Comparative structural, evolutionary, and functional insights into rice γ-glutamyl cyclotransferases reveal differential stability and stress-responsive expression

γ-Glutamyl cyclotransferases (GGCTs) play an important role in glutathione catabolism and redox regulation in plants under diverse stress conditions. In this study, two GGCT isoforms were identified in rice: OsGGCT1 (chromosome 4) and OsGGCT2;1 (chromosome 2), sharing 64.7% nucleotide identity. Phylogenetic analysis showed that both isoforms are conserved and cluster with monocot homologs, suggesting divergence from a common ancestor. Physicochemical characterization indicated differences in stability, with OsGGCT1 exhibiting a lower instability index compared to OsGGCT2;1 . Secondary structure predictions showed random coils as the dominant feature in both proteins, while domain analysis confirmed their belonging to the ChaC family. Promoter cis-regulatory element analysis indicated robust basal transcriptional potential and efficient recruitment of transcriptional machinery. Structural modeling with AlphaFold generated high-confidence 3D structures, which were further validated through stereochemical quality assessments. Molecular docking and molecular dynamics simulations revealed distinct interaction patterns with glutathione: GGCT2;1 displayed stronger binding affinity but greater conformational flexibility, whereas GGCT1 maintained lower structural fluctuations and compactness. Principal component analysis supported these findings, highlighting restricted motions in GGCT1 compared to the dynamic rearrangements in GGCT2;1. Gene expression analysis demonstrated functional divergence under stress conditions: OsGGCT2;1 was strongly induced by arsenate and salt stress, while OsGGCT1 was predominantly upregulated under cold and Fusarium infection. Collectively, these results provide new insights into the structural-functional divergence of rice GGCT isoforms, highlighting their distinct roles in abiotic and biotic stress adaptation.