Genome-wide molecular characterization and expression profiling of the cysteine protease gene family in maize

Cysteine proteases (CPs), a pivotal class of proteolytic enzymes ubiquitously distributed across plant genomes, play critical roles in plant development, senescence, and immune responses. However, systematic investigations of CPs in maize (Zea mays L.) remain limited. In this study, we identified 47 cysteine protease genes (ZmCPs) in the maize B73_V5 genome using bioinformatics approaches. These genes were unevenly distributed across all maize chromosomes and classified into 9 phylogenetically distinct subfamilies, with conserved gene structures and motif compositions within each subfamily. Evolutionary analysis revealed that segmental duplication predominantly drove ZmCPs diversification. Most ZmCPs were predicted as hydrophilic proteins, primarily involved in cellular protein catabolism and organic substance degradation pathways. Tissue-specific expression profiling demonstrated that the majority of ZmCPs exhibited higher transcript abundance prior to the 10-day-after-pollination (10DAP) whole-kernel stage, followed by gradual downregulation. Reanalysis of public RNA-seq datasets identified RD21-clade members as consistently responsive to three lepidopteran insect stressors. qRT-PCR validation further delineated stage-specific defense roles: ZmCP20, ZmCP27, and ZmCP30 were pivotal during the initial phase of Spodoptera frugiperda (FAW) infestation, whereas ZmCP3 and ZmCP4 dominated late-stage defenses. Notably, ZmCP24 functioned as a sustained regulator throughout the defense continuum.This study not only elucidates the dynamic regulatory roles of ZmCPs in maize-lepidopteran interactions but also provides a molecular framework for developing insect-resistant maize cultivars through targeted manipulation of cysteine protease-mediated pathways.