Bioinformatics-Driven Identification of Flooding-Tolerance Genes and Their Structural and Molecular Characterization in Soybean

Abstract Flooding stress is one of the important abiotic factors restricting soybean production globally. Despite numerous efforts, the genetic mechanisms of flooding tolerance are not fully understood. This work used a bioinformatics-driven, integrative approach combining structural analysis, molecular docking, expression profiling, and biochemical phenotyping to prioritize and characterize genes associated with flooding tolerance (FTgenes) during the early stages of soybean seedling development. Functional annotation of 83 previously prioritized FTgenes was used to classify them into five functional categories, with one candidate per category identified for further study: enolase (energy supply); phytocyanin (reactive oxygen species; ROS management); glycoside hydrolase family 17 (GH17; cell wall adjustment); ethylene and sulfur compound biosynthesis-related protein (ESCBp; hormonal signaling); and alfin zinc-finger protein (gene regulation). Comprehensive sequence analyses, phylogenetic relationships, motif predictions, and homology modeling were performed for these five candidate genes. Molecular docking analyses demonstrated that enolase, GH17, and ESCBp had conserved residues necessary for catalysis and forming stable complexes with respective ligands. Expression analysis showed distinct but coordinated transcriptional activation of the FTgenes during short-term flooding stress. Levels of salicylic acid and abscisic acid changed as a function of time upon flooding. In addition, protein levels, chlorophyll a content, total phenolics, flavonoids, and antioxidant capacity were modulated in an organ-dependent manner. Overall, this study has provided evidence for a coordinated network of energy metabolism, ROS homeostasis, cell wall adjustment, hormone signaling, and regulation of gene expression that characterizes the early molecular response of soybean seedlings to flooding stress. These genes provide structurally and molecularly characterized candidate genes for future genetic improvement.