Uncovering the nodulation blueprint by assaying nitrogen response

Supervisors: M. L. Gifford² and N. J. Burroughs¹

¹ Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, UK

² Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, UK

Fixed nitrogen availability is a key limiting factor on plant growth so nitrogen-poor environments have a big effect on agriculture. Fertilisers are used to provide the necessary nutrient when lacking. Natural, cheaper alternatives are needed in the long run. Legumes obtain their fixed nitrogen using nitrogen fixing bacteria in root nodules, so we ask why can non-legumes not do the same?

So far, seeds for relevant tDNA insert lines (for Co-Opted for Nodulation (CON) genes chosen from literature searches) for Arabidopsis thaliana have been obtained, grown, checked for homozygocity of insert and phenotyped by studying root architecture. These will form the basis of both whole root and cell-specific investigations into the gene networks connected to the CON genes using microarrays and transcriptome seqencing, with experiments studying the effects of different nitrogen and Nod factor (a signalling molecule detected by Rhizobia and key in the formation of nodules) concentrations.

Meanwhile, wildtype Medicago truncatula is being used for similar investigations. An experiment involving KNO₃ and Nod factor treatments, with RNA samples taken from whole roots and cells run through fluorescence activated cell sorting (FACS), has been completed and is being repeated to improve experimental reliability, with protocols modified to reduce technical effects of FACS and protoplasting. By comparing this treatments to control, the effects on results of FACS could be seen and data on differential expression in the presence of Nod factor and nitrogen. The next step here is to sort cells by cell-types, leading to further investigation on the cell-specific level, focusing on the inner cortex, where nodule primordia are formed, with both Arabidopsis and Medicago to be studied in this respect. Furthermore, the Arabidopsis knockout lines are currently being crossed with our celltype-specific GFP markers to examine gene expression perturbations in single cell-types caused by the blocked expression of genes found to have roles in root development.

A clustering analysis of pre-existing Medicago expression data (Lohar et al, 2006), collated with more recent knowledge of the plant’s genome using nBLAST, yielded interesting individual and groups of genes for further focus when looking at future data.

The data eventually yielded from microarray and sequencing experiments will feed back into the research forming the systems side of this project.