Molecular dissection of asexual reproduction in the model fern Ceratopteris richardii

The life cycle of all land plants is characterized by the alternation between two multicellular generations: haploid gametophytes and diploid sporophytes. Whereas meiotic recombination and fertilization via sexual reproduction is known to confer adaptive evolutionary benefits, some plant species can reproduce asexually bypassing meiosis and fertilization yet still undergo alternation of generations. In ferns, the two asexual reproductive pathways are termed apogamy and apospory, i.e. the asexual reproduction of sporophytes and gametophytes, respectively. Although understanding the process of asexual reproduction in flowering plants would be of considerable agricultural value, the molecular mechanisms underlying this process have yet to be identified. Similarly, the genes in ferns that underlie apogamy and apospory are unknown. Knowledge of such genes will provide invaluable information not only for the evolutionary study of asexual reproduction in land plants, but also insight into the developmental program for each generation. My PhD research focuses on the investigation of candidate genes involved in asexual reproduction in the model fern Ceratopteris richardii.

Though the molecular mechanisms underlying asexual reproduction are not understood, it is hypothesized that asexual reproduction is the result of altered regulation of certain sexual reproduction-related genes. Therefore, I selected candidate genes for asexual reproduction in C. richardii, CrEMS1, CrKNOX3 and CrANT, based on the functions of their respective homologs during sexual reproduction in two other model systems: Arabidopsis thaliana and Physcomitrella patens. I showed that, in C. richardii, CrEMS1 is expressed during key events of sexual reproduction, including sporogenesis, gametogenesis and embryo development; CrKNOX3 expression is specific for the mature egg cells and CrANT expression is restricted to sperm cells. The presence of CrEMS1 during early embryo development made it a suitable candidate for an embryonic marker for future research.

To study the function of the candidate genes, I overexpressed CrKNOX3 and BnBBM, a flowering plant BBM gene that has similar expression patterns in Brassica to CrANT. The overexpression of CrKNOX3 results in unique cell outgrowth from the antheridia, and overexpression of BnBBM results in spontaneous apogamy induction. Whole mount in situ also showed CrEMS1 expression, marking embryonic identity, in pre-apogamous cells undergoing sugar-induced and overexpression of BnBBM-induced apogamy.

The results of my PhD research provides, for the first time, insight into the molecular mechanisms underlying asexual reproduction in the model fern C. richardii, and invaluable tools, transient and stable Agrobacterium-mediated transformation protocols, for functional genomics research in this fern. The spontaneous apogamy induction on BnBBM-expressing gametophytes and the presence of CrEMS1 in pre-apogamous cells confirm the hypothesis that asexual reproduction is the result of the alteration in expression of sexual-related genes, verifying that the two pathways share a common set of genes.