The S. plumbizincicola SpCTP3 gene was cloned by a PCR amplification using the primers SpCTP3-F/R. The full-length SpCTP3 sequence comprises 1,383 base pairs. The amplified SpCTP3 was incorporated into the pBI121 vector via homologous recombination for the subsequent expression under the control of the CaMV 35S promoter. An Agrobacterium tumefaciens-mediated leaf disc transformation method was used to insert SpCTP3 into P. × canescens plants (Wen et al., 2022). Transgenic plants were identified by a genomic PCR amplification using the primers SpCTP3-F/R and the positive lines were further analyzed by quantitative real-time PCR (qRT-PCR). The relative expression levels were calculated according to the 2^−ΔΔCt method (Jia et al., 2022). The TUBU (Beta tubulin) gene was selected as an internal control. Primer details are listed in Table S1 . Four-week-old transgenic rooted poplar plants and the control group were grown for 4 weeks in Hoagland nutrient solution (i.e., hydroponic system). The nutrient solution was renewed every 3 days. For the heavy metal treatment experiments, plants were grown for 30 days in Hoagland nutrient solution supplemented with 100 μmol/L CdCl₂. The growth chamber conditions were set as follows: 16-h day (25°C)/8-h night (18°C), 50%–60% relative humidity, and a photon flux density of 120 μmol/m²/s. Plants were photographed and their phenotypes were analyzed.

The Cd-treated lines were harvested separately and rinsed thoroughly with tap water. They were then soaked in 20 mmol/L EDTA for 20 min and washed three times with deionized water. The roots, stems, and leaves of each line were collected and dried at 70°C until they reached a constant weight. At least 0.2 g dried material was used to measure the Cd concentration. Briefly, the samples were ground to fine powder, and digested with a mixture consisting of HNO₃:HClO₄ (9:1, v/v) at 120–200°C in a microwave-accelerated reaction system (CEM, Matthews, NC, USA). The final volume was adjusted to 25 mL with distilled water. The Cd concentration was analyzed using the iCAP-7400 inductively coupled plasma optical emission spectroscopy (ICP-OES) system (Thermo Fisher Scientific, California, USA). The heavy metal translocation factor was calculated according to the following formula (Dineshkumar et al., 2019): heavy metal content in the aerial plant parts/heavy metal content in the roots.

Fresh leaves and roots (2.0 g) were homogenized using a precooled mortar and 20 mL precooled extraction solution [50 mmol/L Tris-HCl, 250 mmol/L sucrose, and 1.0 mmol/L C₄H₁₀O₂S₂ (pH 7.5)]. After centrifuging the samples at 1,250 × g for 15 min, the precipitate was collected as the cell wall component. The supernatant was centrifuged at 20,800 × g for 45 min to obtain the precipitate (organelles) and supernatant (soluble fraction). All steps were completed at 4°C. After the collected components were digested, their Cd concentrations were determined by ICP-OES.

Cadmium in different chemical forms was extracted as described by Ouyang et al. (2019). Inorganic titratable acid cadmium (Cd_E) was extracted with 80% ethanol, water-soluble cadmium (Cd_W) was extracted with deionized water, Cd integrated with pectin and protein (Cd_NaCl) was extracted with 1 mol/L NaCl, insoluble CdHPO₄, Cd₃(PO₄)₂, and other Cd-phosphate complexes (Cd_HAC) were extracted with 2% HAC, oxalic acid bound cadmium (Cd_HCl) was extracted with 0.6 mol/L HCl, and the rest was residual cadmium (Cdr). Approximately 0.5 g samples were shaken for the 22-h extraction at 25°C. The proportion of the sample weight and extraction solution volume was adjusted to 1:10 (w/v) before a centrifugation at 5,000 × g for 10 min. The precipitate was resuspended twice with the same extraction solution, shaken at 25°C for 2 h, and then centrifuged at 5,000 × g for 10 min. Finally, the supernatants after three centrifugations were combined. Each pooled solution was then evaporated on an electric plate to a constant weight. After digesting the materials, the Cd concentrations of the three parts were determined by ICP-OES.

