Intermediate Evolutionary State of Motile Sperm and Pollen Tubes in the Extant Gymnosperm Cycas revoluta

Abstract

The transformation of male reproductive cells is a significant event in the evolution of land plant fertilization systems from zooidogamy to siphonogamy. Basal plants such as bryophytes and pteridophytes have motile sperm, whereas most seed plants have non-motile sperm, delivered by a pollen tube. Despite being seed plants, gymnosperm cycads and ginkgo uniquely form highly multi-flagellated and large motile sperm within pollen tubes. However, the evolutionary state of these male reproductive cells remains unknown. We clarified the gene expression profiles of Cycas revoluta pollen tubes and motile sperm that swam toward female reproductive cells. Male cycad cells had fewer genes associated with transcription, translation, and related processes, which is consistent across land plants. Similar to other male gametes, a sperm-specific histone variant may contribute to transcriptional regulation via chromatin condensation. Plasma membrane-localized proteins probably involving male-female interactions revealed that cycad sperm express both factors found in motile plant sperm and seed plant sperm cells, suggesting a transitional state from motile to non-motile male gametes. In contrast, cycad pollen tubes lacked plasma membrane-localized receptor genes previously reported in angiosperms and cell wall-associated factors involved in pollen tube tip growth. These results support the hypothesis that the function of the cycad pollen tube is confined to the haustoria as previously reported. These results indicate a molecular intermediate state of the cycad fertilization mechanism with motile sperm and pollen tubes, providing important insights into the evolution of land plant fertilization. Significant StatementCycads and ginkgo, which possess primitive motile sperm and derived pollen tubes, are important for understanding the evolution of land plant fertilization from zooidogamy to siphonogamy. We explored gene expression in living, capacitated motile sperm and pollen tubes of Cycas revoluta in a tissue-specific transcriptome analysis. Similar to other land plants, the transcriptional repression mechanism has been suggested in these male cells. Plasma membrane-localized genes revealed the characteristics of motile and non-motile sperm, while molecular evidence of pollen tube guidance and tip growth, as seen in angiosperms, was lacking in cycads. A molecular intermediate state of the cycad fertilization mechanism was clarified, providing important insights into the evolutionary process of land plant fertilization.