Male germline regulatory mechanisms integrating cell cycle control with gamete specification.

Plant sexual reproduction is of vital importance both in the natural environment and for crop production. As in animals, plant reproduction relies on the production of male and female reproductive cells (sperms and eggs) that are united at fertilization. This research aims to uncover the underlying processes that control the production and fertility of plant sperm cells that are formed within pollen grains during sexual reproduction. Surprisingly, the molecular pathways that regulate sperm cell formation in flowering plants are largely unknown, even though this process is vital for plant breeding, crop and seed production. We have identified a master regulator gene, termed DUO1 that is vital for sperm cell formation and fertility in the model plant species Arabidopsis thaliana, a close relative of important seed and vegetable crops in the mustard family. The DUO1 gene has an important coordinating role in plant sperm cell development, controlling the activity of a wide network of genes involved in the control of cell division, and in the specialised function of sperm cells at fertilization. In the proposed work we will investigate for the first time how this network of regulatory factors controls plant sperm cell production using a range of technologies involving molecular, genetic and cell biological approaches. We will identify how different sets of genes are coordinated within this network to control cell division and the specialised production of plant sperm cells. This project will provide valuable information on how plants reproduce, allow meaningful comparisons to animal reproduction and generate new knowledge and technology that will allow greater understanding of this economically important part of the plant life cycle. Moreover, collaborative work with rice reproductive biology experts working in China has been established providing a direct route to evaluate the transfer of knowledge in an important food crop in which technologies such as hybrid seed production have significant (~20%) yield advantage. The work will be carried out by a full-time postdoctoral research assistant and a laboratory technician at the University of Leicester. Some aspects of the work will involve collaboration with researchers involving exchange of data and materials with Universities in China and Japan. Some of the specialised high throughput molecular work will be carried out using services provided by the VIB research institute, Belgium.

The germline-specific R2R3 MYB protein, DUO1 is a transcriptional (master) regulator of male germ cell development in Arabidopsis. DUO1 has a uniquely integrative role, promoting germ cell division and cell differentiation to form the twin sperm cells required for double fertilisation. We have developed a well-supported DUO1 network model based on the transcriptomic identification of a number of DUO1 targets that include two zinc finger family proteins, DAZ1 and DAZ2 that we have shown are essential for germ cell division. The aim is to test and to establish a robust model of this essential post-meiotic regulatory network that integrates germ cell division and sperm cell specification. The work programme is focused into the three major objectives that are designed to elucidate the mechanisms that coordinate G2/M transition in male germ cells with gamete specification. 1. To establish the role of DUO1 and DAZ1/DAZ2 in mitotic cyclin expression and G2/M transition, by monitoring CYCB1-family expression in duo1 and daz1/daz2 germ cells using CYCB1-GFP marker lines, and functional complementation of G2/M defects in duo1 and daz1/daz2 germ cells by germline-targeted CYCB1-family expression. 2. To explore the mechanisms by which DAZ1/DAZ2 and MYB3R proteins regulate mitotic cyclin expression and G2/M transition in male germ cells, by the analysis of the proposed active repressor functions of DAZ1/DAZ2, the analysis of MYB3R expression in daz1/daz2 germ cells and the proposed negative regulatory functions of MYB3R proteins on CYCB1;1 expression and G2/M transition. 3. To identify novel targets of the DAZ1/DAZ2 regulon in G2/M transition and functional sperm cell formation, through transcriptomic analysis of isolated DAZ1/DAZ2-deficient germ cells and the functional analysis of candidate negative regulators of germ cell division and differentiation.

Communications and engagement To promote the impact of the research beyond academic community we will prepare press releases through the University of Leicester press office highlighting the research and its outputs. This approach been effective in publicizing some recent highlights arising from our BBSRC supported work. Two independent releases reporting on BBSRC-funded work published in Nature (2008) and PLoS Genetics (2009) were posted on Eureka Alert!, appearing on over 50 news websites within 10 days. To enhance the public understanding of the research and its value we will develop established links with a local school involving radio interviews. To further engage with future scientists the PI, RA and technician associated with the project will also develop a 'Plant Reproductive Biology' Schools Science engagement activity within the HEFCE-funded Centre for Excellence in Teaching and Learning (CETL) at the University of Leicester to promote understanding of the research and the value of plant reproductive systems. Collaboration Collaborative work to explore the DUO1 regulatory network in rice within the framework of a current BBSRC China Partnering Award widens international cooperation associated with the project. Demonstration of the function of germline promoters in rice would impact upon molecular breeding tools and technology. For example, in the development of a male-specific transgenic gametophytic sterility trait that can be applied to maintain the homozygous recessive condition of a male-sterile plant (ms/ms), when using a transgenic restoration approach; thereby decreasing the costs associated with the number of plantings and steps needed for maintenance. This collaborative link thus provides a route for the transfer of knowledge to an important crop in which technologies such as hybrid seed production have a significant (~20%) yield advantage. Two important collaborations have been established with key researchers investigating G2/M transition in the sporophyte. This will enable rapid progress in understanding G2/M transition in germ cells. Dr Peter Doerner (Edinburgh) has provided robust CYCB1-GFP marker lines that represent important resources in the experimental plan. Similarly Prof Masaki Ito (Nagoya University) has provided Promoter-MYB3R reporter lines and cDNA clones for all 5 MYB3R genes. Regular exchange of knowledge during the project will allow us to integrate knowledge of G2/M regulatory networks in sporophytic and gametophytic generations. Exploitation and Application The outputs of the research will establish a substantial knowledge base and new tools for the targeted manipulation and analysis of germline development. By developing a greater understanding of plant gene regulatory networks that control of germ cell proliferation and specification this project will make a significant contribution to the plant science and wider communities. From other perspectives, this project will make a valuable contribution to scientific knowledge, benefitting scientists, teachers and commercial organisations interested in comparing germline development in animals and plants and understanding of more complex developmental programmes, for example in meristems. Collectively, the increased understanding of gametogenesis derived directly and indirectly from the project will have an impact on crop breeding. The work will deliver a range of valuable resources to the plant science and commercial communities, including a unique collection of characterized germline markers, mutant germplasm and system level knowledge of germline regulatory networks. In this way, there will be future economic benefits from funding this work as manipulating gametogenesis and fertility is of major importance to commercial plant breeders, for example in hybrid seed production, the application of 2n pollen, the alteration of hybridisation barriers between species and control of gene flow in transgenic crops.