To determine the net Cd²⁺ flow rate in different lines treated with Cd stress, nine fine roots (approximately 1.5 mm diameter) were selected from each Cd-treated transgenic line and the WT control. The Cd²⁺ net flux was measured according to the noninvasive micro-test (NMT) technique (NMT100 Series; Younger, USA LLC, Amherst, MA, USA) as previously described (He et al., 2015; Chen et al., 2017). Each fine root was equilibrated in the measurement solution (0.05mM CdSO₄, 0.25 mmol/L NaCl, 0.1 mmol/L Na₂SO₄, 0.05 mmol/L KCl, 0.15 mmol/L MES, pH6.0) for 30 min and then transferred to fresh measurement solution before determining the Cd²⁺ net flux. The root Cd²⁺ flow rate was analyzed by moving the electrode between two points (30 μm) on the root surface to measure the voltage difference perpendicular to the root surface.

To analyze antioxidant enzyme activities and the contents of ROS (O^2·− and H₂O₂), 1.0 g leaf and root samples were collected from the WT and transgenic plants and homogenized in 8 mL 50 mmol·L⁻¹ sodium phosphate buffer (pH 7.8) using a prechilled mortar. The samples were then centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was used to measure enzyme activities and the contents of ROS. The superoxide dismutase (SOD) activities were determined using NBT (Nitroblue tetrazolium) photoreduction method as absorbance 560 nm as described by Akbari et al. (2020). The peroxidase (POD) activity was assayed spectrophotometrically using hydrogen peroxide and guaiacol as extracts at 470 nm (Dai et al., 2020). Catalase (CAT) activity was tested in potassium phosphate buffer (pH 7.8) containing 3 mM H₂O₂ at 240 nm. The H₂O₂ content and the O2·⁻ level were determined as described by Alexieva et al. (2001) and Jing et al. (2020). H₂O₂ content was determined using trichloroacetic acid (0.1% w/v) at 630 nm (Alexieva et al., 2001). For determination of O^2·− level, supernatant was mixed with 10 mM of hydroxylamine hydrochloride and left for 20 min followed by the addition of sulfanilamide and naphthylamine. After 20 min of incubation at 25°C, absorbance was measured at 530 nm, and calculations were done using the standard curve of NaNO₂ (Jing et al., 2020).

For titratable acid assay, 0.1 g sample of each treatment was added to water and then ground into a homogenate. The final volume was adjusted to 5 mL for the extraction of titratable acid. The titratable acid content was determined by a titration using sodium hydroxide (0.1 mol/L) and phenolphthalein (1%) as the pH indicator (Huo et al., 2009).

Total RNA was extracted from Cd-treated and WT poplar samples using the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing), after which 800 ng total RNA in 20 μL was used to synthesize cDNA. The qRT-PCR analysis was performed using SYBR Green Premix Ex Taq II (TAKARA, Japan). The primer sequence refers to He et al. (2015) and Ma et al. (2014). Information regarding the qRT-PCR primers is provided in Table S1 . The qRT-PCR reaction mixture was prepared in a volume of 20 μL, with 10 μL of SYBR green master mix, cDNA template (2 μL), ddH₂O (6.8 μL), ROX Reference Dye 0.4μL and 0.4 μL of each of the primer. The qRT-PCR conditions were set as follows; 95°C for 30s, 40 cycles of 95°C for 5 s, and 60°C for 30 s, followed by melting of PCR products step: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. Each sample was analyzed using three technical replicates to ensure the results were reliable. Gene expression levels were calculated according to the 2^−ΔΔCt method, with TUBU selected as the reference gene. Relative gene expression levels were visualized in a heatmap on the basis of Z-score transformed values.

For each experiment, at least three plants were sampled. Statistical analyses were performed using the SPSS software (version 18.0). The significance of the differences between the treated and control samples was analyzed via a one-way ANOVA followed by Duncan’s test (p< 0.05). Data are presented herein as the mean ± standard deviation (SD). The Kruskal Wallis test was used if the data did not meet the assumptions for ANOVA (equality of variances) according to Levene’s test. A principal components analysis (PCA) was performed and the results were plotted using the ggbiplot package of R (version 3.6.4).

