A novel-miR-164 from Atractylodes lancea targets AlNAC1 regulating leaf senescence

A novel miR164-AlNAC1 module adjusting the expression of genes related to chlorophyll synthesis and ROS synthesis, and regulating leaf senescence in Atractylodes lancea. Leaf senescence is a critical developmental process regulated by complex genetic networks. In this study, a novel miRNA belonging to the MIR164 family was identified as a key regulator of leaf senescence in A. lancea by targeting the transcription factor AlNAC1. Bioinformatics analysis revealed that target gene AlNAC1 was closely related to the senescence-associated gene ANAC092 in Arabidopsis. The qRT-PCR and tobacco transient expression demonstrated nov-miR164 negatively regulated AlNAC1. Subcellular localization and transcriptional activity assays revealed that AlNAC1 localized to the nucleus and functioned as a transcriptional activator. Furthermore, EMSA experiments showed that AlNAC1 bound to the G-box motifs on the promoters of AlNYC, AlPAO, and AlRbohC. With the aging of A. lancea leaves, the expression of nov-miR164 decreased, while AlNAC1 and senescence-associated genes (AlSAG21, AlNYC, AlPAO, and AlRbohC) were significantly upregulated. Agrobacterium-mediated transient expression experiment results demonstrated that nov-miR164 negatively regulated AlNAC1 to suppress chlorophyll degradation and ROS accumulation, thereby delaying leaf senescence. Our findings provide new insights into the molecular mechanisms of leaf senescence in A. lancea and highlight the potential of nov-miR164 as a target for increasing crop yield and improving crop longevity.