Transgenic Cynodon dactylon overexpressing CdPIF4 alters plant development and cold stress tolerance

Bermudagrass [Cynodon dactylon (L.) Pers.] is widely used for soil remediation, livestock forage, and as turfgrass for sports fields, parks, and gardens due to its resilience and adaptability. However, low temperatures are critical factors limiting its geographical distribution and ornamental season, even preventing its safe overwintering. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) acts as a hub transcription factor, not only regulating various light responses but also integrating multiple external stimuli to improve plant productivity and architectural adaptation under adverse stress conditions, which makes it potential as a target gene. In this study, we cloned and characterized the CdPIF4 genes in bermudagrass. Expression analysis revealed that it is predominantly expressed in leaves and is regulated by photoperiod and cold stress. Using Agrobacterium-mediated genetic modification, we successfully generated CdPIF4a-overexpressing bermudagrass lines. Under cold stress at 4°C, these transgenic plants demonstrated enhanced cold tolerance, as indicated by higher relative water content, reduced membrane damage, and lower levels of lipid peroxidation levels. Photosynthetic analysis revealed that CdPIF4a-overexpressing plants exhibited higher light energy capture and transfer efficiency at this low temperature, with less energy loss. Additionally, they showed higher antioxidant enzyme activity and lower levels of reactive oxygen species levels. The responsive regulation of cold stress-related genes further validated the role of the CdPIF4a gene in enhancing cold tolerance. This study elucidates that CdPIF4 enhances cold tolerance in bermudagrass through physiological and molecular mechanisms, offering new insights and valuable genetic resources for advancing cold resistance research in bermudagrass and other grass species.