Xyloglucan endotransglucosylase/hydrolase 25 positively regulates the lead tolerance in Raphanus sativus
Tong Han, Yongmei Cui, Yang Jing, Meiying Liu, Xuanchao Chen, Yinghui Song, Xinping Gu, Jiahao Wang, Long Wang
TL;DR
This study identifies and characterizes XTH genes in radish, finding that RsXTH25 improves lead tolerance by reducing damage and boosting plant health.
Contribution
The novel contribution is the functional analysis of RsXTH25 in enhancing lead stress tolerance in Raphanus sativus.
Findings
RsXTH25 is strongly induced by lead stress and its overexpression improves plant tolerance.
Transgenic plants with RsXTH25 show reduced oxidative damage and better photosynthetic performance.
RsXTH25 correlates with transcription factors like RsERF2 and RsHD-ZIP22 under lead stress.
Abstract
Raphanus sativus, an important root vegetable native to China, is widely cultivated for its nutritional value and diverse applications. However, it is highly sensitive to lead (Pb) stress, with Pb predominantly accumulating in the roots. Previous studies have highlighted the pivotal role of xyloglucan endotransglucosylase/hydrolase (XTH) in plant responses to heavy metal stress. Despite this, a comprehensive identification, molecular characterization, and functional analysis of the XTH gene family in R. sativus has been lacking. In this study, 28 XTH genes were identified in R. sativus. Gene structure analysis revealed the presence of eight conserved motifs, along with variations in exon-intron organization and chromosomal distribution across all chromosomes. Phylogenetic analysis of XTH genes from R. sativus, Arabidopsis thaliana and Oryza sativa grouped them into five distinct clades,…
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TopicsPolysaccharides and Plant Cell Walls · Microbial Metabolites in Food Biotechnology · Food composition and properties
Introduction
1
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.
Materials and methods
2
Plant materials
2.1
The seeds of cherry R. sativus inbred line were disinfected, washed and germinated for 3 days, and then cultured for 3 weeks under the photoperiod of 24°C light for 14 hours and 20°C darkness for 10 hours. Transfer seedlings of similar size to plastic containers filled with Hoagland nutrient solution. After one week, the plants were treated with 0 and 200 mg/L Pb(NO3)2. Samples are taken every 4 days. Seedlings grown in Pb-free nutrient solution were used as control. Three replicates were used for each treatment, each with 6-8 plants.
Identification of XTH genes in R. sativus
2.2
To identify XTH genes in R. sativus, genomic nucleotide and amino acids sequences were obtained from the R. sativus genome database. HMMER (version 3.4) was used to screen for sequences containing both the Glycosyl Hydrolase domain (PF00722) and the Xyloglucan Endotransglucosylase (XET) C-terminal domain (PF06955), as annotated in the Pfam database (https://pfam.xfam.org/). The significance threshold for this screening was set at P < e^–5^. This analysis yielded 41 candidate protein. Subsequent domain confirmation using NCBI-CDD identified 28 putative XTH genes in the R. sativus genome (Mukherjee et al., 2009).
Phylogenetic analysis of XTH gene family
2.3
Full-length amino acid sequences XTHs of A. thaliana and O. sativa were retrieved from the Phytozome database and aligned with R. sativus XTH genes using MAFFT (version 7.526) (Katoh et al., 2019). A maximum likelihood phylogenetic tree was constructed with IQ-TREE (version 2.2.2.7) using default parameters and 1000 bootstrap replicates. The resulting tree was visualized and edited using Evolview v2 (He et al., 2016).
Gene structure analysis of XTH gene family in R. sativus
2.4
Conserved motifs in XTH proteins were identified using the MEME (version 5.5.4) Suite. These motifs were then validated against the NCBI-CDD to confirm their biological relevance. Gene structures and chromosomal locations were determined using the GFF3 annotation file from the R. sativus genome and visualized with TBtools II (version 2.016) (Chen et al., 2023).
