Investigation of Reference Genes for qRT-PCR and ARF Gene Family in Michelia compressa (Magnoliaceae) Under Cold Stress

Michelia compressa, a member of the Magnoliaceae family, is an evergreen tree of considerable significance in both landscape gardening and industrial production. However, during its introduction to northern subtropical regions in China, this species often suffers from frost damage, which limits its widespread application. The utilization of housekeeping genes is essential when performing gene family analyses under abiotic stress conditions. Additionally, auxin response factor (ARF) transcription factors (TF) play a crucial role in plant responses to abiotic stresses; however, their specific function in cold stress responses within M. compressa has not been systematically investigated. Ten housekeeping genes were selected from transcriptome data for evaluation using quantitative real-time PCR (qRT-PCR). The optimal housekeeping gene identified through screening was used for verification of gene family analysis. Additionally, key genes underwent functional validation. Analysis conducted with GeNorm, NormFinder, and BestKeeper identified 28S as the optimal reference gene for M. compressa under cold stress. Furthermore, an analysis of the ARF gene family using full-length transcriptome data revealed a total of 48 McoARF genes, which clustered into three groups alongside the Arabidopsis thaliana ARFs. Among these, eight selected McoARF genes exhibited significantly elevated expression levels in leaves under cold stress and demonstrated tissue specificity. Functional validation revealed that transgenic plants overexpressing McoARF13 displayed elevated levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2), and malondialdehyde (MDA), as well as increased activities of peroxidase (POD) and superoxide dismutase (SOD) in leaves under cold stress. This study represents the inaugural screening of housekeeping genes in M. compressa under cold stress conditions, accompanied by an analysis of the ARF gene family. The functional validation of McoARF13 was successfully conducted, offering valuable insights into the molecular mechanisms that underlie cold stress response in M. compressa.