The heavy metal cadmium (Cd) is highly toxic to plants, animals, and humans. Worryingly, Cd can accumulate in vegetables, thereby threatening human health through the food chain (Kubier et al., 2019; Xia et al., 2021). Cd is categorized as a Class I human carcinogen by the International Agency for Research on Cancer (IARC), and has been linked to lung cancer, breast cancer, nephropathy, and osteoporosis, among other conditions (Waalkes, 2003; Akesson et al., 2008; Larsson and Wolk, 2016). Cd is widely used in the production of a variety of industrial products, including batteries, dyes, coatings, electroplating, cadmium quantum dots, alloys, and nuclear fission infrastructure (Turner, 2019). One-sixth of the world’s total Cd production comes from China, where Cd pollution has impacted 2.8 × 10⁹ m² of farmland (Liu et al., 2015). According to the Chinese Ministry of Environmental Protection, Cd has been the most common pollutant exceeding the Ministry’s limits, accounting for 7% of all samples (Zhao et al., 2015). In fact, Cd-polluted soil is beginning to seriously restrict crop production in China, posing a severe threat to the safety and quality of agricultural products. Thus, effective soil remediation techniques must be developed to mitigate or prevent the absorption of Cd by crops.

Cd is a transition metal with low reduction potential, making biological reduction difficult (Nancharaiah et al., 2015). Cd is primarily found in the divalent [Cd(II)] form, which is characterized by high solubility and mobility and which easily accumulates in the food chain (Kubier et al., 2019). Cd(II) can enter plant cells by way of various transporters. Cd(II) uptake occurs mainly via ZIP family (Guerinot, 2000), natural resistance-associated macrophage protein (NRAMP) family, and yellow stripe-like (YSL) family transporters (Thomine et al., 2000; Feng et al., 2017). However, the plants can fix metal ions absorbed into plant tissues. They can chelate Cd, store Cd in their vacuoles, or activate the antioxidant defense system to response to Cd toxicity (Moons, 2003; Kuramata et al., 2009; Zhang et al., 2010). For example, some low molecular weight chelators, such as glutathione (GSH), glutathione synthetase (GS), phosphatidylcholine (PC), and metallothioneins (MT), can bind to Cd and then mediate vacuoles through several transporters (Clemens et al., 2002; Clemens, 2006; Verbruggen et al., 2009). In addition, the plant can reduce the Cd absorption from soil. They can secrete organic acids, such as citric acid, malic acid, oxaloacetic acid, malonic acid, and tartaric acid by the roots (Anjum et al., 2015). Furthermore, microbes in soli can adsorb Cd on the cell surface using electronegative functional groups and exopolysaccharides (EPS), sequestering Cd inside cells using metallothionein and phytochelatin, or producing hydrogen sulfide (H₂S) to coprecipitate with Cd (Xia et al., 2021). Both secreted acids and microbes can lead to the passivation of soluble metal ions in the soil and effectively prevents their absorption by plants (Anjum et al., 2015; Xia et al., 2021).

Soil microbes are important members of all terrestrial ecosystems. Studies suggest that certain microbes can not only passivate Cd in soil but also reduce the absorption of Cd by plants. For example, Cd(II)-resistant Cupriavidus taiwanensis KKU2500-3 can colonize rice tissues and reduce Cd absorption in Cd-polluted soil (Punjee et al., 2018). Both Methylobacterium oryzae CBMB20 and Burkholderia spp. CBMB40 can promote growth and reduce Ni and Cd absorption in tomato (Madhaiyan et al., 2007). In addition, Pseudomonas fluorescens UW4 can reduce Cd stress in lettuce and Enterobacter asburiae NC16 can reduce Cd absorption in wheat (Albano and Macfie, 2016; Zhou et al., 2019). Given these results, the use of microbes is increasingly seen as a potentially low-cost and environmentally friendly method to remediate Cd-polluted soils and to prevent the absorption of Cd by crop plants.

