Stem cells, cell identity and differentiation in plant roots

A four-year BBSRC-supported Industrial CASE PhD studentship is available at the University of Edinburgh and Syngenta starting in September 2013.

Project description:
Plant stem cells, organised in meristems, mediate all post-embryonic growth. In marked contrast to animals however, plants do not develop malignant neoplasia (although infection by Agrobacterium can cause unscheduled growth), and differentiated cells do not resume proliferation; suggesting that the differentiated state in plants is distinct from animals. In this project, we aim to dissect the relationship between ‘stemness’, proliferation and differentiation processes in plants. A better understanding of these processes can be applied in agriculture and plant biotechnology. For example, it is currently not possible to grow pure cell lines of most plant cell types, while only specific cell types produce the plant-based pharmaceuticals that are required for many medicines. Thus, the ability to grow pure cell lines would revolutionise production of large quantities of life-saving drugs.

The specific aims of the project are: isolation and cultivation of specific stem cell types to use in the identification of small molecule agonists or antagonists of stem cell maintenance and cell differentiation.

The basic tools required for the project have already been generated: We have made around 10 plant lines carrying green fluorescent proteins (GFP) expressed under control of cell type-specific promoters, for most cell types in roots. (Root meristems are easy to observe and isolate, and ideal to study differentiation because the temporal stages of this process are spread out along the organ’s longitudinal axis.) We have also begun to generate fluorescent reporter lines (blue and red fluorescent proteins, respectively) that mark proliferating cells, and those that are beginning to differentiate. From plants that carry all three markers, you will prepare protoplasts (plant cells devoid of their cell walls). These protoplasts will be used to isolate specific cell types by cell sorting (using either FACS or LEAP). So for example, to isolate cortex cells, you would separate green-fluorescing from those not fluorescing green, and then separate all green cells into two pools: those that show blue and green fluorescence (cortical cells still proliferating) and those that show green and red fluorescence (non-proliferating cortical cells). By sorting cells by two criteria (cell type and stage in their developmental trajectory), you will be able to separately study cell populations developing towards a specific identity at different stages of this process as well as examine their responses to small molecule agonists or antagonists of cell identity maintenance.

The next step will be to culture these, for which good established protocols are available as a starting point. By monitoring the presence of fluorescent proteins in sorted cells at regular intervals, you will be able to assess whether and for how long their differentiated state is maintained and whether they proliferate. Such cultures will then be the starting material to evaluate the effects of small molecules on maintenance of cell identity and proliferation capacity. By cultivation of protoplasts in a 96-well format, you will be able to proceed in a highly parallel fashion and evaluate large numbers of small molecules. The aim of these studies is to identify molecules that are agonists (promote) or antagonists (inhibit) of stem cell identity or of onset of differentiation markers. Some or all of these screens will be undertaken using high-throughput confocal microscopy available at SBS (Opera system).
This project will provide conceptual and practical skills training in the following areas: stem cell biology, cell biology, growth control, plant signalling mechanisms, cell sorting, fluorescence microscopy, image analysis, chemical genomics, high-throughput screening, large-scale data generation and analysis.