Live Imaging and Laser Disruption Reveal the Dynamics and Cell-Cell Communication During Torenia fournieri Female Gametophyte Development.

The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.