Epigenetic regulation of sexual lineage development in plants

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We find that a fundamental epigenetic pathway, RNA-directed DNA methylation pathway (RdDM), is tailored in plant sexual lineage cells to promote reproductive function and fate. This is the first time that a canonical DNA methylation pathway has been found to be regulated specifically in specific cell types to promote cellular function by gene regulation.

We discovered a novel mode of meiotic recombination control via a meiocyte-specific (meiocyte, the cell going through meiosis) subunit of the general RNA polymerase II transcription factor TFIID.

We revealed the transcriptionally reactivated TEs in the pollen vegetative cells and demonstrated that the vast majority of TE activation is mediated by the demethylase DME, which gains access to heterochromatic TEs due to the natural lack of linker histone H1 in the vegetative cell.
Exploitation Route As most of our staple food comes from the fertilization product of plant sexual reproduction (seeds), this research project
on the epigenetic regulation of male sexual reproductive development will have important impact on crop yield. In addition, male sterility, a phenotype caused by misregulation of male reproductive function, is an important trait for plant breeders as it assists the generation of hybrids by evading the need for laborious emasculation. This research will lead to the discovery of a number of genetic and epigenetic regulators of male reproductive development in the model plant Arabidopsis thaliana. Given the considerable homology in reproductive development regulation between male and female organs, and between Arabidopsis and major crops such as rice and maize, there will be opportunities for translating outcomes of this research into commercial applications in agriculture and plant breeding.
Sectors Agriculture, Food and Drink,Environment

URL https://www.jic.ac.uk/news-and-events/news/2017/12/immortal-plant-cells/
 
Description Our study showed for the first time that a de novo DNA methylation pathway can be employed to regulate cell-specific gene expression and cellular development in plants. This highlights the potential of using this pathway to regulate gene expression for crop improvement, and also shows the potential of the de novo methylation pathway to play a role in animal development, which has applications in combatting human diseases. I have presented our result in a Science Innovation Showcase event open to the industries, which showed interest and I believe recognized the potential.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Healthcare
Impact Types Societal,Economic

 
Description BBSRC DTP Studentship
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description BBSRC Responsive Mode
Amount £779,224 (GBP)
Funding ID BB/S009620/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2022
 
Description EMBO Young Investigator
Amount € 15,000 (EUR)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 01/2019 
End 12/2022
 
Description European Research Council Starting Grant
Amount € 1,500,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 09/2018 
End 08/2023
 
Description Gatsby Grant to Exceptional Researchers
Amount £35,000 (GBP)
Organisation Gatsby Charitable Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 04/2019
 
Description Gatsby PhD Studentship
Amount £107,494 (GBP)
Organisation Gatsby Charitable Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description JIC / CAS (Centre of Excellence in Plant and Microbial Science - CEPAMS)
Amount £99,679 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 08/2017
 
Description JIC Institute Development Grant
Amount £25,000 (GBP)
Organisation John Innes Centre 
Sector Academic/University
Country United Kingdom
Start 06/2015 
End 06/2016
 
Description John Innes Foundation PhD Studentship
Amount £105,206 (GBP)
Organisation John Innes Centre 
Sector Academic/University
Country United Kingdom
Start 09/2015 
End 08/2018
 
Title Bisulfite and RNA sequencing library preparation using small number of plant cells 
Description We and our collaborators at University of Hamburg have modified the published animal single-cell bisulfite and RNA sequencing libary preparation protocols to prepare bisulfite and RNA sequencing libraries from as low as 20 Arabidopsis cells 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Allow the obtainment of methylome and transcriptome from rare cells 
 
Title Isolation of plant cells via FACS 
Description We have developed robust method for protoplasting in Arabidopsis and isolating protoplasts via FACS 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Allows researchers to obtain cells of a specific cell/tissue type, in order to understand their transcriptome, genome or epigenome. 
 
