Elucidating the spatial and temporal control of granule initiation in wheat

This project aims to develop a full mechanistic understanding of how starch granules are initiated in developing wheat grains. Starch is the main calorific component of our staple cereal crops. It is synthesised in plastids (amyloplasts) of the endosperm as semi-crystalline insoluble granules composed of the glucose polymers, amylopectin and amylose. While the synthesis of these polymers is relatively well understood, we are only beginning to understand how starch granule formation is initiated within plastids, and the factors determining granule shape and size. These traits are important determinants of starch functionality, and thus influence crop quality.

Diverse starch granule shapes and sizes are observed across different cereal crops, and this is at least partially determined by differences in granule initiation patterns during grain development. In particular, grains of the Triticeae (wheat, barley, rye) produce two distinct populations of starch granules in the endosperm: large A-type granules, and small B-type granules. These arise from two spatially and temporally separated waves of granule initiation: A-type granules initiate early in grain development in amyloplasts, and B-type granules initiate in late grain development in amyloplast stromules. Our recent research in Arabidopsis leaves identified conserved "initiation proteins" required to initiate the correct number of granules per plastid. Interestingly, we also discovered that these proteins are involved in different aspects of granule initiation in wheat, with some required for correct A-type granule formation, and others for the correct number and timing of B-type granule initiation. How these individual proteins act together in an overall mechanism to orchestrate the initiation of granules during grain development is not understood.

We aim to develop a full mechanistic model of the spatial and temporal control of granule initiation in developing wheat grains. In a genetic approach, we will perform crosses using our mutants of tetraploid wheat defective in individual initiation proteins. Starch granule phenotypes will be analysed in the mutants lacking multiple initiation proteins to reveal functional dependencies. In a complementary biochemical approach, we will profile how the abundance and interactions of initiation proteins change through grain development. This will provide an important opportunity to discover and characterise novel components involved in A- and B-type granule initiation. We will also explore the links between granule initiation and amyloplast structure, using live-cell imaging to visualise amyloplast number and structure in our initiation protein mutants.

Finally, we will investigate the importance of transcriptional regulation in the temporal control of granule initiation. Preliminary data suggest that initiation proteins follow distinct expression patterns. We will explore how transcript levels of initiation proteins change through grain development, and how this is reflected in protein abundance. An inducible promoter system will be used to alter the timing of initiation protein expression, and subsequent effects on the timing of granule initiations will be examined. We will also attempt to identify candidate transcriptional factors that control the distinct expression patterns of initiation proteins.

Overall, our work will reveal mechanisms governing the unique spatiotemporal pattern of granule initiation in wheat, greatly advancing our knowledge of starch synthesis in this important crop. The findings will potentially lead to novel approaches to improve wheat quality by modifying starch granule number and morphology.

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.