Structural and functional characterization of peanut expansin proteins identifies AhEXPA3 as a stress-responsive...

Expansins are cell wall-associated proteins that play important roles in plant growth, development, and environmental responses, yet their structural features and functional significance in peanut remain insufficiently understood. Here, we performed a genome-wide identification and characterization of 70 expansin proteins in cultivated peanut. Phylogenetic analysis classified these genes into four subfamilies (EXPA, EXPB, EXLA, and EXLB), with conserved motif patterns and subgroup-specific exon-intron structures. Collinearity and evolutionary analyses revealed that segmental duplication mainly drove peanut expansin family expansion, with most duplicated gene pairs subsequently undergoing purifying selection. Promoter analysis identified abundant cis-regulatory elements associated with light responses, hormone signaling, and stress responses. Expression profiling indicated that many AhEXP genes were differentially expressed during seed germination and in response to abscisic acid (ABA), salt, and osmotic stresses. Among them, AhEXPA3 was identified as a stress-responsive expansin protein with marked transcriptional induction under abiotic stress conditions. Subcellular localization analysis suggested that AhEXPA3 exhibits an endoplasmic reticulum-associated localization pattern. Functional assays further demonstrated that heterologous expression of AhEXPA3 inhibited seed germination and early seedling establishment under stress conditions in both Arabidopsis thaliana and rice. These findings support a negative regulatory role of AhEXPA3 in stress-responsive seed germination and broaden current understanding of expansin protein function in legumes.