Identification and analysis of the AP2/ERF gene family in Dendrobium officinale based on pan-genome and functional characterization of DofERF109_2

Introduction AP2/ERF transcription factors are key regulators of plant stress responses and developmental processes. Despite their functional significance, limited research has focused on this gene family in the medicinal orchid Dendrobium officinale . Methods Based on the pangenome data of seven Dendrobium officinale individuals from different habitats, we performed a pangenome family analysis of AP2/ERF, including analyses of presence-absence variation (PAV), selection pressure, transposable elements, etc., and conducted functional validation of the screened key members. Results A total of 101, 76, 113, 123, 113, 105 and 113 AP2/ERF genes were identified in the seven Dendrobium officinale individuals, respectively. PAV analysis classified the non redundant members into core (29), softcore (28), dispensable (17) and private (3) genes. Compared with Arabidopsis thaliana , D. officinale AP2/ERFs exhibited a significant evolutionary contraction, although some genes underwent duplication. Most genes experienced negative selection, while a few showed positive selection. Cold and heat stress induced differential expression patterns; genes with stable expression were predominantly core or softcore members. The candidate DofERF109_2 localized to the nucleus. Its transient expression suppressed anthocyanin accumulation in tobacco and downregulated the key enzyme gene DofCHI in D. officinale . Discussion Our pan genome analysis demonstrates that the AP2/ERF family in D. officinale has undergone significant evolutionary contraction compared with Arabidopsis, suggesting lineage specific gene loss or rapid divergence in orchids. Despite this overall contraction, lineage specific duplications (e.g., ERF109 and RAP2.11 ) were observed, which may provide raw material for adaptive evolution. The classification into core, softcore, dispensable and private genes reveals a conserved set likely involved in essential functions, whereas variable genes may contribute to local adaptation. Ka/Ks analysis identified positive selection only in a few genes, often those with recent duplications, supporting neofunctionalization. The widespread presence of transposable elements (68.8% of members) suggests that TE insertion is a common, ongoing process that may generate regulatory diversity without being strongly counter selected. Expression profiling under temperature stress further highlighted functional divergence: cold stress induced gradual upregulation, while heat stress caused downregulation of most genes. Notably, the nuclear localized DofERF109_2 negatively regulated anthocyanin biosynthesis by suppressing DofCHI expression and reducing pigment accumulation. Together, our results provide a comprehensive pan genome resource of AP2/ERFs in D. officinale and identify DofERF109_2 as a candidate negative regulator of anthocyanin synthesis.