RNA extraction and sequencing
2.5
Total RNA was extracted from plant tissue as described in (Liu et al., 2024). RNA quality was assessed with a NanoDrop 2000 spectrophotometer. High-quality RNA samples were then performed for next-generation sequencing using the Illumina platform. Raw sequencing data were subjected to quality control, during which reads containing adapters, poly-N sequences, or low-quality regions were removed. The Q20 and Q30 scores were evaluated using FastQC to further assess data quality. Clean reads were subsequently aligned to the R. sativus reference genome using HISAT2. Gene expression levels were quantified with featureCounts and normalized using the fragments per kilobase of transcript per million mapped reads (FPKM) method. The criteria for filtering differentially expressed genes were |log2FC| ≥ 1 and FDR < 0.05.
Reverse transcription and qRT-PCR analysis
2.6
Total RNA was extracted from R. sativus samples using the FastPure Plant Total RNA Isolation Kit (Vazyme, China) (Yan et al., 2024). High-quality RNA was used for cDNA synthesis, then used to perform qRT-PCR as described in (Liu et al., 2023). Relative gene expression was calculated using the 2^^–ΔΔCT^ method with RsActin as the reference gene (Yan et al., 2023b).
Transformation of hairy roots in R. sativus
2.7
The transformation of hairy roots in R. sativus was described in (Qin et al., 2024). In brief, the Agrobacterium rhizogenes strain MSU440 harboring the recombinant plasmid pCAMBIA1300, stored at -80°C, was revived in LB liquid medium containing 100 mg/L kanamycin and 50 mg/L streptomycin. The culture was incubated until the optical density at 600 nm (OD_600_) reached 0.6. The pellet was collected and resuspended in half-strength MS liquid medium. Under aseptic conditions, rootless seedlings were immersed in the bacterial suspension (OD_600_ = 0.8) supplemented with 200 μM acetosyringone for 10 minutes. The infection was carried out at 28°C on a shaking incubator set to 220 rpm. Following infection, the seedlings were co-cultivated in darkness for 2 days. Subsequently, they were transferred to decontamination medium containing 500 mg/L cefotaxime and cultured in a growth chamber at 25°C under a 16 h light/8 h dark photoperiod. Upon root induction, the transformed seedlings were treated with 200 mg/L Pb(NO_3_)2 for 20 days, after which the growth status of the hairy roots was assessed.
Subcellular localization
2.8
The coding sequences (CDS) of RsXTH25 were inserted into the pCAMBIA1300-GFP expression vector. The resulting fusion constructs were transiently expressed in tobacco leaf epidermal cells through Agrobacterium tumefaciens-mediated infiltration (Wang et al., 2024). At five days post-infiltration, GFP signals were observed using a confocal laser scanning microscope (LSM510, Carl Zeiss, Germany) following standard protocols.
Statistical analysis
2.9
All results are expressed as mean ± standard deviation (SD) from at least three replicates. The data were analyzed using one-way analysis of variance (ANOVA) in SPSS Statistics 29.0, with a P-value < 0.05 considered statistically significant.
Result
3
Identification of XTH gene family in R. sativus
3.1
In order to excavate the XTH (Xyloglucan Endotransglucosylase/Hydrolase) gene family in R. sativus, the Glycosyl hydrolases domain (PF00722) and the Xyloglucan endo-transglycosylase (XET) C-terminus domain (PF06955) were utilized to search for XTH genes via HMMER search. The results identified 45 and 41 genes respectively, among which 41 genes contained both domains. Further domain confirmation through NCBI-CDD revealed a total of 28 XTH genes in R. sativus.
Gene structure and conserved motif identification of XTH gene family in R. sativus
3.2
To further analyze the structural diversity of the RsXTH gene family, the conserved motifs were identified using MEME by TBtools-II. Figure 1 illustrates that the 28 XTH genes collectively harbor 8 conserved motifs. Motif 1, Motif 2-5, and Motif 7 are ubiquitous across all XTH genes. Motif 6 is consistently present from XTH6 to XTH28, whereas motif 8 is only found in a select few XTH genes. RsXTHs show a conserved structure, and the NCBI-CDD analysis notably reveals that all R. sativus XTHs possess the conserved XTH domain (GH16_XET), with an average amino acid length of 298 aa, a maximum of 353 aa, and a minimum of 280 aa. The XTH genes display structural diversity at the DNA level, with exon counts varying between 2 and 4, and intron counts ranging from 1 to 3. As shown in Figure 2, the chromosomal localization analysis indicates that XTHs are not only distributed across all chromosomes but also present on segments RUS00775 and RUS00290.
