[Identification and analysis of the GRF gene family in peanut]

Peanut (Arachis hypogaea L.) is an important oilseed crop widely cultivated in tropical and subtropical regions. The growth-regulating factors (GRFs) are key transcription factors that regulate plant growth and responses to stress. To improve the peanut yield and stress tolerance, it is crucial to investigate the roles of GRFs in growth, development, and stress responses. In this study, we analyzed the physicochemical properties, evolutionary relationships, chromosomal localization, and sequence variations of the AhGRF gene family by bioinformatics methods. Using qRT-PCR, we revealed the expression patterns of AhGRF genes under drought and cold stress conditions. Subcellular localization expression vectors were constructed to determine the cellular distribution of AhGRF2b and AhGRF3b. Finally, yeast two-hybrid (Y2H) assays were performed to identify interacting proteins of AhGRF3b. The results revealed that twenty-four AhGRF genes were identified in peanut, which were unevenly distributed across 16 chromosomes. The deduced proteins ranged from 268 to 630 aa in length, with molecular weights spanning 29 842.27 to 67 980.83 Da. Most AhGRFs were acidic and predicted to be localized in the nucleus. Phylogenetic analysis classified the AhGRF family members into six distinct clades. Multiple sequence alignment demonstrated that the majority of AhGRF genes contained conserved QLQ and WRC domains. Under drought and cold stress conditions, several AhGRF genes, particularly AhGRF2b and AhGRF3b, exhibited significantly upregulated expression, which indicated their responsiveness to abiotic stresses. Transient expression in tobacco showed that AhGRF2b was localized in both the nucleus and cytoplasm, while AhGRF3b was localized in the nucleus. Furthermore, Y2H assays revealed that AhGRF3b may interact with AhCAT3 (catalase), suggesting that AhGRF genes may enhance stress tolerance by regulating reactive oxygen species scavenging. These findings provide a theoretical basis for improving the stress tolerance in peanut breeding programs.