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Phosphoinositide-specific phospholipase C (PI-PLC) is an important enzyme, which is a key player involved in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as environmental stress. However, little is known about its roles in signal transduction during sexual reproduction process. In this study, we cloned and characterized a gene of full-length PI-PLC from ovules of Torenia fournieri, designated as TfPLC1. It was 2,171 bp in length, including an open reading frame encoding a polypeptide of 583 amino acids with molecular mass of 66.02 kDa. The amino acid sequence deduced from the cDNA sequence shows 40–76% similarity to other plant PI-PLCs and contains the characteristic X, Y and C2 domains. Northern blot analysis demonstrated it was predominantly expressed in ovules and flowers. Furthermore, TfPLC1 promoter::GUS transgenic analysis indicated it specifically expressed in ovule, stigma and mature pollen grain. Immunohistochemical staining showed that, in mature stigma, TfPLC1 protein was principally localized in the cells of stigmatic receptive surface. Together, our data suggest that TfPLC1 may play an important role in plant sexual reproduction.

Microinjection of soluble sperm extract and Calcium Green-1 10 kDa-dextran conjugate (CG-1) into the mature central cell of Torenia fournieri induced a significant rise in cytosolic free calcium concentration ([Ca2+]i). The rise reached a maximum at 20 min after injection and then steadily declined. Nevertheless, a relatively high level of [Ca2+]i was maintained even 40 min after injection. Microinjection of sperm extract of maize into Torenia central cells, however, did not trigger any increase in [Ca2+]i, suggesting the possibility of distinct triggers in different species. We also injected caged inositol 1,4,5-triphosphate (InsP3) and caged cyclic ADP-ribose (cADPR) into Torenia central cells to compare the pattern of Ca2+ rise induced by the sperm extract. The results showed that [Ca2+]i elevation triggered by the release of InsP3 after photolysis appears much faster than that induced by sperm extract. The increase in [Ca2+]i reached a maximum at 70–80 s and dropped to the resting level within 300 s after photolysis. Microinjection of cADPR, however, did not induce any changes in [Ca2+]i. The results indicate that sperm extract might contain factors triggering the release of Ca2+ in the central cell.

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes.

A new method is reported to isolate and handle living megagametophytes of Torenia fournieri at any developmental stage. The stages were determined using light microscopy and delimited by correlating floral morphological traits. When significant changes in the osmotic pressure were found during development, enzyme solutions contained different concentrations of osmoticum. Osmotic pressure is lowest in the megaspore, increases until the four-nucleate stage and then gradually decreases until complete embryo sac formation. In enzymatic solutions containing appropriate concentrations of osmoticum, protoplasts of megaspores, two-, four-, eight-nucleate embryo sacs, egg cells, synergids and central cells were successfully isolated. The living protoplasts were collected by micromanipulator, transferred into microdroplets and tested for viability.

New methods were developed to isolate the male gametophyte of Torenia fournieri at any developmental stage. The stages were defined by light microscopic studies and identified by correlating morphological traits of the flower buds. Enzyme solutions were used to isolate gametophytic cells. Preliminary studies were carried out to determine the components of the cell wall, and the optimal osmotic pressures of the appropriate enzyme solutions were adjusted to the different developmental stages. We managed to isolate diploid microsporocytes, haploid microspores, cells of young and mature pollen grains, and sperm cells from growing pollen tubes. Isolated protoplasts were collected in microcapillaries to prepare them for further studies.