Comparative cell-specific profiling to understand the molecular basis of nodulation

Plants must acquire the elemental nutrient nitrogen from their surrounding environment and its availability is often a major limitation to plant growth. To try and cope with this, all plants form lateral roots that explore the soil and increase the surface area on which to take up nitrogen. In addition legumes (peas and beans) have a unique strategy to deal with nitrogen limitation. They enter symbiotic interactions with soil bacteria that are able to fix an atmospheric form of nitrogen that plants cannot take up, and convert it into a useable form. Understanding more about how this happens could allow us to develop nodulation outside legume species, a discovery that would have significant effects on agriculture, the environment and nutrition. For example it would enable farmers to expand the range of crops that they could grow on their land without requiring the use of expensive nitrate fertilisers. Lower fertiliser use would in turn benefit the environment since the fossil-fuel rich process required for its manufacture would be diminished. Adding the ability to fix nitrogen to a commercial non-legume crop, for example wheat, might also increase plant nitrogen content and therefore the nutritive value of such improved crops for consumption. Since there are similarities in the way that lateral roots and nodules form on plant roots it is thought that the plan for 'building' a nodule in legumes comes from a lateral root 'blueprint' that exists in all plants. Despite detailed study of lateral roots and nodules little is known of what this 'blueprint' looks like. The answer might come from the fact that both lateral roots and nodules develop from single types of cells in the root - because of this specificity the important factors that link them have not yet been uncovered. We now have the state-of-the-art technology that will allows us to make the detailed analyses required for such study. We will compare legume vs. non-legume responses to nitrogen and responses during nodulation in single cells using these novel techniques to address how nodulation evolved in legumes.

The ability to nodulate arose several times in legumes and it is thought that a pre-existing lateral root 'blueprint' present in all plants was co-opted for nodulation. Although regulators of nodulation and lateral root development have been characterised, molecular components underlying the proposed co-option have not been uncovered. A likely reason for this is that previous studies of nodulation have been at the whole-root level, and critically both lateral roots and nodules originate from single cell types. In order to identify shared programs of gene regulation the ideal method would therefore be to separate signals from different cells. Recent developments that combine Fluorescently Activated Cell Sorting (FACS) with dynamic treatments to profile environmental responses of individual cell types make a new developmental comparison of the two organs possible. In this systems biology proposal we will compare genomic responses in single cell types of the root between the legume Medicago truncatula and the non-legume Arabidopsis thaliana. The responses will be the effects of nitrogen treatment to induce lateral root development, and Nod-factor treatment to induce nodulation in Medicago. We will measure these responses over a high resolution timecourse and combine the expression data with promoter motif identification and orthology assignment into causative network models. This bioinformatic analysis will enable us to narrow down to gene responses that are shared between different species, different treatments and different cell types, and thus implicate specific genes in co-option. We will then test these genes for predicted effects on lateral root development and nodulation in reverse genetic studies. The best candidates will be characterised for effects on nodulation in Medicago at a completely novel level by profiling single cell types with FACS in inducible gene expression lines. Together this work will shed new light on the evolution of nodulation in legumes.

Plants must acquire the elemental nutrient nitrogen from their surrounding environment and its availability is often a major limitation to plant growth. To overcome this limitation farmers apply nitrogen at a high concentration to their crop plants through the application of fertilisers. The production of nitrate fertiliser is a highly energy demanding process and currently accounts for approximately 2% of the worlds energy usage. The primary energy source for fertiliser production is fossil fuels and as such fertilisers not only account for a significant cost in food production, but also almost 50% of carbon dioxide emissions from agriculture. As energy prices rise the cost of food production increases, mostly due to the link between energy usage and fertiliser production. Reducing agricultural reliance on inorganic fertilisers will dramatically enhance sustainable and affordable food production. This is crucial if we are going to meet the increasing demands for food from an expanding global population. The major natural contributor to biologically available nitrogen in terrestrial systems are nitrogen fixing bacteria in symbiosis with plants. This symbiosis is restricted to a subset of plant species, including legumes such as peas and beans, but absent in many of our major crop plants such as wheat, rice and maize. There is no 'quick fix' to the global nitrogen challenge and biological nitrogen fixation likely holds the key to solving this problem. Our work will use state-of-the-art experimental technologies and analytical methods to uncover how the symbiosis is controlled in individual cells of the legume root and uncover information about how nodulation evolved by comparison to a non-legume root. We need this detailed understanding of the mechanisms that control this symbiosis if we are to use this process to tackle the nitrogen problem.