Genome-wide identification and expression analysis of ferric reductase oxidase (FRO) genes in Gossypium spp. reveal their crucial role in iron homeostasis under abiotic and biotic stress.

Ferric Reductase Oxidase (FRO) genes are pivotal in iron uptake and homeostasis in plants, yet they are not studied in cotton. Here, we identify and analyze 65 FRO homologs (21 GhFRO, 21 GbFRO, 11 GaFRO, 12 GrFRO) across four Gossypium species (G. hirsutum, G. barbadense, G. arboreum, G. raimondii). FRO exhibit conserved ferric reductase activity and conserved domain structures; Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 (PF08030) across species. Physicochemical properties and subcellular localization analysis provided insights into FRO proteins' functional characteristics, mainly localized to the plasma membrane. Phylogenetic analysis delineates 11 groups, indicating both conserved and divergent evolutionary patterns. Gene structure analysis unveils varying exon-intron compositions. Chromosomal localization shows distribution across A and D genomes, suggesting evolutionary dynamics. Synteny analysis reveals paralogous and orthologous gene pairs subjected to purifying selection. The cis-regulatory elements analysis implicates diverse regulatory mechanisms. Expression profiling highlights dynamic regulation across developmental stages, abiotic and biotic stress conditions. GhFRO interacts with Ca⁺⁺-dependent protein kinases-10/28-like (CDPKs10/28-like) and metal transporter Natural resistance-associated macrophage protein 6 (Nramp6) to regulate metal ion transport and iron homeostasis. The three-dimensional protein structure prediction suggests potential ligand-binding sites in FRO proteins. Moreover, qRT-PCR analysis of selected eight GhFROs in leaves treated with stress elicitors, MeJA, SA, NaCl, and PEG for 1h, 2h, 4h, and 6h revealed significant downregulation. Overall, this comprehensive study provides insights into FRO gene diversity, evolution, structure, regulation, and function in cotton, with implications for understanding plant iron homeostasis and stress responses.