Connecting TOR signalling with proliferation through the RETINOBLASTOMA RELATED complex in Arabidopsis root meristem

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Considering that agricultural productivity is fundamentally dependent on how nutrients, carbon and energy underpin plant growth, remarkably little is known about some of the underlying biological processes. Cell growth, which relies on protein translation, constrains cell proliferation. Emerging data in our laboratory suggest that growth signalling by the TARGET OF RAPAMYCIN (TOR) and S6 KINASE (S6K) directly, and through the ribosome biogenesis and translational control orchestrated by EBP1 are connected with the cell cycle control pathways centred on the RETINOBLASTOMA-RELATED (RBR) protein and the downstream transcription factors. Further understanding of the regulatory pathways that link protein translation and cell proliferation to regulate organ growth requires systems approaches in the specific cell types in which these pathways are operational, namely proliferating meristematic cells. In this project, we focus on the Arabidopsis root, where the meristematic and elongation domains are well defined and accessible for microscopic observations to analyze mutants and to collect cell specific quantitative data for computational modelling.In task 1 we shall measure cellular and molecular markers of root growth in seedlings over time as a function of root growth rate. The growth rate will be manipulated by adjusting the sucrose concentration, light intensity. We will start with time course EdU labelling of DNA synthesis to determine the change in cell cycle length. Then we shall set up live root imaging of cell cycle progression using fluorescently labelled nuclei and cell membrane and dual colour cell cycle markers for S-G2 and M phases using confocal microscopy. We shall use translational reporters to determine dynamic changes in protein translation. First we will focus on how Arabidopsis wild-type root meristems respond to sucrose availability and then we shall perform growth quantitation in mutant/silenced lines of TOR, RAPTOR, S6K1 and S6K2, RPS6, eIF3h, EBP1, and use chemical inhibition of TOR.In task 2, we aim to corroborate the data we collect on fixed preparation or by live imaging with the cutting-edge light sheet microscope available in the Laboratory of Plant Morphogenesis directed by Dr Sena at Imperial College London. This instrument has been specifically developed by Dr Sena and colleagues for Arabidopsis root live fluorescence imaging, at high spatial (microns) and temporal (minutes) resolutions and for very long (days) observations. This part of the project will focus on some selected critical parameters and conditions determined in task 1. The outcome of the experiments in tasks 1 and 2 will be dynamic, quantitative, data with cellular resolution on protein translation, cell cycle parameters, cell and meristem sizes, and growth rates under a range of conditions and TOR pathway activities that either stimulate or repress cell proliferation.In task 3 we shall build on our expertise that identified regulatory targets of the TOR signalling pathway that provide regulatory inputs that controls cell cycle transition points and consequently the cell cycle parameters, meristem size and root growth rate. Within task 3 we will collect cell specific dynamic data on the regulatory molecules within the network using transcriptional reporters (promoter-reporter), translational reporters (promoter-UTR-reporter) and functional translational gene fusions (promoter-gene-reporter). We will image the seedling root by confocal microscopy and, for long observations on selected reporters we will also use light sheet microscopy. Additionally, we will determine the phosphorylation state of S6K, RBR specifically in the root meristem and in the elongation zone using domain-specific promoters to express GFP-tagged RBR S6K1. We shall also quantitatively measure RBR-E2FA and RBR-E2FB interactions by co-immunoprecipitation in these domains. These quantitative data will allow modelling the regulatory network.