A dose-sensitive miR164e-WRKY50-JAZ7 module fine-tunes grain size in rice

Polyploidization drives evolutionary innovation and crop domestication by giving rise to novel gene combinations and increasing genetic variation. Tetraploid rice shows promise for yield enhancement owing to its larger grains, but the underlying mechanisms responsible for this trait remain unclear. In this study, we used tetraploid rice CX35-4× and its diploid counterpart CX35-2× to construct the first comprehensive mRNA and microRNA (miRNA) atlas for multiple stages of rice grain development. Transcriptomic analysis revealed that, during key stages of hull development, transcription factors and genes involved in plant hormone signal transduction showed pronounced dose-sensitive effects in tetraploid rice, forming a core regulatory network for polyploid grain development. Our integrated analyses revealed that dose-sensitive miRNAs regulate these key genes, enabling the construction of an miRNA-target network for the control of grain size. Functional validation of dose-sensitive miR164e and WRKY50 confirmed that miR164e inhibits WRKY50 expression by targeting its 3' UTR, negatively regulating grain development. Mutation of miR164e increased thousand-grain weight by 25%, whereas mutation of WRKY50 caused a 16% reduction. DNA affinity purification sequencing identified JAZ7 as a direct WRKY50 target. As a dose-sensitive hormone-signaling component, JAZ7 is transcriptionally upregulated by WRKY50, and loss of JAZ7 function produced an 18% reduction in thousand-grain weight. Our study reveals the dose-sensitive regulatory network that controls grain size in polyploid rice and establishes the novel, dose-sensitive miR164e-WRKY50-JAZ7 module as a key regulatory mechanism. These findings provide new insight into the advantages conferred by polyploidy and identify valuable genetic resources for rice yield improvement.