Title: Reproduction and Seed Development in the Water Lily Nymphaea thermarum – a New Perspective on the Evolution of Flowering Plant Seeds
Abstract: Almost a century of research connects the origin of double fertilization, a major evolutionary innovation of flowering plants (angiosperms), to conflicting parental interests over offspring provisioning during seed development. Because endosperm, the product of the second fertilization event, separates its compatriot embryo (nutrient sink) from the maternal sporophyte (nutrient source) for most of seed development, it is a de facto mediator of nutrient dynamics within a seed. Reciprocal interploidy crosses in a phylogenetically diverse array of lineages have demonstrated that endosperm development and function is subject to parent-of-origin effects stemming from the relative dosage of maternal and paternal genomes. Furthermore, work in a small handful of model systems has revealed that imprinting, an epigenetic phenomena based on chromatin methylation patterns, underlies key components of parent-of-origin effects on seed development. However, neither reciprocal interploidy crosses, nor studies of chromatin methylation, have been performed for any member of the early-diverging angiosperm lineages – hindering our understanding of the evolutionary relationships between interparental conflict, parent-of-origin-effects on seed development, and chromatin methylation. For my dissertation I identified and leveraged the experimental tractability of the water lily Nymphaea thermarum to perform reciprocal interploidy crosses and to document patterns gene expression related to chromatin methylation during reproductive development.
In Chapter 1, we describe the floral biology, female reproductive development, fertilization, and seed ontogeny of N. thermarum, and thereby provide knowledge necessary for further experiments. Flower morphology, with emphasis on the timing of male and female functions, is correlated with key developmental stages of the ovule and the female gametophyte. Development of the seed tissues and dynamics of polysaccharide reserves are also examined. We find that early male and female function indicates that N. thermarum is predisposed towards self-pollination - a phenomenon that is likely to have evolved multiple times within Nymphaea. While we observe other characters common in the Nymphaeales (the formation of distinct micropylar and chalazal developmental domains in the endosperm, and copious perisperm – a storage tissue derived from the maternal sporophyte), we note seed development varies between and among lineages with the Nymphaeales, in a manner that indicates that endosperm ontogeny is intimately related to nutrient dynamics within the seed.
In Chapter 2, we create autotetraploid lines of N. thermarum, and use them (and diploid lines) to perform reciprocal interploidy crosses. Seed component sizes and embryo morphogenesis are compared between crosses over the course of ovule maturation and seed development. We find that changes to maternal and paternal genome dosage alter the development of offspring tissues, but have little impact on nutrient allocation into the seed. Rather, the size of the perisperm (a maternally-derived nutrient storage tissue) is exclusively influenced by maternal ploidy. Altogether, we provide the first evidence that parent-of-origin effects on offspring development may date to the evolutionary origin of flowering plants. We also demonstrate that that the evolutionary transfer of embryo-nourishing function from a genetically biparental endosperm to a genetically maternal perisperm can function as a maternal strategy to recapture control of resource distribution among progeny.
In Chapter 3, we sequence transcriptomes of whole ovules and seeds from three key stages of reproductive development in the waterlily N. thermarum. We first explore general patterns of gene expression, beginning with mature ovules and continuing through fertilization into early- and mid-seed development. We then examine the expression of genes associated with DNA and histone methylation – processes known to be essential for seed development, but variable in terms of mechanistic detail in distantly-related monocots and eudicots. Around 60% of genes putatively homologous to chromatin methylation modifiers are differentially expressed during seed development in N. thermarum, suggesting that the importance of dynamic chromatin methylation in seed development may date back to the earliest phases of angiosperm evolution. However, genes involved in establishing, maintaining, and removing methylation marks associated with genetic imprinting show a mix of conserved and unique expression patterns between N. thermarum and other angiosperms, providing perspective on how the regulation of imprinting has changed throughout angiosperm evolution.