded to the timing of its own fertilization. In the central cell, the first calcium spike correlated to pollen tube discharge as well as the egg cell. But after that, the second calcium spike had not strong correlation with egg cell and central cell fertilizations. About half of our observation didnt detect any calcium spikes after pollen discharge. By contrast, both synergid cells showed calcium oscillation after pollen tube arrival, but before pollen tube discharge, with the receptive synergid cell showing superior calcium elevation. Upon pollen tube discharge a strong calcium spike was observed in both synergid cells. An onset of calcium oscillation in the persistent synergid cell following double fertilization was also observed.

Journal Information

Author: Yuki Hamamura, Moe Nishimaki, Hidenori Takeuchi, Anja Geitmann, Daisuke Kurihara and Tetsuya Higashiyama
Title: “Live imaging of calcium spikes during double fertilization in Arabidopsis
Nature Communications, Published online 22nd August 2014
doi: 10.1038/ncomms5722

Contact

[About Research]
Tetsuya Higashiyama Ph.D.
Director of ERATO Higashiyama Live-Holonics Project
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University
E-mail:
HP: http://www.liveholonics.com/en/

[About Program]
Tsuyoshi Nakamura
Department of Research Project, JST
E-mail:
HP: http://www.jst.go.jp/erato/en/index.html

Japanese


JST, an integrated organization of science and technology in Japan, establishes an infrastructure for the entire process from the creation of knowledge to the return to the society. For more information, visit http://www.jst.go.jp/EN/