Cis-element conservation and divergence in plant reproductive development (CISCODE)

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Changes in the regulatory sequences of key genes are believed to have played a major role in creating phenotypic variation during evolution and in plant domestication. We aim to understand how networks of transcription factors and their target sequences control flower and fruit development, how these networks vary between species, and explore these variations for practical use. We will focus on a key set of regulatory genes, originally identified in Arabidopsis. These include WUSCHEL (WUS), which is required for meristem maintenance. During floral organogenesis, WUS is repressed through the action of AGAMOUS (AG) and SEEDSTICK (STK). AG goes on to direct stamen and carpel development, while STK guides ovule development. Under the control of AG, a further set of genes controls cell differentiation within the carpels, including the development of the dehiscence zone in Arabidopsis and in rapeseed. This network includes SHATTERPROOF (SHP), FRUITFUL (FUL), JAGGED (JAG) and REPLUMLESS (RPL). To identify conserved and divergent aspects of the regulatory networks involving these genes, we will use a comparative genomics approach including Arabidopsis, rice, tomato, Antirrhinum, oilseed rape and Capsella. We will identify target genes using a combination of transcriptional profiling and chromatin Immunoprecipitation-CHIP. We will then identify relevant cis-elements by in silico searches in co-regulated genes or based on sequence conservation (phylogenetic footprinting and shadowing). Functional relevance will be tested in vitro (EMSA, DNase I footprinting assays) and in vivo using reporter gene assays. Modifying these cis-elements and comparing transcriptional activity across species using transient assays and stable transformants will reveal the conservation of the cis-regulatory code. Based on the results, we will use TILLING to screen for cis-regulatory mutations that may alter rapeseed fruit development and reduce seed loss due to premature opening of the fruit.

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