Plants are frequently exposed to abiotic stresses, among which high salt stress posing a serious threat to their growth and development. Salt stress leads to cellular dehydration, membrane damage, and impaired function of membrane-bound enzymes, resulting in metabolic disruption and altered plasma membrane permeability (Zhou et al., 2024). Excessive accumulation of salt ions further induces osmotic and oxidative stress, disturbing plant metabolism and energy homeostasis (van Zelm et al., 2020). Additionally, salt stress interferes with sugar signaling and alters the levels of key sugars, such as sucrose, fructose, and glycolytic intermediates (Zhao et al., 2021). These disruptions negatively affect photosynthesis, inhibit cell division and proliferation, and ultimately suppress overall plant growth and biomass accumulation (Zhao et al., 2021). To cope with salt-alkali stress, plants have evolved diverse physiological and molecular mechanisms, including the regulation of Na⁺ and K⁺ uptake and transport, activation of antioxidant enzymes to scavenge reactive oxygen species (ROS), and enhancement of osmotic adjustment capacity (Agarwal et al., 2013). Under salt stress, plants rapidly accumulate osmolytes such as proline, betaine, and organic acids, which contribute to osmotic balance and mitigate cellular damage (Rao et al., 2023). Moreover, salt stress induces the expression of a wide range of stress-responsive genes involved in osmoprotectant biosynthesis, ion transport, antioxidant defense, and signal transduction, which collectively enhance salt tolerance by modulating metabolic pathways (Kumar et al., 2021). These complex stress-response processes require precise regulation of gene expression and protein turnover. Among them, protein ubiquitination, a key post-translational regulatory mechanism, plays a central role in stress signal transduction and adaptive responses (Karthik et al., 2025). Proteins encoded by the U-box gene family function as critical executors of this regulatory pathway.

In this regard, the U-box gene family, encoding a major class of E3 ubiquitin ligases, has emerged as a critical regulator of plant stress responses. The U-box gene family encodes proteins characterized by the presence of a conserved U-box domain, most of which function as E3 ubiquitin ligases that confer substrate specificity in the ubiquitination pathway (Trenner et al., 2022). This family includes both HECT- and RING-type E3 ligases (Trenner et al., 2022). U-box proteins are widely distributed in plants and play crucial roles in cell cycle regulation, signal transduction, morphogenesis, protein degradation, secretion, and responses to environmental stimuli (Mao et al., 2022). They have been implicated in various developmental processes, including root and reproductive organ formation (Yee and Goring, 2009). For example, in Arabidopsis, PUB25 and PUB26 are key regulators of floral organ development (Li et al., 2021). In rice, loss of OsPUB33 function enhances photosynthesis and sugar transport, promoting cell proliferation and grain filling (Li et al., 2021). Mutation of OsPUB15 disrupts primary root formation, leading to stunted seedling growth or lethality (Wang et al., 2015). In wheat, TaPUB1 negatively regulates the abscisic acid (ABA) signaling pathway through interaction with TaPYL4 and TaABI5, thereby influencing seed development (Zhang et al., 2021b).

Furthermore, U-box genes are also involved in abiotic stress responses. In poplar, overexpression of PalPUB79 enhances drought tolerance by targeting PalWRKY77 for ubiquitination and degradation, thus positively regulating ABA signaling (Tong et al., 2021). Under salt stress, PUB30 mediates the degradation of HB24, downregulating SWEET11 expression, reducing sucrose availability, inhibiting root growth, and contributing to salt stress adaptation (Wang et al., 2023). In wheat, TaPUB1 modulates ion transporter activity to maintain Na⁺/K⁺ homeostasis and enhances antioxidant enzyme activity to mitigate oxidative stress, thereby improving salt tolerance (Wang et al., 2020). Additionally, TaPUB1 has been shown to regulate cadmium uptake and tolerance by modulating the stability of TaIRT1 and TaIAA17 proteins (Zhang et al., 2021a). PUB35 negatively regulates ABA signaling via AFP1-mediated degradation of ABI5, playing a role in salinity and osmotic stress responses (Li et al., 2024).

The U-box domain was first identified in the yeast protein UFD2, comprising a conserved domain of approximately 70 amino acids, marking it as the first known U-box protein (Hatakeyama and Nakayama, 2003). Subsequently, U-box family genes have been identified in various plants, including cotton (Lu et al., 2020), wheat (Yang et al., 2021) and pear (Wang et al., 2021). Recently, Liu et al. (2025) reported a genome-wide characterization of the U-box gene family in Populus alba and analyzed their transcriptional responses to abiotic stresses, providing an important reference framework for PUB genes in poplar. However, the regulatory roles of PUB genes in salt stress, particularly in Populus simonii × P. nigra, remain largely unexplored. In this study, a comprehensive genome-wide identification of PUB genes in poplar was conducted, accompanied by analyses of cis-regulatory elements and gene expression patterns under salt stress conditions. Furthermore, the evolutionary relationships and gene structures of the PUB family were systematically examined. These findings provide a theoretical basis and valuable genetic resources for the identification of candidate genes associated with salt stress tolerance in poplar.

