Abstract
High sugar content is the primary objective in sugarcane (Saccharum spp. hybrids) breeding. Plant microRNA 164 (miR164) plays pivotal roles in plant development and stress responses through the post-transcriptional regulation of its target genes. NAC transcription factors play a crucial role in various plant physiological processes. However, their role in sugar metabolism remains uncharacterized. Our previous work revealed the potential role of the shy-miR164 and a sugarcane NAC transcription factor (ScNAC) in sugar metabolism based on multiomics analysis. In this study, shy-miR164 and ScNAC exhibited an inverse regulatory relationship in sugarcane. Subsequent RLM-RACE and dual-luciferase assays confirmed that shy-miR164 regulates ScNAC expression through direct cleavage of its mRNA. Knockdown of miR164 using short tandem target mimic technology significantly enhanced sugar content in tomato (Solanum lycopersicum L.) fruits, whereas the opposite effect was observed in miR164-overexpressing plants, indicating its involvement in sugar metabolism. Furthermore, heterologous expression of ScNAC in tomato also enhanced fruit sugar content. Taken together, these findings reveal a previously unexplored role of shy-miR164 in sugar metabolism through the direct cleavage of its target gene ScNAC. This work advances our understanding of the mechanisms underlying sugar metabolism and provides candidate targets for improved sugar production in sugarcane through biotechnology.