Reconstructing the evolution of gene function for a master regulator of flowering: LEAFY in the fern Ceratopteris richardii

Abstract

All land plants have a life cycle with alternation of generations that includes both a haploid, gamete-producing gametophyte and a diploid spore-producing sporophyte. Over evolutionary time, land plants have gone from having independent, longer-living gametophytes with ephemeral, dependent sporophytes, as in the bryophytes, to large, independent sporophytes which support small, dependent, short-lived gametophytes in the seed plants. A critical angiosperm innovation is the production of flowers, which enclose unisexual gametophytes within the sporophyte. The evolution of the flower has been hypothesized to have occurred through a combination of cooption of genes present in the seed plant common ancestor, and expansion of gene families involved in flower development. A common approach to learning about the evolution of flowering genes is to investigate their homologs in representatives of earlier diverging lineages of non-flowering plants. For instance, ferns represent a lineage in a key phylogenetic placement representative of early-diverging vascular plants and sister to all seed plants, with independent and photosynthetic gametophytes and sporophytes. In more derived lineages, where gametophytes are small and dependent, studying the dependent stage is more challenging and less informative, leaving open questions about the role of genes of interest across both life stages. In this dissertation, I focus on the homosporous fern Ceratopteris richardii (C. richardii), to investigate gene function across the plant life cycle. This research aims to broadly inform understanding of the functional evolution of key reproductive genes in land plants. The goal of Chapters 1 and 2 is to investigate the role of the floral meristem identity gene LEAFY (LFY) in C. richardii gametophytes and sporophytes. My work uncovered new roles for the two C. richardii LFY paralogs, CrLFY1/2, in gametophyte reproduction, particularly in sperm cells and in the lateral meristem that generates gametangia. In the sporophyte, I found a conserved function in early embryogenesis, in the first division of the fern zygote, a role previously described for a moss species but not in any vascular plant. Together, these findings highlight a novel haploid reproductive function for LFY homologs, suggesting that this may have been an ancestral role that was coopted by sporophytes as they became the dominant stage of the plant life cycle. In Chapter 3, I investigate the developmental genetics of the fern gametophyte. C. richardii gametophytes develop as either hermaphrodites, with a multicellular meristem that produces both egg (in archegonia) and sperm (in antheridia), or as ameristic males (developing from a transient apical cell), which produce only sperm. The first spores to germinate develop into hermaphrodites, which secrete the hormone antheridiogen, inducing surrounding gametophytes to develop as males. By comparing transcriptomes of mature male and hermaphrodite gametophytes via RNA-seq, I find a total of 22,719 expressed genes, over half of which (12,424) are differentially expressed between the two sexes, 47.8% upregulated in males and 52.2% upregulated in hermaphrodites. Gene ontology analysis indicated that microtubule genes have high mutual correlation to CrLFY1, suggesting a potential novel role of CrLFY1 in regulating aspects of sperm development.