Project Outline

Like animals, plants reproduce through having sex- namely, the fusion of haploid male and female sex cells. Although this process is superficially similar between animals and plants, the two lineages each invented it separately as a form of convergent evolution¹.

As with animals, the male and female sex cells of plants (for convenience, also called sperm and eggs) are produced by meiosis, halving the cell’s genetic material to create haploid cells and allowing recombination (shuffling of sections between chromosomes) to occur. In modern plants we are used to thinking about ‘male’ meiosis a flower’s stamens (to generate sperm-carrying pollen) and ‘female’ meiosis in the flower gynaecium (to generate the egg-carrying embryo-sac). Plant reproduction has two key differences from animals:

1. In most plants their sex is not genetically predetermined, producing both male and female reproductive organs in a context-dependent manner.
2. The single haploid cells produced by meiosis do not immediately become sex cells, but divide by mitosis into multicellular haploid tissues that produce the sex cells. In flowering plants development of this haploid ‘generation’ follows predetermined genetic programmes.

Genes that control male and female meiosis in plants have mostly been identified through studies in the model flowering plant, Arabidopsis thaliana². Many genes are similar between them, but some are sex-specific and so are thought to determine ‘male’ or ‘female’ identity. However, older plant groups (the mosses, clubmosses and ferns) use a different, older form of plant reproduction, from which the modern sexual mechanisms of plants evolved. Critically, this ancestral mechanism had only type of meiosis1, leading to a single type of haploid organism that makes both male and female sex cells. This ancient mechanism is called homospory. How separate male and female meiosis (heterospory) evolved from this remains a complete mystery, but is one of the fundamental first steps in how plants first evolved seeds³. This project aims to investigate how heterospory first evolved, using a new genetic model in one of these homosporous plant groups- the fern Ceratopteris richardii⁴. This project will attempt to answer this question using primarily ‘wet lab’-based experiments:

• Characterise the process of meiosis in a homosporous fern by advance microscopy for the first time. To do so, we will use different DNA staining techniques, a BrdU time course and immunolocalization of meiotic specific proteins using antibodies raised from plants.
• Identify the genes expressed during fern meiosis through RNA-seq and other molecular methods (qPCR, Fluorescent In Situ Hybridization), and test what their role in fern meiosis is through fern genetic engineering.
• Test to see whether fern meiosis is more similar to male or female meiosis in Arabidopsis by comparing them through bioinformatics.
• Test to see if genes found in fern meiosis can have the same function in Arabidopsis male and/or female meiosis through genetic engineering of Arabidopsis, swapping out the Arabidopsis gene for the fern copy.

References

1. Niklas KJ, Kutschera U. (2009). The evolution of the land plant lifecycle. New Phytologist 185: 27-41. https://doi.org/10.1111/j.1469-8137.2009.03054.x
2. Wang Y, Copenhaver GP (2018). Meiotic recombination: Mixing it up in plants. Annual Review of Plant Biology 69: 577-609. https://doi.org/10.1146/annurev-arplant-042817-040431
3. Linkies A, Graeber K, Knight C, Leubner-Metzger G. (2010). The evolution of seeds. New Phytologist 186: 817-831. https://doi.org/10.1111/j.1469-8137.2010.03249.x
4. Plackett ARG, Huang L, Sanders HL, Langdale JA (2014). High-Efficiency Stable Transformation of the Model Fern Species Ceratopteris richardii via Microparticle Bombardment. Plant Physiology 165: 3-14. https://doi.org/10.1104/pp.113.231357

Techniques

• Genetic transformation of two plant genetic models (Arabidopsis thaliana and Ceratopteris richardii) using two separate plant transformation techniques (Agrobacterium-mediated transformation and microparticle bombardment)
• Manipulation of candidate gene function through functional knockout (RNAi or CRISPR) and heterologous expression of candidate genes.
• Detailed phenotypic analysis of transgenic lines, with associated statistical analysis where appropriate.
• A wide range of molecular biology techniques to build transgenic constructs (gene cloning and DNA assembly), genotype plant backgrounds (DNA purification and PCR) and examine candidate gene expression, (RNA extraction, quantitative real-time PCR and next-generation sequencing).
• Brightfield and advanced fluorescence microscopy to characterize the stages of fern meiosis.
• DNA staining via Fluorescent in situ hybridization (FISH) to locate chromosome regions and/or whole chromosomes in Ceratopteris during meiosis, and Bromodeoxyuridine (BrdU) staining assays to study DNA replication during meiosis.
• Immunolocalization of meiotic specific proteins using plant specific antibodies raised during the last decades at the lab.
• Bioinformatic and phylogenetic analysis