Targeted Regulation of AhGRF3b by ahy-miR396 Modulates Leaf Growth and Cold Tolerance in Peanut

Peanut (Arachis hypogaea L.) is an important oil and cash crop, but its growth and productivity are severely constrained by low-temperature stress. Growth-regulating factors (GRFs) are plant-specific transcription factors involved in development and stress responses, yet their roles in peanut remain poorly understood. In this study, we identified AhGRF3b as a direct target of ahy-miR396 using degradome sequencing, which demonstrated precise miRNA-mediated cleavage sites within the AhGRF3b transcript. Expression profiling confirmed that ahy-miR396 suppresses AhGRF3b via post-transcriptional cleavage rather than translational repression. Functional analyses showed that overexpression of AhGRF3b in Arabidopsis thaliana promoted leaf expansion by enhancing cell proliferation. Specifically, leaf length, width, and petiole length increased by 104%, 22%, and 28%, respectively (p < 0.05). Under cold stress (0 °C for 7 days), transgenic lines (OE-2 and OE-6) exhibited significantly better growth than Col-0, with fresh weight increased by 158% and 146%, respectively (p < 0.05). Effect size analysis further confirmed these differences (Cohen's d = 11.6 for OE-2 vs. Col-0; d = 6.3 for OE-6 vs. Col-0). Protein-protein interaction assays, performed using the yeast two-hybrid (Y2H) system and 3D protein-protein docking models, further supported that AhGRF3b interacts with Catalase 1 (AhCAT1), vacuolar cation/proton exchanger 3 (AhCAX3), probable polyamine oxidase 4 (AhPAO4), and ACT domain-containing protein 11 (AhACR11), which are involved in reactive oxygen species (ROS) scavenging and ion homeostasis. These interactions were associated with enhanced CAT and PAO enzymatic activities, reduced ROS accumulation, and upregulation of stress-related genes under cold stress. These findings suggest that the ahy-miR396/AhGRF3b module plays a potential regulatory role in leaf morphogenesis and cold tolerance, providing valuable genetic resources for breeding cold-tolerant peanut varieties.