Transcriptome Analysis Reveals the Role of OlMYB35 in Drought Response of Opisthopappus longilobus

Cliff habitats are characterized by limited and heterogeneous water availability, requiring plants to develop adaptive strategies to cope with drought stress. Opisthopappus longilobus, a cliff-endemic Asteraceae species restricted to the Taihang Mountains of northern China, has evolved under persistent water-limited conditions and represents a valuable model for investigating the molecular mechanisms underlying drought adaptation. However, the transcriptional regulatory networks involved in its drought response remain largely unexplored. In this study, we performed RNA sequencing of O. longilobus leaves under control and drought conditions to investigate drought-responsive regulatory networks. Six RNA-seq libraries were generated, and a total of 5260 differentially expressed genes (DEGs) were identified in response to drought stress. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that these DEGs were mainly associated with phytohormone signal transduction, stress-responsive regulation, defense responses, metabolic reprogramming, and transcriptional regulation. Notably, multiple transcription factor families, including MYB, ERF, and ABF, were enriched among drought-responsive genes, suggesting their involvement in drought adaptation. Furthermore, quantitative RT-PCR was used to validate the RNA-seq results. Among the drought-responsive transcription factors, an R2R3-MYB transcription factor, OlMYB35, was identified as a candidate regulator and was further demonstrated to play a positive role in drought response through transient transformation assays. Taken together, this study provides new insights into drought-responsive regulatory mechanisms in O. longilobus and identifies OlMYB35 as a promising candidate gene for further functional validation and potential application in stress-resilient chrysanthemum breeding.