Functional analysis of AP2/ERF family members in flavonoid biosynthesis of wolfberry

Flavonoids, a group of major bioactive ingredients, contribute to the nutritional and medicinal properties of wolfberry ( Lycium barbarum ). APETALA2/Ethylene response factors ( AP2/ERFs ) are widely distributed in plants and play crucial roles in regulating growth, development, and stress responses. However, the knowledge of AP2/ERF genes in wolfberry remains limited, and specific AP2/ERF family members involved in flavonoid biosynthesis have not been identified. Here, we systematically identified and characterized AP2/ERF proteins in wolfberry and identified key AP2/ERF family members involved in regulating flavonoid biosynthesis. LbAP2/ERF genes were identified via BLASTP and HMM analysis, using Arabidopsis AP2/ERF as queries and the L. barbarum genome. Gene duplication was analyzed with DupGen_finder, fruit RNA-seq was tested to determine expression profiles across five developmental stages, and LbAP2/ERF089 function was verified using transient overexpression and dual-luciferase assays. A total of 148 genes belonging to the L. barbarum AP2/ERF ( LbAP2/ERF ) family were identified, with dispersed duplication likely being the primary driver of their amplification. The LbAP2/ERF genes exhibit distinct expression profiles during different stages of fruit development, indicating their potential importance in wolfberry fruit development. Based on the metabolite and gene network analysis, a series of LbAP2/ERF genes involved in flavonoid biosynthesis were identified, including LbAP2/ERF089, LbAP2/ERF011, LbAP2/ERF068 , and LbAP2/ERF099 . Functional analysis further revealed that LbAP2/ERF089 positively regulates flavonoid synthesis by activating the expression of biosynthetic genes LbLAR and LbDFR . Overall, our results provide new insights into the transcriptional regulation of flavonoid and anthocyanin biosynthesis and offer valuable genetic resources for enhancing the nutritional and medicinal value of wolfberry.