Title： Comprehensive characterization of the genes in AP2/ERF family and their involvement in salt–alkali stress response during Nelumbo nucifera seed germination

Background: Nelumbo nucifera Gaertn. is an important aquatic crop valued for its ornamental, nutritional, and ecological traits. However, soil salinization and alkalization impose major constraints on its growth and productivity. The AP2/ERF transcription factor family plays central roles in plant responses to abiotic stresses, but their functions in salt–alkali tolerance of N. nucifera remain largely unknown. Results: We identified 101 AP2/ERF transcription factors in the N. nucifera genome, exhibiting diverse physicochemical properties and structural variations. Phylogenetic analysis grouped them into five subfamilies, showing both evolutionary conservation and species-specific divergence compared to Arabidopsis thaliana . Biochemical assays during seed germination under salt–alkali stress revealed dynamic changes in malondialdehyde, proline, superoxide dismutase, peroxidase, and catalase activities, indicating that AP2/ERF genes may enhance stress tolerance by regulating proline metabolism and reactive oxygen species scavenging. Transcriptome analysis of seedlings treated with 150 mM salt–alkali solution for 5 and 10 days identified 7,350 differentially expressed genes, including 29 AP2/ERFs with significant expression changes. Among them, 13 genes, such as AP2-9 , ERF23 , ERF15 , ERF31 , ERF34 , and DREB21 , were consistently upregulated under stress, suggesting their key roles in stress adaptation. Furthermore, qRT-PCR validation confirmed the upregulated expression of AP2-9 , ERF23 , ERF34, and DREB21 in both 5-day and 10-day salt–alkaline treatments, corroborating the transcriptome results and supporting their involvement in the regulation of salt–alkali stress responses during seed germination. Conclusions: This study provides the first comprehensive characterization of the AP2/ERF gene family in N. nucifera and highlights their involvement in salt–alkali stress responses. These findings advance our understanding of the molecular mechanisms underlying stress adaptation in N. nucifera and provide a theoretical basis for breeding salt–alkali tolerant varieties, thereby enhancing the ecological and economic value of saline–alkaline wetlands.