Plant fertility is vitally dependent upon sexual reproduction and the differentiation of male (sperm) and female (egg) gametes. Despite the importance of this process for future food security we have limited knowledge of the mechanisms involved in gamete development. This research aims to uncover fundamental mechanisms that underlie key decisions in gamete development and the role of gene networks in plant and crop fertility.
Through screens in the genetic model Arabidopsis thaliana, we have identified key regulators of sperm development including the transcription factor DUO1, which is widely conserved in important food crops such as maize, wheat, rice and tomato (Kim et al. 2008; Brownfield et al. 2009). Our work has established a regulatory framework for male germline development (Berger and Twell, 2011), and in recent work we have discovered DAZ1, a novel class of zinc finger transcription factor, which forms an important regulatory node downstream of DUO1 (Borg et al., 2014).
Project Aims & Description This project will explore the functional conservation and mechanisms by which the DUO1-DAZ1 regulatory module coordinates cell division and sperm differentiation. Studies of gene function will include genes from Arabidopsis and from several crop species including tomato and cereals. Genetic and molecular analysis will be combined with comparative transcriptome studies to uncover co-expression and co-function networks and their impact on sperm cell differentiation and plant fertility. The project seeks to establish the conservation of DUO1-DAZ1 function in crop plants, to identify DAZ1 target genes, and to model how the target genes are integrated with the wider germline gene networks.
The research is expected to deliver novel information and tools of potential value in plant breeding applications such as hybrid seed production and the control of gene flow.
Possible timeline Year 1. Construct novel germline mutants using CRISPR-Cas9 technology and identify germline targets of the DUO1-DAZ1 regulon based on bioinformatic analysis of transcriptome data.
Year 2. Use genetic and molecular methods to analyse germline mutants and establish in vitro/in vivo DNA binding assays for DAZ1.
Year 3. Devise a network model for the contribution of DAZ1-targets to male germline development and analyse gene function by gene-editing and manipulation of protein function.
Techniques that will be undertaken during the project Manipulation of gene expression and protein function by transgenic analysis and CRISPR-Cas9 gene-editing; Fluorescence and confocal laser scanning microscopy; Comparative transcriptome analysis (microarray & RNA-seq data); co-expression and co-function network analysis;Transient gene expression assays and quantitative analysis (qRT-PCR)
Borg, M., Brownfield, L., Khatab, H., Sidorova, A., Lingaya, M. and Twell, D. (2011) The R2R3 MYB transcription factor DUO1 activates a male germline-specific regulon essential for sperm cell differentiation in Arabidopsis. Plant Cell 23:1-16.
Borg, M., Rutley, N., Kagale, S. Hamamura, Y., Gherghinoiu, M., Kumar, S., Sari, U., Esparza-Franco, MA., Sakamoto, W., Rozwadowski, K., Higashiyama, T. and Twell, D. (2014). An EAR-dependent regulatory module promotes male germ cell division and sperm fertility in Arabidopsis. Plant Cell 26:1-17.
Brownfield, L., Hafidh, S., Borg, M., Sidorova, A., Mori, T. and Twell, D. (2009) A plant germ cell-specific integrator of cell cycle progression and sperm specification PLoS Genet. 5: e1000430.
Kim, H.J., Oh, S-A., Brownfield, L., Ryu, H., Hwang, I., Twell, D*. and Nam, H-G*. (2008) Control of plant male germline proliferation by SCFFBL17 degradation of cell cycle inhibitors. Nature 455, 1134-1137.
Berger, F. and Twell, D. (2011) Germline specification and function in plants. Annu Rev Plant Biol 62:461-484.