Decoding the genomic landscape of class III peroxidase family in Saccharum officinarum and the immune role of ScPOD01

• Sugarcane POD activity increased broadly during Sporisorium scitamineum infection. • A total of 174 POD genes were identified in the Saccharum officinarum genome. • Structural diversity and evolutionary mechanisms of PODs were elucidated. • The expression of POD genes is tissue-specific and stress-responsive. • ScPOD01 , a S. scitamineum -induced POD , activated plant immunity via HR/SA pathways. Class III peroxidases (PODs) are crucial for plant growth and stress responses, yet few have been characterized in sugarcane ( Saccharum spp.). In this study, elevated POD activity was detected across eight sugarcane genotypes during their interaction with Sporisorium scitamineum , suggesting a role in smut defense. To further explore this gene family, 174 SoPOD genes, classified into seven subgroups, were identified in S. officinarum LA-purple. Family expansion primarily was assumed to result from whole-genome/segmental duplications (83.90%), with Ka/Ks < 1 in 94.74% of gene pairs. SoPOD promoters were enriched in stress- and hormone-related cis -regulatory elements. Transcriptome data revealed that SoPOD expression was tissue-specific and responsive to cold, drought, and S. scitamineum infection. Notably, SoPOD30 was exclusively upregulated in the resistant cultivar after infection but undetectable in the susceptible one. Its homolog ScPOD01 was subsequently isolated from sugarcane hybrid cultivars and shown to possess a canonical four-exon/three-intron gene structure and plasma membrane localization. Real-time quantitative PCR demonstrated ScPOD01 induction by S. scitamineum infection across all eight cultivars and under various abiotic stresses (abscisic acid, methyl jasmonate, hydrogen peroxide, polyethylene glycol, and sodium chloride). Interestingly, the transient expression of ScPOD01 in Nicotiana benthamiana activated immune responses, as evidenced by intensified DAB staining and upregulation of hypersensitive reaction- and SA-related genes, however its prokaryotic expression did not enhance bacterial stress tolerance. These findings uncover the evolutionary diversification and expression specificity of the sugarcane POD family and elucidate the immune function of ScPOD01 , laying a foundation for understanding POD roles in sugarcane disease resistance.