Chinese cabbage (Brassica rapa L.) is one of the most popular and widely cultivated vegetables in China. Unfortunately, B. rapa is known to accumulate Cd in its roots and edible tissues (He et al., 2013; Wu et al., 2022). Recently, the B. rapa Cd stress response has been studied using transcriptomic and proteomic approaches (Sun et al., 2023; Yu et al., 2023). Notably, several Cd-resistant microbes have been found to reduce Cd absorption by B. rapa, including Pseudomonas sp. B7 (Wu et al., 2022), Enterobacter sp. A11, and Comamonas sp. A23 (Wang et al., 2020). However, the mechanism by which these microbes alter Cd dynamics and toxicity in B. rapa is unclear. In this work, we isolated a highly Cd(II)-resistant bacterium (Stenotrophomonas sp. CD2) from Cd-contaminated soil and studied its ability to immobilize Cd(II) in medium and in soil. In addition, we evaluated the ability of strain CD2 to ameliorate Cd accumulation in B. rapa. Finally, we utilized transcriptomics to discover the mechanisms underlying the ability of “CD2” to reduce Cd accumulation in B. rapa.

Chinese cabbage (B. rapa.) is one of the most popular and widely cultivated vegetables in China. However, the risk of Cd contamination of this popular crop is increasing as more farmland becomes polluted with this toxic heavy metal (Zhao et al., 2015). Here, we tested the ability of the Cd-resistant bacterial strain CD2 to reduce Cd(II) accumulation in B. rapa (Figure 2). Strain CD2 was identified as belonging to the genus Stenotrophomonas (Supplementary Figure S1) and was found to be able to immobilize 0.1 mmol/L Cd(II) within 36 h (Figure 1). Several bacterial species and strains have been reported to immobilize Cd(II). For example, Pseudomonas sp. B7 can remove 0.1 mmol/L Cd(II) within 60 h (Wu et al., 2022). Enterobacter sp. A11, Comamonas sp. A23, and a co-culture of the two, can remove 0.1 mmol/L Cd(II) in around 48 h (Wang et al., 2020). Notably, the immobilization ability of strain CD2 was stronger than that of these bacteria. Microbes resist and immobilize Cd(II) through several mechanisms (Ammendola et al., 2014; Ziller and Fraissinet-Tachet, 2018; Gallardo-Benavente et al., 2019; Shi et al., 2020; Xia et al., 2021). Our research suggests that strain CD2 relies on a combination of intracellular sequestration and extracellular adsorption mediated by biofilm and EPS. In addition, strain CD2 was found to produce H₂S, which could lead to the production of CdS precipitates. The genome of strain CD2 contained a multitude of efflux proteins such as CusA, CzcD, and MntH; Cd(II)-binding proteins such as CadW; and genes involved in the production of biofilms, EPS, and H₂S (Supplementary Table S1). Therefore, our phenotypic observations were in good agreement with our genotypic analysis.

Inoculation with strain CD2 was found to effectively reduce the Cd content in the roots and aerial tissues of B. rapa. Specifically, “CD2”-inoculation reduced the Cd content of roots by 51.16% and of aerial tissues by 55.56%, compared to uninoculated Cd-exposed plants. Moreover, the residual Cd content in the aerial tissues (edible parts) met food security standards (0.2 mg/kg) in ‘CD2-inoculated plants (Codex Alimentarius Commission, 2010; European Union, 2011; Ministry of Health (MOH), 2012). In addition, adding strain CD2 to the soil significantly increased the nonbioavailable Cd content and reduced the bioavailable Cd content. This suggests that strain CD2 may prevent Cd accumulation in plants by reducing the bioavailable Cd(II) content of the soil itself. Notably, this mechanism has been reported in other microbe-plant systems (Shi et al., 2020; Wang et al., 2020; Wu et al., 2022; Lou et al., 2024). The addition of strain CD2 to the soil effectively reduces the Cd accumulation in B. rapa during pot experiments. The future research will primarily focus on evaluating the efficacy of strain CD2 in mitigating Cd accumulation in plants when applied to real-world soil contaminated with Cd.

