Sucrose-associated SnRK1a1-mediated phosphorylation of Opaque2 modulates endosperm filling in maize

During maize endosperm filling, sucrose not only serves as a source of carbon skeletons for storage-reserve synthesis, but also acts as a stimulus to promote this process. However, the molecular mechanism details about sucrose and endosperm filling are poorly understood. Here, we found that sucrose promoted the expression of endosperm-filling hub Opaque2 (O2), coordinating with storage-reserve accumulation. A protein kinase called SnRK1a1 attenuated O2-mediated transactivation, but sucrose released the suppression. SnRK1a1 phosphorylated O2 at Serine 41 (S41), negatively affecting its protein stability and transactivation ability. Mutation of SnRK1a1 resulted in larger seeds with increased kernel weight and storage reserves, while overexpression of SnRK1a1 had the opposite effect. Overexpression of the native O2 (O2-OE), phospho-dead (O2-SA) and phospho-mimetic (O2-SD) variants all increased 100-kernel weight. Although O2-SA seeds exhibited smaller kernel size, they synthesized higher starch and proteins, thereby resulting in larger vitreous endosperm and increased test weight. O2-SD seeds displayed larger kernel size, but had unchanged levels of storage reserves and test weight. O2-OE seeds represented an admixture of O2-SA and O2-SD, showing elevated kernel dimensions and nutrient storage. Overall, this study discovered a novel mechanism to modulate endosperm filling and S41 in O2 offered potential for engineering efforts to enhance storage-reserve accumulation and yield in maize.