Comparative in-silico analysis of the bHLH protein family in rice Oryza sativa subsp. japonica cv. Nipponbare

The basic Helix-Loop-Helix (bHLH) proteins are key regulators of gene expression, development and responses to environmental stimuli in plants. In this study, we performed a comprehensive computational analysis of six bHLH proteins (OsbHLH056, OsbHLH057, OsbHLH058, OsbHLH059, OsbHLH062 and OsbHLH063) in Oryza sativa subsp. japonica cv. Nipponbare. Their physicochemical properties, secondary and tertiary structures, domains, phylogenetic relationships, subcellular localisation and protein-protein interactions were investigated. Results showed that OsbHLH062 had the highest molecular weight (29,686.27 kilodalton (kDa) and amino acid number (265), while OsbHLH058 had the lowest (7,896.08 kDa; 77 amino acids). Isoelectric point analysis indicated five proteins were acidic, while OsbHLH058 was basic. All proteins were predicted to be unstable, reflecting the flexibility essential for regulation. Aliphatic index values (61.13–85.26) suggested moderate thermo-stability. The secondary structure was dominated by α-helices, which enhance structural stability and the extinction coefficients suggested enrichment of cysteine, tryptophan and tyrosine residues. Phylogenetic analysis showed OsbHLH057 as the earliest ancestor among the six proteins. Subcellular localisation predictions identified nuclear targeting for all proteins, with OsbHLH059 showing the highest nuclear localisation probability. Protein-protein interaction analysis highlighted potential partners, implying roles in diverse cellular pathways. This study provides valuable insights into the molecular characteristics, structure and interactions of rice bHLH proteins. These findings form a foundation for experimental validation and functional characterisation. Understanding these proteins may enable the development of innovative strategies to enhance abiotic stress tolerance and crop productivity in rice.