Lead (Pb), a heavy metal element widely present in the environment, does not contribute to the structural composition of plant tissues or participate in cellular metabolic activities (Carocci et al., 2016; Henry et al., 2015). However, its excessive accumulation can exert severe toxic effects on plants. In agriculture, soil Pb pollution can degrade soil fertility, consequently reducing crop yields (Aslam et al., 2021; Yan et al., 2023a). Moreover, environmental Pb is readily absorbed by plants through their roots and leaf surfaces, leading to its accumulation and distribution in various plant tissues. This can result in agricultural products exceeding permissible Pb limits, thereby compromising product quality and safety (Collin et al., 2022; Levin et al., 2021; Sun and Luo, 2018). Plants have evolved various mechanisms to resist lead (Pb) stress, with the cell wall playing a crucial role as the first line of defense (Collin et al., 2022). As a major structural component, the cell wall can act as a barrier to restrict Pb entry into cells by immobilizing Pb ions through adsorption onto its components, such as pectin, cellulose, hemicellulose, and lignin (Wu et al., 2020). Among the components of the cell wall, hemicellulose, plays a crucial role in responding to Pb stress by binding Pb ions, thereby immobilizing them and limiting their toxicity to plant cells (Zhang et al., 2020b).

Xyloglucan (XyG) is an essential hemicellulose polymer in the primary cell walls of dicotyledonous plants and non-commelinid monocotyledonous plants (Hrmova et al., 2022; Yan et al., 2019). Numerous genes are involved in the biosynthesis of xyloglucan, including those encoding glycosyltransferases that assemble its β-1,4-glucan backbone and side-chain modifications (Hayashi and Kaida, 2011; Pauly et al., 1999; Pauly and Keegstra, 2016). Among these enzymes, xyloglucan endotransglucosylase/hydrolase (XTH) enzymes play a key role in modifying xyloglucan by cleaving and reconnecting its chains, thereby regulating cell wall remodeling and expansion (Julian and Zabotina, 2022; Stratilová et al., 2020; Suo et al., 2024). To date, the XTH gene family has been identified across numerous plant species, with the number of XTH genes varying significantly among them. In monocotyledonous plants, rice, wheat, and pineapple contain 29, 71, and 24 XTH genes, respectively (Han et al., 2023; Li et al., 2019; Yokoyama et al., 2004). In contrast, dicotyledonous plants such as Arabidopsis, poplar, and sweet potato have 33, 38, and 36 XTH genes, respectively (Yang et al., 2023; Yokoyama and Nishitani, 2001; Zhang et al., 2023). In Arabidopsis, Pb stress obviously induces the expression of XTH18, and mutation of XTH18 enhanced the tolerance of plant to Pb stress (Zheng et al., 2021). In addition, the expression of XTH5, XTH20, and XTH31 are rapidly and consistently upregulated in Arabidopsis roots under Pb stress. This imply that XTHs might play a crucial role in plant response to Pb stress (Zheng et al., 2021). However, the role of XTH genes in response to Pb stress in other plant species is still poorly understood.

R. sativus, a widely cultivated root vegetable of the Brassicaceae family, is propagated through seeds and valued for its nutritional and medicinal properties. R. sativus roots are abundant in bioactive compounds, including glucosinolates, anthocyanins, and antioxidants, which exhibit anti-inflammatory, anticancer, and antioxidant activities, underscoring their significant economic and health benefits (Gamba et al., 2021; Khan et al., 2022). However, R. sativus is highly sensitive to lead (Pb) stress, which disrupts root system development, impairs water and nutrient uptake, and induces oxidative cellular damage. These effects lead to stunted growth, reduced yields, and economic losses, posing a challenge to sustainable production (Tang et al., 2021; Wang et al., 2015, 2013). Despite the known impacts of Pb stress, the molecular mechanisms underlying R. sativus responses to this heavy metal remain poorly understood.

To date, the identification of the XTH family in R. sativus under lead (Pb) stress remains largely unexplored. In this study, we first analyzed the homologous evolution and protein domains within the XTH gene family. Subsequently, we observed a significant upregulation of RsXTH25 in R. sativus roots under Pb stress conditions. Functional analysis of RsXTH25 further revealed its role in mediating Pb stress tolerance. In addition, several transcription factors, such as RsERF2, RsHD-ZIP22, and etc., exhibited strong positive correlations with RsXTH25, implying their roles in regulating Pb-induced RsXTH25 expression. These findings provide a foundational basis for future research into the molecular mechanisms of Pb stress responses in R. sativus and the development of genetically improved R. sativus varieties with enhanced Pb tolerance.

Xyloglucan endotransglucosylase/hydrolase (XTH) is a key enzyme involved in the remodeling of plant cell walls, capable of cleaving and reconnecting xyloglucans (Eklöf and Brumer, 2010). Genome-wide analyses have identified varying numbers of XTH genes across different plant species, including 33 in A. thaliana, and 38 in poplar, showing its extensive involvement in various stages of the plant life cycle (Cheng et al., 2021; Ishida and Yokoyama, 2022; Yan et al., 2019). Therefore, studying the XTH gene family is of great significance for understanding plant growth and development. However, the comprehensive characterization of the XTH gene family in R. sativus remains elusive. Here, our study has unveiled the presence of 28 XTHs in R. sativus.

