Studies On Dynamics Of Actin Cytoskeleton In The Embryo Sac Of Torenia Fournieri During Fertilization

The research on the cytoskeleton has become a hotspot for the biologists, and in recent years great achievements have been made in this field. Embryo sacs are usually surrounded by layers of nucellus, integument and ovary tissues. This poses great difficulty for the direct observation of the female gametophyte. Hence, information concerning actin organization and changes during double fertilization is scarce. While there is such a kind of plant which has a semi-naked embryo sac, it is a excellent material to study the mechanism of fertilization in angiosperm because of the distinct advantage it owns. We studied actin dynamics in the embryo sacs of Torenia fournieri during development and fertilization.By meliorating the fixation metho we succeeded in labeling the actin cytoskeleton in the embryo sac of Torenia fournieri and studied the actin dynamics during development and fertilization by confocal laser scanning microscopy. Before the completion of cellularization ,the actin filaments distributs asymmetrically in the embryo sac just as short bundles; lots of actin bundles aggregates in the micropylar part of the embryo sac. From anthesis to 2 days after flower opening , actin filaments mainly distributes lengthways in the periphery of the central cell, and they become thicker and denser as the embryo sac became mature. The actin filaments in the central cell become fragmented after pollination , it becomes more evident after the pollen tube enters the embryo sac, the actin filaments fragment into many dots and the quantity of the actin filaments obviously decrease. After the pollen tube discharges its contents, A fluorescent actin region appears in the chalazal end of the degenerated synergid. Concurrently, an actin corona also appers near the degenerated synergid. After fertilization the corona fade, the actin filaments in the endosperm reorganize into clear network. The F-actin in the degenerated synergid spreads and forms a new actin corona. When endosperm nucleus are moving to the chalazal part of the embryo sac, the actin filaments in the endosperm are reorganizing; the endosperm nucleus are surrounded by dense actin filaments and there are some long actin filaments connect the endosperm nucleus and the chalazal part of the embryo sac. The actin cytoskeleton in the egg cell changes greatly after pollination. Before pollination there are lots of ultrashort actin bundles gather in the cortex of the egg cell. After pollination the intensity of F-actin fluorescence decreases greatly, there are only a few actin filaments in the cortex of the egg cell and actin cytoskeleton mainly organizes into patches in the cortex of the egg cell.Labeling actin cytoskeleton with GFP-talin is a new technique developed in recent years. This method can study the actin dynamics in living cells without doing any harm to the normal shape and function of the cell, so it is a rather perfect method. Since there is no any report about organ specific promoters of Torenia fournieri, we cloned two promoters ACT11 and AtSERK1 which can express well in the embryo sac of Arabidopsis, and constructed the plant expression vectors by fusing them with the chimeric gene GFP-talin . This provides a basic work for study the actin dynamics and functions in the living embryo sacs of Torenia fournieri during development and fertilization.