Transcriptomic analysis can identify key genes and expression patterns, as well as clarify the mechanisms, associated with the plant response to heavy metal stress. Several metabolic pathways have been implicated in the plant response to Cd(II) stress (Thomine et al., 2000; Anjum et al., 2015). Surprisingly, we found that different functions and pathways were activated in uninoculated and “CD2”-inoculated Cd(II)-exposed plants, according to GO and KEGG enrichment analyses. According to the GO enrichment analysis, inoculation with “CD2” affected the expression of genes related to cell wall biosynthesis in roots and membranes (plasma, thylakoid, chloroplast, and photosynthetic) in aerial tissues. Similar results have been reported in Cd-stressed maize and mustard (Brassica juncea) (Peng et al., 2015; Li et al., 2023). Studies suggest that the biosynthesis of cutin, suberine, wax, and phenylpropanoid confers abiotic stress resistance in plants. This is because these lipophilic cell wall barriers mediate the flux of gasses, water, and solutes (Pollard et al., 2008). The phenylpropane metabolic pathway precedes the biosynthesis of flavonoids, which participate in the abiotic stress response through a wide range of chemical reactions (Gan et al., 2022). We found that “CD2”-inoculation resulted in an increase in DEGs related to cutin, suberine, wax, and phenylpropanoid biosynthesis (Figure 5B). These results suggest that inoculation with “CD2” increases the Cd-stress resistance of B. rapa by regulating secondary metabolism.

In plants, excessive Cd accumulation results in reactive oxygen species (ROS) metabolism disturbance. In response, plants have evolved antioxidant mechanisms to maintain physiological homeostasis (Li et al., 2022). We identified several Cd-responsive DEGs related to oxidative stress resistance in both the GO and KEGG enrichment analyses. In uninoculated Cd-exposed roots, these DEGs were enriched in the GO terms catalase (2 upregulated), antioxidant (4 upregulated and 1 downregulated), glutathione transferase activity (2 upregulated and 1 downregulated), and response to hydrogen peroxide (3 upregulated and 4 downregulated). Meanwhile, in uninoculated Cd-exposed aerial tissues, these DEGs were only enriched in glutathione transferase (2 upregulated and 2 downregulated) (Supplementary Table S2). Following inoculation with “CD2”, Cd-exposed roots were found to be enriched in hydrogen peroxide (4 upregulated and 5 downregulated) while Cd-exposed aerial tissues were found to be enriched in glutathione transferase (10 downregulated). KEGG enrichment analysis indicated that glutathione metabolism was enriched significantly in both the roots and aerial tissues of uninoculated Cd-exposed plants, but only in the aerial tissues of “CD2”-inoculated Cd-exposed plants. In addition, “plant-pathogen interaction” was enriched significantly in the roots of “CD2”-inoculated Cd-exposed plants (Figure 5B). The ‘plant-pathogen interaction’ KEGG category includes important signaling molecules which mediate the interaction between microbes and plants. Two genes in this category (CPK and RBOH) were upregulated in the roots of Cd-stress plants (Supplementary Table S3). Both of these genes confer oxidative stress resistance through regulating Ca²⁺ dynamics. Together, these results indicate that Cd-induced oxidative stress was more severe in roots than in aerial tissues. This may be related to the relatively lower content of Cd in aerial tissues than in roots. Moreover, inoculation with “CD2” appears to reduce oxidative stress in B. rapa through a ‘plant-pathogen interaction’-related mechanism.

