Overexpression of PavGRF5 in sweet cherry negatively regulates cold stress tolerance in transgenic Arabidopsis

• Elucidated the role of PavGRF5 in regulating physiological changes in plants under low-temperature stress. • Through genetic transformation, we demonstrated that PavGRF5 enhances cold tolerance in transgenic plants, providing a valuable genetic resource for molecular breeding aimed at improving cold resistance. • PavGRF5 and PavGIF1 synergistically participate in the cold stress response in sweet cherry (Prunus avium). Growth-regulating factors (GRFs) are essential for the growth, development, and stress responses of organisms. However, studies on GRF genes in sweet cherry ( Prunus avium L.) remain limited, and the underlying mechanisms by which they respond to low-temperature stress are still unclear. PavGRF5 was found to be down-regulated under cold stress induction. In this study, we cloned PavGRF5 , a GRF family member significantly suppressed under low-temperature stress, and conducted genetic transformation experiments in Arabidopsis thaliana to investigate its functional role in response to cold stress. Our results demonstrated that Arabidopsis lines overexpressing PavGRF5 exhibited impaired growth under low-temperature conditions. Compared with the wild type (WT), these transgenic lines exhibited significantly elevated levels of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ), as well as increased accumulation of reactive oxygen species (ROS). Moreover, the activities of key antioxidant enzymes-including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT)-were markedly reduced in transgenic plants. Consistently, the contents of osmoprotectants such as proline, soluble proteins, and soluble sugars were also significantly decreased. We employed virus-induced gene silencing (VIGS) and antisense oligonucleotides (AsODN) to suppress the expression of the PavGRF5 gene in sweet cherry. Our findings demonstrated that PavGRF5 expression was significantly downregulated in the silenced lines, whereas the expression of low-temperature positively regulated genes in sweet cherry were markedly increased. Transcriptional activation assays revealed that PavGRF5 does not possess intrinsic transcriptional activity. Using yeast two-hybrid and luciferase complementation assays, we confirmed a direct physical interaction between PavGRF5 and PavGIF1. Notably, cold stress suppressed the expression of both PavGRF5 and PavGIF1, potentially alleviating the negative regulatory effects of the PavGRF5–PavGIF1 module during the low-temperature response in sweet cherry. We cloned the PavGRF5 promoter, which contains a cis-acting element responsive to low-temperature. Analysis of PavGRF5 promoter activity revealed Ruby reporter gene expression at the stem–leaf junctions of transgenic Arabidopsis seedlings. Taken together, our findings elucidate a negative feedback regulatory mechanism involving PavGRF5 in the response to low-temperature stress, offering novel insights into the molecular basis of abiotic stress tolerance in sweet cherry.