Genome-wide identification of peroxidase genes and functional analysis of MtPRX76 on lignin synthesis in Medicago truncatula.

Peroxidases (PRXs) are involved in diverse physiological processes, including cell elongation and lignification. However, studies on PRX genes and their tissue specificity in Medicago truncatula remain limited. In this study, 117 MtPRX genes were identified through bioinformatic analysis and classified into five distinct groups. Segmental duplications were identified as the major driving force for MtPRX expansion. Evolutionary analysis revealed closer phylogenetic relationships between MtPRX and GmPRX in soybean. Expression of MtPRXs were detected in roots, stems, leaves, flowers, seeds, and leaf buds, with members exhibiting distinct tissue-specific expression patterns. Tnt1 insertion mutants of the tissue-specific gene MtPRX76, designated mtprx76-1 and mtprx76-2, showed significantly reduced gene expression levels and decreased lignin content. Transcriptome analysis identified 3015 and 3564 differentially expressed genes (DEGs) in mtprx76-1 and mtprx76-2, respectively. GO and KEGG enrichment analyses revealed that the phenylpropanoid biosynthesis pathway was the most significantly enriched. Furthermore, transcriptional levels of 14 key regulatory genes involved in lignin biosynthesis were significantly downregulated in both mutant lines. These results demonstrate that MtPRX76 functions as a positive regulator influencing lignin biosynthesis. This study systematically characterizes the member features, sequence structures, evolutionary relationships, and tissue-specific expression patterns of the MtPRX gene family, and tissue specific expression patterns, while functionally validating MtPRX76. These findings establish a theoretical basis for understanding Class III PRX gene functions and breeding low lignin germplasm in alfalfa.