Description
  
 

Objectives

This project will use genomics technologies in a comparative context to harness the information present in the diversity of species to fill in the gaps in our understanding of model and crop plants. We will focus on a fundamental, economically important and experimentally tractable biological system, plant reproduction, and use genomic and post-genomic tools to model and manipulate the regulatory network at the centre of the reproductive process. Using a comparative approach, we aim to understand how evolutionary variation in non-coding DNA regions has led to variation in reproductive processes in (crop) species. To derive maximum benefit from a broad comparative analysis, we will focus on a key set of genetic interactions characterised in depth in the reference species Arabidopsis thaliana.
Our final aim of this project is to achieve an understanding of the cis-regulatory elements controlling reproduction in plants, to understand the evolutionary variation in this network and to benefit from this information to predictably manipulate the system.

To achieve this aim, our specific experimental objectives are:

  1. To define the cis-regulatory code underlying key steps in reproductive organ formation, floral determinacy and seed and fruit production.
  2. To create a framework for the extrapolation of genomic and post-genomic knowledge from well characterised model systems to other species.
  3. Conversely, to use comparative tools to inform our understanding of model systems.
  4. To exploit our enhanced understanding to create regulatory mutations for crop improvement.
  5. To benefit from synergies in bioinformatics, genomics resources in multiple plant species, and post-genomic methods to analyze the interaction between cis-elements and regulatory proteins in vivo.

Background

The expanding world population depends on agricultural crops, mainly as cereals and fruits. Improvements of crop plants to achieve better yields under suboptimal growth conditions, such as those found in many developing countries, will be essential to keep up with population growth and reduce the impact of high yield farming on the environment (1).

Most agricultural products, such as seeds and fruits, are derived from the reproductive process of flowering plants. Therefore, crop improvement requires a detailed understanding of flower and fruit development. Research on reference species, such as Antirrhinum or Arabidopsis, has revealed interconnected regulatory networks based primarily on transcription factors that guide the patterning and growth of flowers and fruits (2,3). The genetic interactions between these genes and many of their target genes have been identified, but the combinatorial code of cis-elements that mediates these interactions is still mostly unknown.

The present project is focused on a set of transcription factors (TFs) that occupy key positions in these regulatory networks (Fig 1). The homeodomain TF WUSCHEL (WUS) controls the stem cell population that sustains development of all new plant organs, in part through the regulation of Arabidopsis Response Regulator (ARR) genes, which modulate the cellular responses to the growth regulator cytokinin (4). During floral organogenesis, WUS is repressed through the action of AGAMOUS (AG) and SEEDSTICK (STK), both encoding MADS-domain proteins (5), and by TFs of the HD-ZIP family. AG goes on to play a key role in specifying stamen and carpel identity, while STK guides ovule development (6, 7). Under the control of AG and STK, elaborate genetic networks guide the development of these structures (8, 9). Of particular interest here is the genetic network that controls cell type identity and 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) (10).

Figure 1: Schematic representation of the key transcription factors involved in reproductive organ and fruit development. Only the Arabidopsis genes are indicated.
  • P1: Angenent
  • P2: Colombo/Tonelli
  • P3: Sablowski/Østergaard
  • P4: Davies
  • P5: Yves Van de Peer
  • P6: Lohmannn
  • P7: Morelli
The proteins encoded by the regulatory genes above have largely been conserved during evolution and can be identified in distantly related species (11). More often than changes in protein function, it is believed that changes in the expression pattern of regulatory genes have played a major role in creating phenotypic variation during evolution and during plant domestication (12). By studying the cis-regulatory code of flower development in a range of species, we will not only be able to infer how different structures can be made using the same set of basic regulators, but we will also be able to obtain a deeper insight into the regulatory networks in Arabidopsis. Previous analyses of evolutionary changes in non-coding regions have given insight into the regulation of floral regulators, showing that it is possible to derive understanding on a functional level from phylogenetic sequence analyses (13, 14).

One example of how changes in the regulatory interactions shown in Fig. 1 may underlie differences between species is the control of fruit opening in Arabidopsis and Brassica. In both species, a specialised structure called the replum separates the valve margin tissues that eventually cause the dried fruit to open and release the seeds. In Arabidopsis, replum formation depends on RPL, which functions at least in part by restricting the expression of genes promoting valve margin development such as SHP1/2 and JAG (Fig 1). In Brassica, replum formation is reduced in comparison with Arabidopsis. This may be due to changes in the expression of RPL or in the regulation of targets such as JAG and SHP. Understanding the basis for this difference will not only reveal how regulatory changes underlie developmental differences between species, but would have practical application in further reducing seed loss due to pod shattering in Brassica.

Research plan

To achieve the objectives we will focus on a group of key regulators of the development of reproductive organs and fruits (Figure 1). These genes provide a good starting point for a comparative analysis because they have been well-characterised in model species and their interactions are partly known. Each partner will study a subset of interactions underlying a specific biological process: P1 will focus on AG-SHP-FUL; P2 on STK-MYB-NFY; P3 on AG-JAG-RPL-SHP; P4 on WUS-AG, P6 on WUS-ARR and P7 will concentrate on HD-ZIPIII-WUS. Each set of interactions will be analysed through a set of approaches described below in each of the work packages (WPs). All data (including sequences, micro array data, images of GUS patterns etc) will be easily accessible to all partners in a central database that will be coordinated by P5.

References:

  1. Welch, R. M. Biotechnology, biofortification, and global health. Food Nutr Bull 26, 419-21 (2005).
  2. Zik, M. and Irish, V. F. Flower development: initiation, differentiation, and diversification. Annu Rev Cell Dev Biol 19, 119-40 (2003).
  3. Lohmann, J. U. and Weigel, D. Building beauty: the genetic control of floral patterning. Dev. Cell 2, 135-42. (2002).
  4. Leibfried, A., et al.. WUSCHEL controls meristem function by direct regulation of cytokinin inducible response regulators. Nature 438, 1172-1175. (2005)
  5. Ferrario, S.et al. Control of floral meristem determinacy in petunia by MADS-box transcription factors. Plant Physiol 140, 890-8 (2006).
  6. Pinyopich, A. et al. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424, 85-8 (2003).
  7. Favaro, R. et al. MADS-box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell 15, 2603-11 (2003).
  8. de Folter, S. et al. Transcript profiling of transcription factor genes during silique development in Arabidopsis. Plant Mol Biol 56, 351-66 (2004).
  9. Gomez-Mena, C. et al. Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development 132, 429-38 (2005).
  10. Dinneny, J. R. and Yanofsky, M. F. Drawing lines and borders: how the dehiscent fruit of Arabidopsis is patterned. Bioessays 27, 42-9 (2005).
  11. Irish, V. F. and Litt, A. Flower development and evolution: gene duplication, diversification and redeployment. Curr Opin Genet Dev 15, 454-60 (2005).
  12. Doebley, J. (2004). The genetics of maize evolution. Annual Review of Genetics 38, 37-59
  13. Hong, R. L. et al. Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. Plant Cell 15, 1296-309 (2003).
  14. De Bodt, S. et al. Promoter Analysis of MADS-Box Genes in Eudicots Through Phylogenetic Footprinting. Mol Biol Evol 23, 1293-1303 (2006).
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  16. de Boer E. et al. Efficient Biotinylation and single step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci USA 24, 7480-7485 (2003)
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