| Summary: | Abscisic acid (ABA) is a key phytohormone involved in regulating plant growth and responses to environmental stress. As receptors of ABA, pyrabactin resistance 1 (PYR)/PYR1-like (PYL) proteins play a central role in initiating ABA signal transduction. In this study, a total of 30 <i>PopPYL</i> genes were identified and classified into three sub-families (PYL I–III) in the pan-genome of 17 <i>Populus</i> species, through phylogenetic analysis. Among these subfamilies, the PYL I subfamily was the largest, comprising 21 members, whereas PYL III was the smallest, with only four members. To elucidate the evolutionary dynamics of these genes, we conducted synteny and <i>Ka</i>/<i>Ks</i> analyses. Results indicated that most <i>PopPYL</i> genes had undergone purifying selection (<i>Ka</i>/<i>Ks</i> < 1), while a few were subject to positive selection (<i>Ka</i>/<i>Ks</i> > 1). Promoter analysis revealed 258 <i>cis</i>-regulatory elements in the <i>PYL</i> genes of <i>Populus euphratica</i> (EUP) and <i>Populus pruinosa</i> (PRU), including 127 elements responsive to abiotic stress and 33 ABA-related elements. Furthermore, six structural variations (SVs) were detected in <i>PYL_EUP</i> genes and significantly influenced gene expression levels (<i>p</i> < 0.05). To further explore the functional roles of <i>PYL</i> genes, we analyzed tissue-specific expression profiles of 17 <i>PYL_EUP</i> genes under drought stress conditions. <i>PYL6_EUP</i> was predominantly expressed in roots, <i>PYL17_EUP</i> exhibited leaf-specific expression, and <i>PYL1_EUP</i> showed elevated expression in stems. These findings suggest that the drought response of <i>PYL_EUP</i> genes is tissue-specific. Overall, this study highlights the utility of pan-genomics in elucidating gene family evolution and suggests that <i>PYL_EUP</i> genes contribute to the regulation of drought stress responses in EUP, offering valuable genetic resources for functional characterization of <i>PYL</i> genes.
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