Ligand-receptor-like kinase signalling in Arabidopsis root development

Food security represents a major global issue, and it was put forward by the crop scientist Jonathan Lynch that a 'second green revolution' should be a priority. This should focus on root architecture, since root branching critically influences nutrient and water uptake efficiency. However, root architecture has not been a trait selected for by plant breeders in major cereal crops. In addition, in multicellular organisms, cell-cell communication plays a crucial role in patterning, growth and interaction with the environment. For example, a tight control of formative cell divisions - which are mainly asymmetric (stem) cell divisions - to produce tissues and organs and to prevent over-proliferation is crucial for the postembryonic growth and development of plants. This control of formative cell divisions is not only critical in plants, but also in animals to prevent irregular divisions or cancers. We recently identified a protein, the membrane-associated receptor-like kinase ACR4, which is crucial in the control of root architecture and which is a key regulator of formative cell divisions in the root of the model plant Arabidopsis thaliana. ACR4 has originally been described in maize for its role in other processes, and provides an obvious starting point for the detailed analysis of the role of receptor-like kinase signaling pathways in root architecture and in registering and conveying positional information. This research proposal aims to further characterize this ligand/receptor signaling mechanism that can integrate mobile signaling molecules to control formative cell divisions during organogenesis and, as such, provides an excellent tool to study short range, cell-cell communication during growth and development. First, we will identify those transcriptional changes that affect cell identity in the main and lateral root downstream of a ligand-driven cue from neighbouring cells. These data will provide us with a large number of transcriptional markers for other experiments. Second, unequivocal identification of the ligand is crucial to fully understand receptor-like kinase-mediated signaling pathways. One important aspect is how ligand-dependent directional cues from surrounding cells modulate receptor-like kinase signaling. We will identify and localize ligands for the ACR4 receptor-like kinase. Third, the three dimensional structure of eukaryotic membrane proteins has only been determined very limitedly. We will determine the folding and structure of purified receptor domain protein and use that for detailed analyses. Fourth, temporary and reversible phosphorylation of proteins is one of the most important post-translational modifications with a regulatory function, such as the control of transcriptional changes. Therefore, we will identify the substrates of ACR4 during main and lateral root development. Sixth, with the knowledge that we will gain on ligand-receptor-like kinase signaling and what is known about hormone signaling and transcriptional networks in the same processes, we will build a comprehensive model.

The past decade researchers have mainly focused on the analyses of phytohormone gradients and mobile transcription factors to explain patterning. Especially in the root, model processes, such as the root meristem and lateral root initiation are fairly well understood on the level of transcriptional changes and hormonal control, but very little attention has been given to an alternative way of cell-cell communication using ligand-receptor-like kinase systems. Lately more of these receptor-like kinases and potential ligands have been described in root meristem maintenance and patterning, making it a perfect moment to start investigating how these receptor-like kinases and their respective ligands control the same and different developmental processes and what their common and independent targets are. In brief, in this project, I will use different and interdisciplinary strategies, based on state of the art transcriptomics, chemical genomics, proteomics, interactomics, associomics and more in depth analysis on the single gene/protein/ligand level to put forward a model describing ligand/receptor-mediated cell-cell communication during formative cell divisions, and, more specifically, to identify key components in the ACR4-dependent signalling cascade. The aspects this project plans to address have been rarely covered focusing on one receptor-like kinase, and will allow elucidating the mechanisms and structural aspects behind one crucial signalling pathway, such as: 1. Identification of downstream transcriptional changes, to clarify if signalling pathways across developmental processes are conserved. 2. Identification of substrates involved in development on a genome-wide scale, which - to our knowledge - has only been done for BRI1. 3. Identification and physical interaction of ligands with their receptor, for which only a small number have been demonstrated.

With this project we will elucidate the modes of action of ligand-receptor-like kinase signalling in roots. Next to other control mechanisms, these signalling pathways are important for understanding root architecture. This ambitious project demands skills in Arabidopsis molecular genetics, plant molecular cell biology, proteomics, and I have gained invaluable experience of a systems approach whilst working at the Department of Plant Systems Biology (Gent, Belgium). The multidisciplinary expertise, resources and tools that have been assembled for this project uniquely position us to create, test and validate the role of ligand-receptor-like kinase signalling in root development. The knowledge gained from this study in the model system Arabidopsis thaliana will help scientists understand this basic signalling mechanism and how best to manipulate root growth and enhance crop yield. These outputs provide practical solutions for improving crop performance and deliver food security, a strategic priority area supported by the BBSRC. The project will also generate a number of new and innovative experimental tools and data resources. Furthermore, the results will be integrated in the available models for root development. All these aspects of the research project would be of interest to a wide spectrum of researchers from other disciplines. In this respect, I will also attempt to create new links by participating in workshops to provide training or information dissemination, and by attending international meetings. Currently, an ongoing collaboration with Dr. Remco Sprangers (Max Planck for Developmental Biology, Tübingen, Germany) will lead to the purification and 3D analysis of the receptor domain. But also within the Centre for Plant Integrative Biology (CPIB), a BBSRC/EPSRC Centre for Integrative Systems Biology, and the University of Nottingham, this interdisciplinary project can benefit from the available tools and technology. For example, the proteomics facility headed by Dr. Susan Lidell (University of Nottingham) will be assisting in the phosphoproteomics part of the project. I will disseminate results at scientific conferences, and through published journal articles. The institute routinely prepares press releases for high profile papers and I also regularly publish articles in review journals. Finally, I will regularly make public presentations about topical issues such as GMO's to increase public awareness, but also provide the public with a balanced view on the pros and cons of GMO's.