Epigenetic regulation of sexual lineage development in plants

A key characteristic of life is the ability to reproduce. Reproductive strategies are major contributors to evolutionary fitness and can vary substantially between species. Like humans, most flowering plants reproduce sexually by mating with another individual; however, unlike humans, plants typically possess both male and female organs, and many plant species, including major crops, are capable of self-fertilization. Sexual reproduction in flowering plants is important to mankind as it produces the seeds that comprise most of our staple food. With decreasing arable land, an exploding population and global climate change, feeding the world in the 21st century will require a step-change in the efficiency of seed production, and this can only come from a deeper understanding of plant reproductive development.

Sexual reproduction in plants is carried out by two highly specialized families of cells, here called the male and female sexual lineages (SLs). A fundamental but still unresolved question that has always fascinated me is how SL function and fate are installed and maintained precisely in these cell lineages. My DPhil and postdoctoral studies focused on how genetic and 'epigenetic' pathways contribute to SL function and fertility. 'Epigenetic' regulation - such as DNA methylation - is named after its ability to alter gene expression by modifying the state of DNA without changing its genetic sequence. Recently, I discovered that the RNA-directed DNA methylation (RdDM) pathway regulates SL development in Arabidopsis plants by controlling the expression of several hundred genes. Consistent with the importance of the RdDM pathway in SL development, its mutations cause defects in SL development in both Arabidopsis and maize.

My proposed research integrates plant developmental, molecular, genetics and epigenetics biology to investigate how RdDM installs reproductive function and fate in the male SL of the model plant Arabidopsis thaliana. To detect changes in the DNA methylation and gene expression, I have developed state-of-the-art techniques such as fluorescence-activated cell sorting and micromanipulation to isolate all types of male SL cells to high purity. I will use whole-genome sequencing of these SL cells from RdDM mutants to pinpoint the function of the SL-specific RdDM pathway, and a combination of genetics and developmental biology to determine how the genes controlled by the SL-specific RdDM regulate SL development. Finally, through a combination of genomics, developmental biology and mutant screens, I will decipher the mechanism by which RdDM is directed to genes in the SL.

This multi-disciplinary program of work will deepen our understanding of male SL development and function by identifying a number of key genetic and epigenetic regulators. Due to the significant parallels between male and female SL development, and because discoveries in the model plant Arabidopsis have been routinely translated into major crops such as rice and maize, these insights will be widely applicable and may be used to improve crop fertility and yield. At a more generic level, my work will demonstrate, for the first time, how epigenetic pathways can be tailored in a specific lineage of cells to convey precise biological functions. This kind of developmental regulation likely affects many biological processes in a wide range of cell types and tissues. I therefore believe that my work will lay a foundation for the study of epigenetic regulation of plant development. Many DNA methylation mechanisms are highly conserved between Arabidopsis and mammals, and recent evidence points to a role for DNA methylation in directing the differentiation of human cell lines. Insights from this work thus have the potential to shed light on the regulation of lineage development by DNA methylation in mammals, which is important to combat DNA methylation-related human diseases such as cancer.

In flowering plants, reproduction is carried out by two specialized cellular sexual lineages (SLs). SLs initiate as meiocytes, each producing four spores via meiosis; these spores then divide and differentiate into gametes and their companion cells. Although past studies have identified a network of genes required for SL function, the genes required for the initiation of SLs are few, and it is unknown how these few genes execute the massive shift of transcription repertoire in the transition between somatic and reproductive development.

My preliminary studies suggest that a SL-specific RNA-directed DNA methylation pathway (RdDM) promotes male SL development by regulating the expression of key reproductive and somatic genes, with RdDM mutations causing meiotic defects. In this proposal I will investigate the mechanism by which male SL development is regulated by the SL-specific RdDM in the model plant Arabidopsis thaliana. First, I will use reverse genetics and Illumina sequencing to precisely determine the effect of RdDM on the SL-specific expression of genes in the 4 types of male SL cells, and will explore how RdDM regulates SL development through these genes by characterizing the functions of two novel candidate genes. Second, I will decipher the mechanism underlying SL-specific RdDM activity using a combination of genomic, forward genetic and developmental biology approaches to identify novel SL-specific proteins and non-coding RNAs that target RdDM.

My proposed research will lead to the discovery of novel genetic and epigenetic regulators of SL development and function, which can be exploited to improve crop yield. My work will also greatly expand our knowledge of epigenetic regulation of plant development, as it demonstrates, for the first time, how a DNA methylation mechanism is adapted by a specific lineage of cells to promote their biological function - a mode of regulation that will likely be relevant to developmental processes outside the SL.

The outcomes of this research will be of significant benefit to farmers and plant breeders as it directly relates to crop yield, and thereby to the UK public in general. My chosen model organism, Arabidopsis thaliana, belongs to an economically important plant family, Brassicaceae, with many crops such as rapeseed and cabbage. Given the conservation of reproductive development regulation between Arabidopsis, Brassicaceae plants and other major crops such as rice and maize, findings from this research have the potential to improve crop yield that is of interest for agricultural biotechnology and plant breeding companies, and to enable production of male-sterility lines invaluable for plant breeders. The commercial exploitation of potential findings can eventually benefit farmers, and in the long run, the general UK public by contributing to UK's economical competitiveness.

Global warming poses threats to agricultural productivity in many aspects. During the flowering plant life cycle, the reproductive phase, especially on the male side, is one of the most sensitive to hot or cold temperature stresses. Sterility and barren seed set can be caused by even a single hot day or cold night in many crops such as tomato and maize. In particular, my proposed work focuses on the male reproductive development regulation by a specific DNA methylation pathway, the RNA-directed DNA methylation pathway (RdDM). Recent implication of RdDM in heat tolerance indicates outputs from my research might provide insights into the mechanism underlying the impairment of male fertility by elevated temperature, with potential applications in developing novel approaches for protecting crop fertility facing weather extremes, which are expected to be more frequent globally and in the UK.

Plant breeding combined with biotechnology plays a major role in agricultural improvement. Besides explicit findings of new genes and non-coding RNAs that control reproductive development, this proposed research will generate a wealth of methylomic and transcriptomic data on 4 essential reproductive cell types. These data are not only invaluable for scientists, but may also be used by breeders and the agrobiotech industry as markers for breeding or research, which in the long term will impact positively on agricultural productivity and UK's economy.

Elucidating the mechanism of a basic epigenetic pathway, RdDM, this proposed research has impacts beyond crop improvement. In humans, an analogue of a plant RdDM component, Hiwi protein, and its associating DNA hypermethylation are linked with cancers. It is thus conceivable that insights generated from this proposed work may facilitate our understanding of the role of Hiwi-associated DNA methylation in cancer, which can be translated to promote public health and quality of life.

In summary, the proposed project will have strong social and economic impacts involving food security, economic competitiveness and public health, owing to the important relevance of reproductive development to crop yield, and the emerging significance of epigenetic mechanisms on a range of biological processes in plants and mammals.