Bilateral NSF/BIO-BBSRC - Translational landscape to link cell growth with proliferation in the root meristem

Our life depends on growing plants. Projected population increases together with anticipated disruptions to agricultural production by climate change create a pressing need to achieve step-change improvements in agricultural production to guarantee security of global food supplies. Increases in the application of nitrogen fertilizers underpinned the "green revolution" but are unsustainable. Work described in this proposal will contribute to an alternative route to increased agricultural production, which could be described as a "second green revolution". According to this strategy, agricultural productivity is increased through use of crops in which growth responses are optimized to sustain the increase in biomass in what would otherwise be limiting environments. Plant growth fundamentally depends on maintaining growth and proliferation of cells, which occurs in the meristems. The rate of cell production must be aligned with developmental cues, available energy, nutrient supplies and environmental conditions. Cytoplasmic growth in meristematic cells is largely constrained by protein synthesis and is coupled to cell division to maintain cell size homeostasis. There are evolutionarily conserved sensing and intracellular signalling mechanisms that inform cells on the available nutrient supply. Central to this is the so called TARGET OF RAPAMYCIN (TOR) protein, so named after an antifungal compound produced by a bacterium that was discovered in the Easter Island, Rapa Nui. TOR is central for cell growth mainly through the regulation of protein synthesis and connecting protein synthesis and cell proliferation, but these regulatory mechanisms are not well understood in plant cells. TOR is a master regulator and also functions through other output pathways. One main route of TOR function is through stimulating ribosomes to increase the translational capacity of cells for protein synthesis. Recent findings unexpectedly show that a canonical ribosomal protein target also functions as a transcriptional regulator (repressor). We found that this is in association with a key controller of the cell cycle, the RETINOBLASTOMA RELATED (RBR) protein, named after the cancer in the eye when mutated in humans. RBR and its partner proteins are thought to constitute a switch that controls cell proliferation and cell growth and can be flicked by environmental conditions. In this project we shall use root meristematic cells to systematically uncover transcriptionally and translationally regulated genes that function to connect cell growth and proliferation. We will then design experiments through which we can precisely observe the molecular behaviour of the components of the switch in time, as we alter the growth conditions, while at the same time following changes in growth through microscopic movies. These types of experiments will produce a wealth of data that allow building a comprehensive knowledge of the regulatory network. With additional help from carefully optimized computer models, we can learn the functioning of this cellular decision making circuitry and make predictions at different environmental and nutrient conditions what is the extent of cell proliferation and therefore root growth. Having achieved to construct such a predictive model we will test its performance in different real life situations, such as what happens to root growth in dark, or under limited nitrate or sucrose. We might also find that we missed some components, and this will prompt us for further experimentation. Having perfected the model we can start adapting it to other growth-altering conditions, such as stress, or to other parts of the plant important for crop yield, such as fruits or seeds.

Cell growth and protein translation constrain cell proliferation. Emerging data indicate that growth signalling pathways (e.g. TOR and S6K) affect both translation and the commitment to cell proliferation through the RETINOBLASTOMA RELATED (RBR) pathway. The hypothesis is that translational control of specific mRNAs, for example through the untranslated regions, provides important regulatory links to couple cell growth and cell proliferation. Key pieces of evidence are that mutations of translation/ribosome associated proteins such as TOR, S6K, eIF3h and EBP1 deregulate the cell cycle.
This project aims to establish how growth-stimulating regulatory pathways (TOR, S6K) affect the Arabidopsis translatome as well as cell proliferation in the seedling root. Aim 1 will yield quantitative data on cell cycle parameters and translation over time while manipulating growth by environmental and genetic perturbation. In aim 2 the translation state of mRNAs will be measured in three ways, by ribosome immunopurification targeting root meristematic cells and cells in the elongation zone, by ribosome footprinting, and by density gradient fractionation of polysomes, followed by RNA sequencing. Together with mRNA transcript data, these techniques will reveal complementary aspects of translational control by the signalling pathways. In aim 3, we derive biochemical parameters of translation for all mRNAs. Aim 4 is to computationally identify cis-regulatory sequence motifs in mRNAs. Our final aim 5 is to condense the regulatory pathway to the principal components. We will collect time/spatially resolved data on the cell proliferation switch. Together with data from previous aims, we will use graph theoretical methods to generate a Bayesian network model of the cell proliferation switch.
Establishing the integration of protein translation and cell cycle control in the root tip model system will lay the groundwork for future modelling of cell proliferation in other plant organs.

Our principal aim is to deliver excellent science that will provide profound novel insights into one of the most fundamental aspects of plant biology, namely how protein synthesis drives cell growth and proliferation, and how this impacts on biomass and plant yield. Uncovering the underlying regulatory machinery, and building predictive models that test our understanding of these processes, may improve our ability to optimize plant function by genetic improvement. While our model will necessarily focus on a tractable model system, the response of the Arabidopsis root tip to photosynthate, it is clear that aspects of the model will apply to other organs, such as fruits or seeds, to crop plants, where the same machinery is highly conserved, and to other signals, such as stress, or nitrogen status.
Besides disseminating the immediate intellectual merit of the work through publication, we also propose a strategy for translating the results for the benefit of agricultural biotechnology, through partnerships with industry, joint academic-industrial grant proposals, and exploring the protection of intellectual property. The public will benefit in the long term from economic activity, market forces on consumer prices, and sustainable agricultural production.
The immediate impact of this research will be enhanced knowledge and understanding of the fundamental process, which will be communicated to public in ways that enrich societal understanding of fundamental biological processes and scientific methods, such as genetic modifications. The PIs have skills and developed channels to disseminate this knowledge in schools and during University open days. An existing partnership with a local artist will be developed to produce documentation and to engage the public in creative ways.
Finally, the training and professional mentoring of the postdocs and PhD students employed in this multidisciplinary and collaborative project will develop the scientific workforce in academia or industry in a growth area that suffers from a shortage of skilled personnel.