Pan-genome characterization of the maize 4CL gene family and its dynamic responses to abiotic stress

1.Pan-genome analysis across 26 maize inbred lines identified 13 Zm4CL genes (nine core and four near-core) classified into three evolutionary clades.2.Structural variations (SVs) are significantly associated with the expression and altered conserved protein domains of key Zm4CL genes.3.Zm4CL genes exhibit distinct tissue-specific expression patterns and dynamic enzymatic and transcriptional responses to stresses, particularly cold and drought.4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway and plays important roles in plant growth, development, and responses to environmental stresses. However, a comprehensive pan-genome analysis of the 4CL gene family in maize is still lacking. In this study, 13 Zm4CL genes were identified from a maize pan-genome comprising 26 diverse inbred lines, including nine core genes and four near-core genes. Phylogenetic analysis classified these genes into three evolutionary clades, while Ka/Ks analysis indicated that most members have been maintained under purifying selection, although several genes exhibited greater evolutionary divergence and relatively relaxed evolutionary constraints. Structural variation (SV) analysis revealed significant associations between SVs and the expression of Zm4CL2 and Zm4CL3, while sequence comparisons suggested that SVs were also associated with alterations in conserved protein domains in some genotypes. Transcriptome analyses revealed distinct tissue-specific expression patterns and diverse transcriptional responses to abiotic and biotic stresses. Enzyme activity assays showed that cold stress significantly increased 4CL activity at 12 h, whereas heat, salt, and alkali stresses caused an initial decrease followed by recovery, while drought had no significant effect. Time-course RT-qPCR further validated dynamic expression changes of representative Zm4CL genes under cold and drought stresses. Overall, this study provides a comprehensive pan-genome framework for understanding the evolutionary conservation, regulatory diversification, and stress-responsive characteristics of the maize Zm4CL gene family, providing valuable resources for future functional studies and the genetic improvement of stress tolerance in maize.