The U-box gene family is broadly distributed in plants and plays a critical role in regulating growth and development, particularly under abiotic stress conditions (Trenner et al., 2022). In this study, 103 U-box genes were identified in the poplar genome and classified into six subfamilies. Notably, members of Group VI possess fewer introns, which may facilitate more efficient gene expression tailored to specific physiological functions. Conversely, Group V members contain a higher number of introns, potentially enabling more complex regulatory mechanisms and contributing to functional diversity. Motif analysis revealed nine conserved motifs with potential functional implications. Motif 1 corresponds to the conserved U-box domain responsible for E3 ubiquitin ligase activity, while other motifs likely contribute to substrate recognition, protein-protein interactions, or regulatory specificity (Van Roey et al., 2014). Furthermore, the presence of a subset of U-box proteins extracellularly indicates potential additional roles outside the nucleus, possibly in cell signaling or interactions with the cell wall and extracellular matrix (Trujillo, 2018). Similar localization patterns have been observed in cabbage, supporting the notion that such distribution is conserved among plant species (Wang et al., 2023). This conserved subcellular targeting underscores the functional versatility of U-box proteins, allowing them to participate in diverse cellular pathways critical for plant growth and adaptation.

The evolution of gene families is largely driven by DNA segmental duplications, tandem duplications, and gene conversion events, all of which contribute to genetic diversification and adaptive potential under stress conditions (Flagel and Wendel, 2009). Our analysis revealed that segmental duplication (28 pairs) was far more prevalent than tandem duplication (4 pairs) within the U-box gene family, indicating that segmental duplication is the primary mechanism driving its expansion in poplar. This phenomenon was also observed in the late embryogenesis-abundant (Cheng et al., 2021a) and xyloglucan endotransglucosylase/hydrolase (Cheng et al., 2021b) gene families of poplar. Furthermore, collinearity analysis with six other species demonstrated that poplar U-box genes share more syntenic gene pairs with dicotyledonous species than with monocots, suggesting greater evolutionary conservation within dicots. These duplicated genes likely contributed to the functional diversification of the U-box gene family during evolution.

Cis-acting elements in promoter regions are critical regulators of gene expression (Cui et al., 2023). Characterization of these elements enables the identification of genes involved in developmental and stress response pathways (Marand et al., 2023). We found that the promoter regions of poplar U-box genes are enriched with MYB binding sites associated with drought responsiveness, gibberellin response elements, endosperm expression motifs, and abscisic acid (ABA) response elements (Zhang et al., 2025). The abundance of these cis-elements indicates a significant involvement of U-box genes in hormone signaling pathways and stress responses. Moreover, the presence of shoot- and root-specific elements, cell cycle regulatory elements, and seed-specific motifs suggests that U-box genes may modulate poplar’s adaptation to abiotic stress by influencing developmental processes.

The U-box gene family plays a vital role in the development of plant tissues and organs and exhibits distinct tissue expression patterns. For example, in Coffea canephora, 40 genes are expressed in leaves, 33 in stems, and 14 in fruit tissues (Liu et al., 2024). Tomato U-box genes exhibit strong expression in meristems and leaf tissues, implicating their role in vegetative growth (Sharma and Taganna, 2020). In the present study, a tissue-specific expression analysis of 103 PpPUB genes revealed distinct spatiotemporal expression patterns in roots, stems, and leaves. Based on transcript levels, these genes were classified into five expression groups, underscoring their potential involvement in tissue-specific physiological processes. Similar expression patterns have also been observed in Setaria italica (Zhou et al., 2024).These findings provide valuable insights into the functional diversity of PpPUB genes during poplar growth and development.

U-box family genes play critical roles in plant responses to abiotic stress. For example, MYB-related elements in twelve SbPUB genes in sorghum regulate flavonoid biosynthesis, a key pathway contributing to salt tolerance (Cui et al., 2023). In contrast, overexpression of GmPUB8 in soybean leads to hypersensitivity to salt and drought stresses (Wang et al., 2016), highlighting the diverse functional roles of U-box genes across species. In this study, 40 differentially expressed PtrPUB genes were identified in roots and 21 in leaves under salt stress, with 14 genes co-expressed in both tissues. Functional annotation based on homologous Arabidopsis genes suggests that several poplar U-box genes may be involved in developmental regulation during stress. For example, AT3G52450 (homologous to Potri.010G103100.1) and AT2G35930 (homologous to Potri.006G202600.1) participate in water stress responses and negatively regulate PAMP-triggered immunity (Trujillo et al., 2008). AT1G60190 (homologous to Potri.012G078900.1) and PUB18 are implicated in repressing salt stress responses, thereby modulating salt tolerance (Bergler and Hoth, 2011). AT2G23140 (homologous to Potri.002G037700.1) is broadly expressed and regulates root development, pollen tapetum formation, and ROS-mediated chloroplast degradation (Wang et al., 2013). Additionally, AT2G45910 (homologous to Potri.001G239100.1) promotes seed germination and flower development (Cao et al., 2006). Collectively, these findings underscore the pivotal role of U-box genes in coordinating plant stress responses and developmental processes.

In summary, we identified 103 U-box gene family members in poplar, distributed across 18 chromosomes. Promoter analyses revealed abundant cis-acting elements related to abiotic stress response, growth, development, and hormone signaling. Collinearity analysis indicated four pairs of tandem duplications and 30 pairs of segmental duplications, highlighting the evolutionary dynamics of this gene family. RNA-Seq data revealed differential expression of U-box genes under salt stress, with 40 and 21 genes showing significant regulation in roots and leaves, respectively. These results collectively suggest that poplar U-box genes play essential roles in mediating responses to salt stress. This study provides a valuable theoretical foundation and gene resource for future efforts aimed at identifying and characterizing salt tolerance genes in poplar.