Cold stress is one of the major abiotic stressor that profoundly impacts plant growth. Cotton, a widely cultivated variety, is particularly susceptible to cold stress. Unraveling the responses to cold stress is critical for cotton demand. In this investigation, we conducted comparative physiological and transcriptomic analyses of the cold-tolerant variety XLZ16 and cold-sensitive variety XLZ84 at seedling emergence stage under cold stress. Following exposure to cold stress, XLZ16 exhibited a markedly higher growth phenotype and increased activity of antioxidant enzymes, while simultaneously showing reduced cellular oxidative damage and apoptosis. Furthermore, the levels of auxin (IAA), cytokinin (CTK), and salicylic acid (SA) significantly increased during cold stress, whereas the contents of catendorsterol (TY), brassinosterone (CS), and jasmonic acid (JA) significantly decreased. Integrated with stoichiometric analysis, these findings definitively demonstrated significant differences in antioxidant capacity and hormone content between the two varieties during their response to cold stress. A total of 6207 potential cold-responsive differentially expressed genes (DEGs) were identified through transcriptome sequencing analysis. Enrichment analyses of these DEGs revealed that pathways related to "hormones biosynthesis and signaling" as well as "circadian rhythm" were associated with cold response. Notably, the hub gene Gh_D12G2567 (GhJAZ3), encoding jasmonate ZIM-domain (JAZ) proteins, was found to influence the JA signal transduction pathway and regulate cotton growth under cold stress within the MEred module network. Furthermore, suppressing the expression level of GhJAZ3 by virus-induced gene silencing led to the reduction of cold resistance, implying GhJAZ3 as a positive regulator of cold tolerance. This study provides valuable insights into the response mechanisms of cotton under cold stress. It also serves as a reference and foundation for further enhancing cold tolerance of new cotton varieties.
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.