Characterization of phenylalanine ammonia-lyase genes in soybean: genomic insights and expression analysis under abiotic stress tolerance

• PAL is the key enzyme in the phenylpropanoid pathway. • Nine PAL genes were identified and characterized in soybean genome. • Synteny analysis links GmPAL genes to dicots with shared evolutionary origin. • GmPAL3/4/5 and GmPAL7 significantallyt upregulated under stresses, key for soybean stress resilience. • GmPAL genes provide insights for soybean improvement to environmental stresses. Phenylalanine ammonia-lyase (PAL) is a critical enzyme in the phenylpropanoid pathway, playing essential roles in plant development and responses to environmental stresses. Despite its importance, an inclusive characterization of the PAL gene family in soybean ( Glycine max ) has yet to be fully explored. This study identified nine PAL genes within the soybean genome, which were randomly distributed across six chromosomes including Gm02, Gm03, Gm10, Gm13, Gm19 and Gm20. All genes comprised of Lyase_aromatic domain with different physicochemical properties. Phylogenetic analysis grouped PAL proteins into three main clades, with closeness to dicots species such as Arabidopsis thaliana, Citrullus lanatus and Vitis vinifera. GmPAL Gene structure analysis revealed 10 conserved motifs and 2 exons across all genes. Cis -regulatory element analysis highlighted key elements associated with plant growth and development (13%), hormone signaling (36%), light responsiveness (37%), and stress responsiveness (14%). Synteny analyses revealed 11 segmentally duplicated GmPAL genes, which underwent purifying selection. Furthermore, GmPAL collinearity analyses demonstrated significant homology with dicot species, suggesting a shared evolutionary origin and potential functional conservation. Protein-protein interaction and 3D modeling confirmed GmPAL proteins involvement in phenylpropanoid regulation. Furthermore, 89 gma-miRNAs from 32 different families were predicted that targeted all genes. TF analysis revealed significant associations in stress regulation. GO and KEGG analysis linked GmPAL genes to biological processes and the phenylpropanoid pathway. FPKM-based expression profiling revealed tissue-specific and stress-responsive expression patterns for GmPAL genes, under various conditions. Furthermore, qRT-PCR confirmed diverse expression of all GmPAL genes in soybean leaves under abiotic stresses under cold, heat, drought, salinity, metal ion toxicity, and hormone treatments. Notably, GmPAL3/4/5 and GmPAL7 were significantly upregulated under all stresses, while CdCl₂ uniquely upregulated all GmPAL gene expressions, highlighting their potential for soybean stress resilience studies. This comprehensive study provides valuable insights into the structure, evolution, and functional regulation of GmPAL genes, offering a foundation for future research and their potential application in improving soybean resilience to environmental stresses.