Phylogenetic tree illustrates the evolutionary relationships of the XTH gene family in R. sativus (RsXTH), A. thaliana (AtXTH) and O. sativa (OsXTH), clustering the genes into five distinct groups (Groups I–V) ( Figure 3 ). The grouping reflects conserved evolutionary patterns, with notable gene expansion in R. sativus, likely due to segmental and tandem duplication events, as seen in other plant species such as soybean (Song et al., 2018). These duplications may have contributed to the diversification of RsXTH genes, enabling functional specialization. For example, genes within Group V (RsXTH22, RsXTH24, RsXTH27) or Group III (RsXTH5, RsXTH6, RsXTH15) could have undergone sub-functionalization or neofunctionalization, adapting to distinct biological roles. The clustering of RsXTH genes with AtXTH genes highlights conserved motifs and potential shared functions, such as regulating cell wall remodeling and environmental stress responses (Eklöf and Brumer, 2010; Hara et al., 2014). These findings provide a foundation for exploring the specific roles of RsXTH genes, especially under abiotic stress conditions like lead toxicity, and offer valuable insights into the evolution and functional diversity of the XTH gene family in R. sativus.

Plants can regulate their resistance to heavy metals such as Pb through structural modifications of the cell wall (e.g., polysaccharides, pectin, cellulose, lignin, and hemicellulose) and localized reinforcement (Krzesłowska, 2011; Krzesłowska et al., 2016). When plants are exposed to Pb stress, numerous cell wall-related genes are either upregulated or downregulated, indicating that cell wall modification serves as a common defense strategy for plants to adapt to Pb stress (Zheng et al., 2021). For example, in A. thaliana, the expression levels of XTH5, XTH18, XTH20, and XTH31 were rapidly and consistently upregulated in response to Pb stress (Zheng et al., 2021). In Chinese cabbage, BrXTH9_1 and BrXTH16 were both upregulated under Pb stress in varieties with low (LPA) and high (HPA) Pb accumulation. Interestingly, BrXTH9_1 showed a stronger induction in the HPA variety, whereas BrXTH16 exhibited a more pronounced upregulation in the LPA variety (Gao et al., 2023). This suggests that different XTH members may have similar or distinct functions in responding to Pb stress, even within the same species or across different species. Here, we found that several RsXTH genes (RsXTH10, RsXTH11, RsXTH16, RsXTH23, RsXTH24, RsXTH25, and RsXTH27) were obviously upregulated exposed to Pb stress. However, five RsXTH genes (RsXTH1, RsXTH2, RsXTH3, RsXTH4, and RsXTH5) were notably downregulated in response to Pb stress ( Figures 4 , 5 ). One possible explanation for the downregulation of these RsXTH genes is that it reflects a trade-off mechanism. Under Pb stress, plants may reallocate limited resources to prioritize essential survival processes, such as detoxification and antioxidant defense. Consequently, the expression of genes associated with growth and development, including certain XTH genes, is downregulated. This hypothesis is consistent with previous studies indicating that, during stress conditions, plants commonly suppress non-essential metabolic pathways to maintain vital physiological functions (Zhang et al., 2020a).

Although Pb stress can significantly induce changes in XTH expression and xyloglucan content, as reported in many studies (Gao et al., 2023; Zheng et al., 2021), there is still limited research on the specific biological functions of XTH in response to Pb stress. To the best of our knowledge, there is currently few reports showing that knocking out XTH18 in A. thaliana significantly enhances Pb tolerance (Zheng et al., 2021). Here, we found that RsXTH25 positively regulated Pb stress response in R. sativus based on the following evidences: firstly, Pb stress obviously induced RsXTH25 expression ( Figures 4 , 5 ). Secondly, transiently overexpressing of RsXTH25 significantly enhanced Pb tolerance ( Figures 6B–D ). Thirdly, transiently overexpression RsXTH25 alleviated the oxidative damage caused by Pb stress ( Figures 6H, I ). Our study clearly confirmed the critical role of XTH in R. sativus response to Pb stress. However, it is worth noting that several other RsXTH genes were also induced by Pb stress, and whether they are involved in the response of R. sativus to Pb stress remains to be investigated in future studies.

In addition, our study successfully identified potential upstream transcription factors involved in the regulation of RsXTH25 under lead (Pb) stress. Cluster analysis revealed that the gene module containing RsXTH25 was enriched with multiple transcription factor families, including ERF and WRKY, which are known to play key roles in abiotic stress responses (Jiang et al., 2017; Wu et al., 2022). This suggests that these transcription factors may contribute synergistically to enhancing plant tolerance to Pb stress. Notably, transcription factors such as RsERF2 and RsHD-ZIP22 exhibited strong positive correlations with RsXTH25 expression ( Figure 8 ). It is hypothesized that these factors participate in Pb stress responses, such as cell wall remodeling and reactive oxygen species (ROS) scavenging, by directly binding to the RsXTH25 promoter or indirectly regulating its expression via intermediate genes (Zhao et al., 2021). Future studies will be essential to validate these regulatory interactions and elucidate their biological functions, providing a theoretical foundation for the genetic improvement of Pb-tolerant crops.

In this study, phylogenetic analysis showed that 28 RsXTH genes were classified into five groups and distributed across 9 chromosomes. The analysis of conserved domains indicated a high similarity between RsXTHs and XTH proteins in other species. RsXTH25 was significantly upregulated under Pb stress and positively regulates Pb stress response. Moreover, transcription factors such as RsERF2 and RsHD-ZIP22 showed strong positive correlations with RsXTH25, suggesting that they may function as upstream regulators mediating its expression under Pb stress.