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 (<i>Lycium barbarum</i>). <i>APETALA2/Ethylene response factors</i> (<i>AP2/ERFs</i>) are widely distributed in plants and play crucial roles in regulating growth, development, and stress responses. However, the knowledge of <i>AP2/ERF</i> genes in wolfberry remains limited, and specific <i>AP2/ERF</i> family members involved in flavonoid biosynthesis have not been identified. Here, we systematically identified and characterized AP2/ERF proteins in wolfberry and identified key <i>AP2/ERF</i> family members involved in regulating <i>flavonoid biosynthesis. LbAP2/ERF</i> genes were identified via BLASTP and HMM analysis, using Arabidopsis AP2/ERF as queries and the <i>L. barbarum</i> genome. Gene duplication was analyzed with DupGen_finder, fruit RNA-seq was tested to determine expression profiles across five developmental stages, and <i>LbAP2/ERF089</i> function was verified using transient overexpression and dual-luciferase assays. A total of 148 genes belonging to the <i>L. barbarum AP2/ERF</i> (<i>LbAP2/ERF</i>) family were identified, with dispersed duplication likely being the primary driver of their amplification. The <i>LbAP2/ERF</i> 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 <i>LbAP2/ERF</i> genes involved in flavonoid biosynthesis were identified, including <i>LbAP2/ERF089, LbAP2/ERF011, LbAP2/ERF068</i>, and <i>LbAP2/ERF099</i>. Functional analysis further revealed that <i>LbAP2/ERF089</i> positively regulates flavonoid synthesis by activating the expression of biosynthetic genes <i>LbLAR</i> and <i>LbDFR</i>. 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.