Combining genetic and modelling approaches to understanding vascular development in Arabidopsis

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Background: In higher plants, post-embryonic tissues are derived from pluripotent stem cell populations present in niches referred to as meristems. Meristems are composed of central organizing cells with adjacent dividing stem cells that provide cells for new organs. Meristems that are responsible for length-wise plant growth are located at the root and shoot apices. Radial expansion also occurs via divisions in meristematic tissues, referred to as the cambium and procambium, which are present in the vascular tissue. Plant vascular tissue is constituted of two conductive tissues, the xylem and phloem. Xylem is required for water transport, and phloem is required primarily for nutrient transport. A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue, which represents a large renewable resource of energy and biomaterials.Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, is a signalling module characterised by a peptide ligand called TDIF that is expressed in the phloem, and its cognate receptor, PXY which is procambium expressed. The non-cell autonomous interaction of these factors results in particularly complex outputs. This ligand-receptor pair is thought to regulate the rate of cell division in the procambium, exclude xylem identity from the division zone and also control vascular organization by influencing the orientation of cell division. TDIF influences these outputs via interactions with a number of other signalling components and transcriptional regulators. While the identification of targets and interactors that regulate various aspects of PXY signalling outputs has been successful, it remains unclear as to how these complex outputs are integrated. Aims: In order to understand how the three outputs of PXY signalling (i.e. vascular organisation, cell division and exclusion of xylem identity from the procambium) are integrated, we aim to generate quantitative data that will be used to make a logical network model that simulates PXY signalling. Our model will assist in generating hypotheses that will be tested using genetics and molecular biology.Methodology: The student will initially combine the use of histology, microscopy and imaging methodologies with computational analysis (such as cell profiler software) to determine precise numbers of xylem phloem and procambium cells present in vascular tissue during inflorescence development. These analyses will be combined with gene expression data that the student will generate using quantitative RT-PCR. Wild-type Arabidopsis and pxy mutants will be used, as will lines where TDIF has been removed. TDIF is derived from 3 genes, and we have collaborated with Zack Nimchuk (University of North Carolina) who has generated a triple mutant using CRISPR/Cas9 to entirely eliminate the ligand. This data will be used build a mathematical/computational model of the system. The model will take the form of a spatially distributed logical network, generated by the student under the supervision of co-supervisor Natasha Savage (University of Liverpool). This model will then be used to produce testable hypotheses.Subsequent experiments will be guided by modelling data. We envisage model predictions will be tested by manipulating gene expression in planta. For example, if the model predicts that negative feedback of components is critical for maintaining the system, we will manipulate expression using heterologous and/or inducible promoters that are not susceptible to such feedback and assessing the consequences for vascular development. Other manipulations could be used to test a variety of emergent properties including morphogen gradients, rates of cell division etc.Timetable of Activities:Month 0-12: Generate growth data and gene expression data for Arabidopsis vascular tissu