Phylogenetic relationship, and gene structure of RsXTH gene family. (A) The phylogenetic relationship and motif distribution of RsXTHs. (B) The conservative structural domains of RsXTHs. (C) The distribution of coding sequences in RsXTHs.
Chromosomal distribution of RsXTHs in R. sativus.
Phylogenetic analysis of XTH gene family
3.3
The full-length amino acid sequences of XTH genes from R. sativus, A. thaliana and O. sativa were aligned by MAFFT (Supplementary Table S2). Subsequently, a phylogenetic tree was constructed using IQ-TREE with the maximum likelihood estimate method. As illustrated in Figure 3, all XTH genes can be categorized into five distinct groups. Notably, Cluster 3 and Cluster 5 contain the highest number of XTH genes, with a high degree of sequence similarity among the genes within each cluster. Further statistical analysis reveals that XTH genes from R. sativus and A. thaliana are almost distributed across other branches. In contrast, the XTH genes of O. sativa, a monocotyledonous plant, show a significant clustering in cluster 2. This finding highlights the widespread presence and potential functional diversity of the XTH gene family in monocotyledonous and dicotyledonous plants.
Transcriptome analysis of R. sativus under Pb treatments. (A) Principal component analysis (PCA). (B-D) The volcano plot of differential expression genes (DEGs) under Pb treatments. (E) The heatmap of RsXTHs under Pb treatments.
Transcriptome analysis of R. sativus seedlings under Pb treatment
3.4
To explore the function of RsXTHs in R. sativus seedlings under Pb stress, transcriptome sequencing was conducted on seedlings exposed to a Pb concentration of 200 mg/L. The transcriptome data yielded high-quality results, as shown in Supplementary Table S3. Principal component analysis (PCA) revealed a close correlation among the three replicates for each sample, confirming the data’s reliability (Figure 4A). Through transcriptomic differential expressed genes (DEGs) analysis, a series of genes showing differences were found under different Pb treatment conditions (Figures 4B-D; Supplementary Tables S4, S5). Further expression analysis of RsXTHs under Pb stress showed that several RsXTH genes, including RsXTH10, RsXTH11, RsXTH16, RsXTH27, RsXTH23, RsXTH24, and RsXTH25, were significantly upregulated in response to Pb treatment. In contrast, RsXTH1, RsXTH4, RsXTH3, RsXTH2, and RsXTH5 exhibited strong downregulation. Some genes, such as RsXTH6, RsXTH7, and RsXTH9, maintained relatively stable expression levels, suggesting minimal responsiveness to Pb stress (Figure 4E).
Quantitative RT-PCR analysis of RsXTHs under Pb treatments. The expression of RsXTH11 RsXTH23, RsXTH24, RsXTH25, and RsXTH27 in R. sativus roots exposed to Pb stress was detected using qRT-PCR. Error bars indicate standard deviation (SD, n = 3). Different letters indicate significant differences according to one-way ANOVA (P < 0.05).
To further validate the transcriptome results, qRT-PCR analysis was conducted on five RsXTH genes. As shown in Figure 5, the expression levels of RsXTH11, RsXTH23, RsXTH24, RsXTH25, and RsXTH27, were significantly upregulated under Pb treatment. Among them, RsXTH25 exhibited the most substantial increase, with transcript levels peaking at day 8. These expression patterns were consistent with the RNA-seq data.
