Elucidating the spatial and temporal control of granule initiation in wheat

The initiation of the two distinct types of starch granules in the developing wheat endosperm is both spatially and temporally separated. Large A-type granules are initiated at the early stages of grain development, while small B-type granules are initiated later and at least partially in amyloplast stromules. We recently discovered proteins required for proper granule initiation in wheat. SS4, BGC1 and ISA1 establish the correct granule number per amyloplast in early grain development. BGC1 also promotes B-type granule initiations in late grain development, while MRC represses B-type granules in early grain development. These roles are consistent with the timing of their expression. How these proteins act together to orchestrate A- and B-type granule initiation during grain development is not understood. Here we will explore how the spatiotemporal control of granule initiation is coordinated at the transcriptional and post-translational levels. Using our mutant collection in the tetraploid wheat Kronos, we will look for genetic dependencies and interactions between granule initiation proteins by analysing the phenotypes of multiple mutants lacking several components of granule initiation. As the orthologs of these initiation proteins in Arabidopsis act via protein-protein interactions, we will use immunoprecipitations to identify their interaction partners in wheat endosperm during A- and B-type granule initiation, and characterise new partner proteins using TILLING mutants. RNAseq and quantitative proteomics will be used to relate changes in protein interactions with changes in transcript and protein abundance through grain development. Finally, we will explore the importance of transcriptional regulation on the timing of granule initiation, using inducible promoters to express BGC1 and MRC at different developmental stages. Overall, this work will reveal the mechanisms coordinating the spatiotemporal pattern of granule initiation during grain development.