OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408.

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.