Effect of RsXTH25 on Pb tolerance in R. sativus. (A) Transcript levels of RsXTH25 in the hairy roots of OE-RsXTH25 transgenic plants. (B) Phenotypic analysis of R. sativus seedlings under Pb treatment. Scale bar = 1 cm. (C–I) Quantitative analysis of shoot fresh weight (C), root fresh weight (D), chlorophyll a content (E), chlorophyll b content (F), total chlorophyll content (G), malondialdehyde (MDA) content (H), and electrolyte leakage rate (I) in transgenic and wild-type roots under Pb treatment. Error bars in (A) and (C–I) represent standard deviation (SD, n = 3). Different letters indicate statistically significant differences as determined by one-way ANOVA (P < 0.05). Capital letters denote comparisons among control groups, while lowercase letters denote comparisons among Pb-treated groups.
Transient overexpression of RsXTH25 enhances the tolerance to Pb stress in R. sativus
3.5
Given that RsXTH25 was consistently induced by Pb stress and exhibited the highest fold change, we selected it for further functional analysis. To validate the role of RsXTH25 in regulating Pb tolerance in R. sativus, we generated transgenic hairy roots by infecting R. sativus seedlings with Agrobacterium rhizogenes. The transcript levels of RsXTH25 in the hairy roots of two OE-RsXTH25 lines were approximately fivefold higher than those in control plants transformed with the empty vector (Figure 6A). The fresh shoot and root weights of the transgenic lines were significantly higher than those of the wild type under Pb treatment (Figures 6B-D). To further evaluate Pb stress tolerance, we examined several physiological parameters. Transgenic seedlings maintained significantly higher levels of chlorophyll a, chlorophyll b, and total chlorophyll under Pb stress than wild-type plants (Figures 6E-G), indicating improved photosynthetic performance. Additionally, we assessed oxidative damage by measuring malondialdehyde (MDA) content and electrolyte leakage. As shown in Figures 6H, I, Pb exposure led to significant increases in both parameters across all lines; however, the increases were less pronounced in OE-RsXTH25 lines than in wild-type plants. These findings suggest that transient overexpression of RsXTH25 enhances Pb tolerance in R. sativus by alleviating Pb-induced oxidative damage and maintaining chlorophyll stability.
Subcellular localization of RsXTH25. Confocal microscopy images of N. benthamiana epidermal cells co-expressing the RsXTH25-GFP fusion protein and the plasma membrane marker pm-rk. Scale bars = 20 μm.
XTH proteins have been reported to localize to the endoplasmic reticulum, cell wall, or plasma membrane (Genovesi et al., 2008; Zhu et al., 2012). To gain insights into the functional role of RsXTH25, its subcellular localization was examined. As shown in Figure 7, the RsXTH25-GFP fusion protein was detected at the cell periphery, co-localizing with the plasma membrane marker pm-rk, indicating that RsXTH25 is localized to the plasma membrane.
Upstream transcription factor analysis of RsXTH25 under Pb Treatment. (A) Mfuzz clustering of the gene expression in R. sativus under Pb Treatment. (B) Classification of transcription factors in cluster 3. (C) Top 20 transcription factors positively correlated with RsXTH25 expression.
Upstream transcription factor analysis of RsXTH25 under Pb treatment
3.6
To explore potential upstream regulators of RsXTH25 in response to Pb stress, we conducted a comprehensive transcription factor analysis. Using mfuzz clustering analysis (Figure 8A), we identified that RsXTH25 was grouped into cluster 3. Further analysis of all genes in this cluster revealed 333 transcription factors (Figure 8B), predominantly from the ERF, WRKY, NAC, and bHLH families, which are known to be involved in stress responses. To refine the candidate list, we performed Pearson correlation analysis to assess the expression relationships between these transcription factors and RsXTH25. As shown in Figure 8C, several transcription factors, including RsERF2, RsHD-ZIP22, RsB3_1, and RsGRAS5, exhibited strong positive correlations with RsXTH25 and are therefore considered potential upstream regulators.
Phylogenetic relationships among XTH proteins in R. sativus, A. thaliana and O. sativa. The phylogenetic tree was investigated using the IQ-tree software. The RsXTHs were indicated with red stars, while the AtXTHs were indicated with blue circle and the OsXTHs were indicated with orange square.
Discussion
4
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.
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