Behavior of Male Gamete Fusogen GCS1/HAP2 and the Regulation in Arabidopsis Double Fertilization.

In the sexual reproduction of flowering plants, two independent fertilization events occur almost simultaneously: two identical sperm cells fuse with either the egg cell or the central cell, resulting in embryo and endosperm development to produce a seed. GCS1/HAP2 is a sperm cell membrane protein essential for plasma membrane fusion with both female gametes. Other sperm membrane proteins, DMP8 and DMP9, are more important for egg cell fertilization than that of the central cell, suggesting its regulatory mechanism in GCS1/HAP2-driving gamete membrane fusion. To assess the GCS1/HAP2 regulatory cascade in the double fertilization system of flowering plants, we produced Arabidopsis transgenic lines expressing different GCS1/HAP2 variants and evaluated the fertilization in vivo. The fertilization pattern observed in GCS1_RNAi transgenic plants implied that sperm cells over the amount of GCS1/HAP2 required for fusion on their surface could facilitate membrane fusion with both female gametes. The cytological analysis of the dmp8dmp9 sperm cell arrested alone in an embryo sac supported GCS1/HAP2 distribution on the sperm surface. Furthermore, the fertilization failures with both female gametes were caused by GCS1/HAP2 secretion from the egg cell. These results provided a possible scenario of GCS1/HAP2 regulation, showing a potential scheme for capturing additional GCS1/HAP2-interacting proteins.

Behavior of filamentous temperature-sensitive Z2 (FtsZ2) in the male gametophyte during sexual reproduction processes of flowering plants.

Filamentous temperature-sensitive Z (FtsZ) is a critical division protein in bacteria that functions in forming a Z-ring structure to constrict the cell. Since the establishment of the plastid by endosymbiosis of a cyanobacterium into a eukaryotic cell, division via Z-ring formation has been conserved in the plastids of flowering plants. The FtsZ gene was transferred from the cyanobacterial ancestor of plastids to the eukaryotic nuclear genome during evolution, and flowering plants evolved two FtsZ homologs, FtsZ1 and FtsZ2, which are involved in chloroplast division through distinct molecular functions. Regarding the behaviors of FtsZ in nonphotosynthetic cells, the plastid localization of FtsZ1 proteins in the cytoplasm of microspores and pollen vegetative cells but not in generative cells or sperm cells has been reported. On the other hand, the significant accumulation of FtsZ2 transcripts in generative cells has been reported. However, the synthesis of FtsZ2 in the male gamete has not been investigated. Additionally, FtsZ2 behavior has not been analyzed in pollen, a nonphotosynthetic male tissue. Here, we report FtsZ2 protein behaviors in the male gamete by analyzing the localization patterns of GFP-fused protein at various pollen developmental stages and in gametes during the fertilization process. Our results showed that FtsZ2 localization coincided with that of plastids. FtsZ2 protein in male gametes was almost absent, despite the presence of the transcripts. Moreover, transmission of paternal FtsZ2 transcripts to the zygote and endosperm was not observed.