Epigenetic control of the plant male gametophyte and germline

Like animals, plants have a reproductive system which relies on the production of male and female reproductive cells - sperms and eggs - and a fertilization process. As almost all major crops require this sexual reproduction system to produce seeds, the importance of understanding the developmental biology of plant fertility and reproduction is critical. In animals, much recent work has shown that short information-carrying molecules of RNA ('small RNAs'/sRNAs) are essential for the development of male reproductive cells, a role which was never previously anticipated . The pathways in cells that generate and process these RNA molecules are consequently of great importance. In plants, there is now rapidly accumulating evidence to show that these RNA molecules have essential roles in regulating normal development and health of the plant, through controlling how genes are expressed, the way chromosomes are assembled and defence against parasites. Surprisingly, the role of sRNA pathways in regulating the reproduction of plants has not been studied - even though evidence is available to suggest that they are likely to fulfil the same kinds of important functions. For example, in the formation of the male reproductive cells (pollen) it is well established that there is a major change in the expression of many genes, a cycle of RNA abundance, and dramatic changes in the architecture of the chromosomes. Further, one biotechnology technique called RNAi (which relies on theses RNA pathways to work) has been shown to be effective in plant gametophytes. Our proposal will investigate - for the first time - the presence and function of these RNA processing pathways in the plant male gametophyte and germline (all contained in the pollen grain). Using a range of new technologies, we will use viral proteins that disrupt these pathways to determine the effect of perturbing their normal function on the development and fertility of the pollen. This will also allow us to find which genes in pollen may be regulated by small RNAs. We will investigate whether the genes that encode the various parts of the RNA processing pathways are active in the different cells that compose the pollen. Using a new sequencing strategy (454) we shall then attempt to sequence and identify all the sRNA molecules in the gametophyte, which will tell us immediately which genes are likely to be involved in this system. Finally we shall set up experiments to test whether a cellular system for processing a specific class of small RNA ('microRNA') is functional. In terms of benefits, this project will provide valuable information on how plants reproduce, allow meaningful comparisons to animal reproduction and generate new technology that will allow greater understanding of this economically important part of the plant life cycle.

In animals, epigenetic machinery is essential for generating and maintaining the germline and germ cells. Angiosperms lack strict germlines but after meiosis form male and female haploid cell lines (the gametophytes) which generate sperms and eggs. Male gametophyte development involves a switch to a significantly different gene expression programme. While dramatic DNA methylation and chromatin changes occur on a global scale during gametophyte formation, data are sparse and conflicting concerning the small RNA (sRNA) pathways, which are so critical to epigenetic gene regulation in the sporophyte. We will determine the contribution of sRNA pathways to development of the male gametophyte and germline of Arabidopsis thaliana by inhibiting sRNA processing systems through expression of a range of viral suppressors of eukaryotic gene silencing systems. To identify which sRNA pathways are active during gametophyte and germline development we shall use microarrays and RTPCR to monitor expression of genes encoding key elements of the small RNA processing and RNA-dependent DNA methylation pathways, at different stages of development. We will then use artificial microRNA constructs, and (ii) RNA-dependent DNA methylation and transcriptional gene silencing, using hairpin constructs directed to non-endogenous promoter constructs to confirm that the different cell types of the gametophye and germline are capable of (i) mature microRNA biogenesis and microRNA-directed cleavage and/or translational inhibition transcripts. Finally we will explore the 'totality' of the impact of sRNA systems on gametophyte and germline development by determining - for the first time - the 'sRNA transcriptome' at three key developmental stages. Established bioinformatics approaches will be used to identify (i) genomic and mRNA targets of gametophytic/germline sRNAs, and (ii) trends in sRNA expression during development.