Genomic DNA was extracted from the WT and transgenic lines using the CTAB method. The transgenic lines were verified using SpCTP3-F/R primers ( Table S1 ). Of the 13 putatively transformed lines, 11 were confirmed as transgenic ( Figure S1 ). The PCR results showed that SpCTP3 was expressed in the transgenic poplar plants ( Figure S1 ). Total RNA was extracted from some of the transgenic lines and reverse transcribed to cDNA for the qRT-PCR analysis. Three independent lines (OE-3, OE-6, and OE-7) with high SpCTP3 expression levels were selected for the subsequent analyses.

There was no significant difference in the dry weights of the untreated WT and transgenic plants. After the Cd treatment, the dry weight of the above-ground parts of the transgenic and WT plants decreased by an average of 28.49% and 12.96% (relative to the control), respectively. The dry weight of the root system decreased slightly after the Cd treatment, but this decrease was not significant. Accordingly, SpCTP3 expression has no effect of the biomass of transgenic poplar plants exposed to Cd stress ( Figure 1 ).

To further functionally characterize SpCTP3, we measured the Cd content of specific parts of the WT and transgenic plants treated with Cd for 30 days ( Figure 2 ). Notably, the root and leaf Cd contents were higher for the SpCTP3-overexpressing transgenic lines than for the WT controls. In contrast, there were no obvious differences in the stem Cd contents. The distribution of Cd was in the order of roots, leaves, and stems. The leaf Cd content was 48.58%–82.49% higher for the transgenic lines than for the WT controls. Similarly, the translocation factor was higher for the transgenic lines than for the WT plants. These observations indicated that compared with the WT plants, the transgenic poplar plants were better able to transport Cd from the roots to the aerial parts.

In this study, Cd²⁺ was differentially distributed in various subcellular components. Most of the Cd in the roots and leaves of the transgenic lines was stored in the soluble fraction, followed by the cell wall and organelles ( Figure 3 ). In the WT plants, 36.12% and 56.77% of the Cd were present in the cell wall and soluble fraction, respectively; the rest of the Cd was detected in the organelle fraction. In the WT leaves, 44.47% and 47.52% of the Cd content was distributed in the cell wall and soluble fraction, respectively. The cell wall Cd content was lower for the transgenic lines than for the WT plants, whereas the opposite pattern was observed for the Cd content in the soluble fraction.

Cadmium absorbed by plants is present in cells in diverse forms and affects plant growth and development. We extracted various forms of Cd and determined the Cd contents using an ICP-OES system. The Cd_W content was highest in the roots and leaves of the transgenic and WT plants ( Figure 3 ). The Cd in the WT roots was mostly detected as Cd_W (59.19%), followed by Cd_NaCl (29.11%) and then Cd_HCl, Cd_HAc, and Cd_E. The Cd in the transgenic roots was also mostly detected as Cd_W (average of 75.59%), although the Cd_HAc content was also relatively high. The Cd in the leaves of the transgenic and WT plants was predominantly in the Cd_E, Cd_NaCl, and Cd_HAc forms. Compared with the WT control, the Cd treatment of the transgenic plants decreased the Cd_W and Cd_NaCl contents, while increasing the Cd_HCl and Cd_HAc contents. Hence, the overexpression of SpCTP3 altered the Cd distribution and chemical forms in the transgenic plants.

To clarify the changes in Cd uptake by the roots of transgenic and WT poplar plants exposed to Cd stress, we used the NMT technique to analyze the Cd²⁺ flow rate at 120 μm from the root tip after the Cd treatment. The Cd²⁺ flow rate was 72.927–163.48 pmol/cm²/s for the transgenic lines and 44.707–61.058 pmol/cm²/s for the WT plants ( Figure 4 ). The net influx of Cd²⁺ was higher for the transgenic roots than for the WT roots.

An exposure to Cd stress results in different degrees of oxidative stress in plants. The O 2 · − and H₂O₂ concentrations were compared between the WT and transgenic poplar plants ( Figure 5 ). For both the WT and transgenic samples, the Cd treatment induced a significant H₂O₂ and O 2 · − burst. The leaf O 2 · − content was 2-times higher in the transgenic plants than in the WT plants. In the roots, the H₂O₂ and O 2 · − contents increased in the transgenic plants by varying degrees, resulting in levels that were significantly higher than those in the WT plants. Additionally, H₂O₂ and O 2 · − accumulated more in the leaves than in the roots, although this phenomenon was significantly less pronounced in the WT plants than in the transgenic plants.

