Compound starch granule formation in grass seeds is associated with distinct temporal patterns of gene expression

There is extensive interspecies variation in starch granule morphology in the endosperm of grass species, but the factors underpinning this variation are poorly understood. The two major granule morphology types among species are simple and compound granules, where simple granules arise from a single initiation per amyloplast, and compound granules form from multiple granule initiations. Here, we carried out an extensive survey of seed starch morphology, examining specimens from 105 species within the Pooideae using electron microscopy. This not only expanded our current knowledge of the diversity of starch granule types but also confirmed the homoplasy of simple and compound starch granules. We then selected species representing independent origins of simple (Brachyelytrum japonicum, Phaenosperma globosa, and Brachypodium distachyon) and compound granules (Nardus stricta, Melica altissima, and Calamagrostis brachytricha) for analysis of starch structure and gene expression. There were no clear patterns in starch content, amylopectin structure, or amylose content that could distinguish the two granule types. However, comparative transcriptomics over seed development revealed gene expression patterns associated with granule type, most notably increasing expression of STARCH SYNTHASE 3a (SS3a), STARCH BRANCHING ENZYME 1 (SBE1), and limit dextrinases (LDAs) between 3 and 9 days post anthesis in species with compound granules. Mutation of these genes in rice, which natively produces compound granules, resulted in altered granule shape and/or size. Our work highlights differences in gene expression that could contribute to natural variation in granule morphology within the Pooideae while providing important new starch phenotype and gene expression datasets that can be widely used to study endosperm development.