Understanding the molecular and genetic mechanisms regulating senescence in wheat.

Monocarpic senescence in wheat is a tightly regulated process that is essential in determining grain quality levels. However, despite its importance, relatively little is known about the molecular mechanisms that underpin senescence in wheat itself. In this thesis, we have taken advantage of new genetic and genomic resources in wheat to carry out forward and reverse genetics approaches with the aim of identifying novel senescence regulators and further characterizing genes with known roles in senescence. Initially, we carried out a forward genetic screen of the Kronos TILLING population and identified a novel dominant chlorosis phenotype. Using a combination of reduced representation sequencing techniques, we were able to map the locus underpinning this phenotype to a 4.3 Mb region containing 59 genes. We then turned out attention to further characterization of the NAM-1 genes, which are known positive regulators of wheat senescence. We identified missense mutations in highly conserved residues of the NAC domain of NAM-A1 within the Kronos TILLING population. Mutations in these residues not only disrupted protein function, leading to delayed senescence, but they specifically inhibited protein-protein binding. This suggested that the residues may be essential for NAC domain function across species. Using overexpression and heat-shock-inducible transgenic constructs, we then demonstrated that premature expression of NAM-B1 is not sufficient to induce premature senescence. This suggested that NAM-B1 may interact with one or more proteins to drive senescence. We carried out a yeast two-hybrid screen against NAM-B1 and identified seven candidate interactors. One of these genes, the NAC transcription factor NAC3-B1, is part of a triad which also positively regulates senescence and whose predicted regulatory targets are enriched for senescence-related processes. We therefore hypothesized that the NAM-1 and NAC3-1 genes may work together to regulate senescence onset and progression in wheat