Genome-Wide Analysis of the PIN-FORMED Gene Family in Populus euphratica and Functional Characterization of PePIN1a Through Heterologous Expression in Arabidopsis thaliana

PIN-FORMED (PIN) auxin efflux carriers are key regulators of polar auxin transport, a process that controls plant organ morphogenesis and adaptive development. Nevertheless, genome-wide information on the PIN gene family in Populus euphratica Oliv. is limited, and how PIN homologs function during its distinctive heterophyllous leaf development remains unknown. Here, we systematically identified PIN genes throughout the P. euphratica genome and examined the subcellular localization and developmental effects of heterologously expressed PePIN1a in Arabidopsis thaliana. Bioinformatic analyses revealed 32 PePIN genes on 13 chromosomes and scaffolds, with segmental duplication contributing substantially to family expansion. Phylogenetic reconstruction resolved three PePIN subfamilies whose members exhibited conserved gene structures, protein motifs, and functional domains. Analysis of cis-acting elements indicated that most PePIN genes contained multiple elements responsive to hormones, light, and abiotic stress. Transcriptomic comparisons of four leaf morphotypes (linear, lanceolate, ovate, and broad-ovate) revealed relatively high PePIN1a expression in leaf tissues. Its greater expression in broad-ovate than in linear leaves prompted its selection for further functional validation. Subcellular localization assays revealed peripheral fluorescence from 35S::PePIN1a-GFP, consistent with association with the plasma membrane. In A. thaliana, PePIN1a overexpression significantly increased the number of rosette leaves relative to wild-type plants, while expression in the pin1 mutant rescued the reduced-leaf-number phenotype. These results systematically characterize the P. euphratica PIN family and show that PePIN1a alters rosette leaf number and exhibits PIN1-like activity in A. thaliana. PePIN1a is therefore a candidate for further studies of heterophyllous leaf development in P. euphratica. Because functional assays were conducted only in A. thaliana, its native role in P. euphratica heterophylly still requires validation.