Cis-element conservation and divergence in plant reproductive development

Most agricultural products are derived from fruits of flowering plants, such as rice and corn. Since fruits originate from flowers, crop improvement requires a detailed understanding of flower and fruit development. Research on reference species, such as Antirrhinum or Arabidopsis, has revealed genes that control key steps in the development of flowers and fruits. These genes encode transcription factors, which regulate other genes that contain specific DNA sequences within their regulatory regions. It is believed that variation in these regulatory sequences and in their interaction with key transcription factors have played a major role in creating the changes in flower and fruit development seen during evolution and in plant domestication. We aim to understand how networks of transcription factors and their target regulatory 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. One of them is WUSCHEL (WUS), which controls the stem cell population that sustains development of all new plant organs. During floral organogenesis, WUS is repressed through the action of AGAMOUS (AG) and SEEDSTICK (STK). AG goes on to play a key role in specifying stamen and carpel identity, 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 structures that in some species eventually allow the fruits to open and release seeds. This network includes SHATTERPROOF (SHP), FRUITFUL (FUL), JAGGED (JAG) and REPLUMLESS (RPL). We will initially use Arabidopsis to fill gaps in our knowledge of how these genes regulate each other and additional target genes during development. Each of the European partners in this project will focus on a subset of the genes mentioned above. In all cases, we will first identify the regulatory sequences that are targeted in vivo by the transcription factors encoded by these genes. We will then verify whether these target sequences are conserved across species and test their importance for the expression of the genes that contain them. We will then check whether variations in regulatory sequences can explain some of the developmental differences seen across species. In our case, we will check whether changes in the regulation of SHP, FUL, JAG and RPL are involved in the differences in fruit development between Arabidopsis and rapeseed. Based on the results, we will then perform a targeted screen for changes in regulatory sequences that may alter rapeseed fruit development for practical use, specifically, to reduce seed loss due to premature opening of the fruit.

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 foorprinting 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.