Description Christine Mezard 
Organisation French National Institute of Agricultural Research
Department INRA Versailles
Country France 
Sector Academic/University 
PI Contribution We are evaluating the effect of AXR1 on the DNA methylation in male meiocytes
Collaborator Contribution They identified the AXR1 gene, whose mutation affects recombination during meiosis and generally the DNA methylome in somatic tissues.
Impact Too early to say
Start Year 2019
 
Description Egg and central cell methylomes 
Organisation Seoul National University
Department Department of Biological Sciences
Country Korea, Republic of 
Sector Academic/University 
PI Contribution I designed the experiment together with my collaborators, and I and my team did all the genome analysis.
Collaborator Contribution UC Berkeley and Seoul National University isolated the egg and central cells from Arabidopsis, UC Berkeley and Tokyo Metropolitan University isolated these from rice, Hamburg University performed bisulfite sequencing library preparation
Impact Park et al. 2016 PNAS 113(52):15138-15143
Start Year 2012
 
Description Egg and central cell methylomes 
Organisation Tokyo Metropolitan University
Country Japan 
Sector Academic/University 
PI Contribution I designed the experiment together with my collaborators, and I and my team did all the genome analysis.
Collaborator Contribution UC Berkeley and Seoul National University isolated the egg and central cells from Arabidopsis, UC Berkeley and Tokyo Metropolitan University isolated these from rice, Hamburg University performed bisulfite sequencing library preparation
Impact Park et al. 2016 PNAS 113(52):15138-15143
Start Year 2012
 
Description Egg and central cell methylomes 
Organisation University of California, Berkeley
Department Department of Plant and Microbial Biology
Country United States 
Sector Academic/University 
PI Contribution I designed the experiment together with my collaborators, and I and my team did all the genome analysis.
Collaborator Contribution UC Berkeley and Seoul National University isolated the egg and central cells from Arabidopsis, UC Berkeley and Tokyo Metropolitan University isolated these from rice, Hamburg University performed bisulfite sequencing library preparation
Impact Park et al. 2016 PNAS 113(52):15138-15143
Start Year 2012
 
Description Egg and central cell methylomes 
Organisation University of Hamburg
Country Germany 
Sector Academic/University 
PI Contribution I designed the experiment together with my collaborators, and I and my team did all the genome analysis.
Collaborator Contribution UC Berkeley and Seoul National University isolated the egg and central cells from Arabidopsis, UC Berkeley and Tokyo Metropolitan University isolated these from rice, Hamburg University performed bisulfite sequencing library preparation
Impact Park et al. 2016 PNAS 113(52):15138-15143
Start Year 2012
 
Description JIC-CAS CEPAMS grant 
Organisation Chinese Academy of Sciences
Department Institute of Genetics & Developmental Biology
Country China 
Sector Academic/University 
PI Contribution I came up with the idea and hypothesis, and designed the experimental strategy. Upon receiving the funding, I employed a postdoc who is performing the major epigenetics and biochemistry work for this joint project.
Collaborator Contribution My partners in the Chinese Academy of Science bring the mass spectrometry expertise that is required for the joint project.
Impact N/A
Start Year 2015
 
Description Meiosis-specific TFIID in promoting recombination 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We found a TFIID component is meiocyte specifically expressed, and performed meiocyte extraction and RNA-seq with wildtype and mutants.
Collaborator Contribution Identification of the allele and recombination phenotyping
Impact Not yet
Start Year 2017
 
Description Methylomes of megaspore mother cells 
Organisation National Laboratory of Genomics for Biodiversity
Country Mexico 
Sector Public 
PI Contribution We designed the experiemnts together with our collaborator, prepared and sequenced the libraries, and analysed the data.
Collaborator Contribution They isolated the megaspore mother cells from wild type Arabidopsis and mutants
Impact A publication by mid 2018
Start Year 2015
 
Description Robust maintenance of CG methylation in pollen 
Organisation University of California, Berkeley
Department Department of Plant and Microbial Biology
Country United States 
Sector Academic/University 
PI Contribution We designed and performed the experiments, and analysed data.
Collaborator Contribution UC Berkeley contributed to data analysis.
Impact Hsieh et al. 2016 PNAS 113(52):15132-15137
Start Year 2015
 
Description ASM poster 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact PhD student Billy Aldridge presented a poster on the JIC annual science meeting, which sparked lots of interest and discussion. He won the runner-up award, and an opportunity for presenting his research orally during the annual science meeting next year (2018)
Year(s) Of Engagement Activity 2017
 
Description Science Innovation Showcase 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact An event showcasing John Innes Centre science to industry, aiming to develop and promote new relationships between scientists and industry, breaking down any barriers and enabling productive collaborations.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/news-and-events/whats-on/science-innovation-showcase/