Transporter proteins are responsible for sequestering Cd into vacuoles, thereby preventing the transport of Cd from roots to shoots (Zhang et al., 2010). Several upregulated proteins related to vacuolar sequestration were identified in the roots of Cd-exposed B. rapa, including three in uninoculated plants and two in “CD2”-inoculated plants. These results suggest that vacuolar sequestration may inhibit Cd transport in both inoculated and uninoculated plants. In plants, Cd(II) uptake and accumulation are also affected by ion channel proteins and transporter proteins such as ZIPs, ABC transporters, and ATPases. We observed that Cd exposure changed the expression of ABC transporters, ATPases, sodium/calcium exchanger proteins, and potassium channel proteins in uninoculated or “CD2”-inoculated plants (Supplementary Table S3). Additionally, inoculation with “CD2” resulted in the downregulation of a ZIP family Zn-transporter and three blue copper proteins (BCPs) in roots (Supplementary Table S3). BCPs can bind Cd(II) or participate in Cd(II) efflux (Wang et al., 2023). Thus, it appears that inoculation with “CD2” may negatively impact the expression of Zn transporters and BCPs, resulting in reduced Cd accumulation in B. rapa.

A growing body of research indicates that TFs are important regulators of the plant response to abiotic stress. In particular, the WRKY, MYB, bHLH, ERF, ZIP, and C2H2 TFs have been implicated in the Cd stress response (Tokumoto et al., 2019; He et al., 2021). We found that the ERF, MYB, C2H2, NAC, WRKY, and G2-like TFs were highly responsive to Cd in the roots and aerial tissues of both uninoculated and “CD2”-inoculated plants (Figure 6), suggesting that these TFs represent an innate Cd stress-resistance mechanism in B. rapa. In addition, inoculation with “CD2” resulted in the upregulation of AP2, Dof, WOX, Trihelix, B3, EIL, and M-type MADS TFs in both roots and aerial tissues. Dof TFs are specific to plants, and play important roles in growth, development, seed germination, photoresponse, storage protein accumulation, biological stress, carbon and nitrogen metabolism, secondary metabolite biosynthesis, and stress response (Zou and Sun, 2023). In Arabidopsis thaliana, Dof4.2 regulates the accumulation of flavonoids under stressful conditions (Skirycz et al., 2007). According to KEGG analysis, inoculation with “CD2” resulted in induction of the phenylpropane metabolic pathway, which precedes stress-reducing flavonoid biosynthesis (Gan et al., 2022). Therefore, inoculation with strain CD2 may help defend plants against Cd stress through a mechanism mediated by Dof and flavonoid biosynthesis. The more experiments would be carried out in the future to detect the function of Dof and flavonoid biosynthesis pathway in resisting Cd.

In this study, the highly Cd(II)-resistant Stenotrophomonas sp. CD2 was isolated from Cd-contaminated soil. This strain was found to be able to immobilize Cd(II) through both intracellular sequestration and extracellular adsorption related to the production of biofilm, EPS, and H₂S. Application of “CD2” to Cd-polluted soil resulted in reduced bioavailability of Cd(II) and reduced Cd(II) accumulation in B. rapa. Moreover, the Cd content of the aerial tissues (edible parts) of “CD2”-inoculated plants was found to meet food security standards. Transcriptomic analysis revealed that uninoculated B. rapa resisted Cd-induced ROS toxicity mainly through the expression of catalase and glutathione transferase, while “CD2”-inoculated plants exhibited an increase in ‘plant-pathogen interaction’ pathways. Both uninoculated and inoculated plants were able to prevent the transportation of Cd from roots to shoots by vacuolar sequestration. In addition, inoculation with “CD2” was found to inhibit the expression of Zn transporters and BCPs; alter the expression of various TFs (in particular, Dof) and the biosynthesis of phenylpropane, cutin, suberine, and wax. The results of this study suggest that Stenotrophomonas sp. CD2 may prove to be a useful inoculant to prevent Cd accumulation in B. rapa.

The bacterial strain Stenotrophomonas sp. CD2 has been deposited as a patent strain in China Center for Type Culture Collection (http://www.cctcc.org/) under the accession number of CCTCC M 20231348.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/genbank/, CP102248 https://www.ncbi.nlm.nih.gov/, PRJNA1064471.

XF: Conceptualization, Writing – review & editing, Project administration, Data curation. KY: Data curation, Investigation, Methodology, Writing – original draft. QP: Data curation, Formal analysis, Writing – original draft. RL: Data curation, Formal analysis, Writing – original draft. YZ: Conceptualization, Project administration, Writing – original draft.