Cadmium significantly affected the CAT, SOD, and POD activities ( Figure 6 ). The CAT activity levels were significantly higher in the transgenic lines than in the WT plants after the Cd treatment. Compared with the WT control, the CAT activity was respectively 1.37-fold and 2.98-fold higher in the roots and leaves of the transgenic lines by Cd stress. Furthermore, there was no significant difference in the SOD activities of the Cd-treated transgenic and WT leaves, but the root SOD activities were slightly lower in the transgenic lines than in the WT control following the Cd treatment. In addition, the POD activity in the leaves was slightly higher in the transgenic lines than in the WT plants, but there was no significant difference in the root POD activity.

Titratable acids are often considered to be important for the resistance of plants to heavy metal pollution because they contribute to the chelation of metal elements and convert metal ions to less toxic or non-toxic chelated states. Titratable acids were extracted from the SpCTP3-overexpressing and WT plants and the total organic acid content was determined via acid-base titration ( Figure 7 ). The Cd treatment induced a substantial change in the titratable acid content. More specifically, the titratable acid content in the transgenic roots and leaves increased significantly, peaking at 20.76 μmol·g⁻¹ in the roots (i.e., 31.87% higher than the titratable acid content of the control roots) and 30.81 μmol·g⁻¹ in the leaves. Thus, the overexpression of SpCTP3 increased the titratable acid content of transgenic lines under Cd stress conditions.

On the basis of the physiological data ( Figure 8 ), differences in the Cd uptake by the roots and the subsequent translocation to the shoots may be a possible reason for diversity in the Cd accumulation between the WT and transgenic poplar plants. Therefore, we focused on the transcriptional regulation of some transporter genes involved in Cd²⁺ transport and detoxification. The expression levels of all 12 genes related to Cd transport and detoxification were differentially up-regulated in the roots after the Cd treatment ( Figure 8 ). In the roots, the up-regulated expression of ZIP2 (encoding zinc-regulated transporter 2), ZIP6.2, and IRT (encoding an iron-regulated transporter-related protein) was greater in the transgenic lines than in the WT control. Among the leaves, the most up-regulated genes were IRT, YSL2 (encoding a yellow stripe-like protein), and CAX (encoding a cation exchanger). Moreover, the Cd treatment significantly up-regulated the expression of HMA4 (encoding heavy metal ATPase 4) only in the transgenic roots. Similar expression trends were observed for other genes in the leaves and roots, in which NAS (encoding a nicotianamine synthase), MTP1 (encoding metal tolerance protein 1), and Nramp1.3 (encoding natural resistance-associated macrophage protein 1.3) expression levels increased significantly in the Cd-treated transgenic lines.

To determine the response patterns of the WT and transgenic poplar plants to Cd²⁺, a PCA was conducted using the physiological data related to the exposure to Cd²⁺ (e.g., Cd concentration as well as ROS, antioxidant, and titratable acid contents) ( Figure 9 ). The PCA results indicated that 88.11% of the total variance could be explained by the effects of the Cd stress on the WT and SpCTP3-overexpressing transgenic poplar plants, with principal components 1 and 2 respectively explaining 74.68% and 13.43% of the Cd-induced variance. There was a significant difference between the SpCTP3-overexpressing transgenic and WT poplar plants under Cd stress conditions.

The soluble fraction of cells consists mainly of vesicles, which contain many proteins, sugars, and organic acids that bind to heavy metals to decrease their effects and limit the amount of heavy metals entering organelles, thereby protecting cells from Cd stress (Li et al., 2014; Ouyang et al., 2019). For example, in Capsicum annuum roots, the Cd content in the soluble fraction can be as high as 77%–87% (Xin and Huang, 2014). Additionally, Cd is mainly distributed in the soluble fraction of the leaves and roots of Cd-tolerant radish varieties (Xin et al., 2017). We obtained similar results. In the WT control, the soluble fraction had the highest Cd content (56.77%), followed by the cell wall. In the transgenic lines, the rank-order for the Cd content was as follows: soluble fraction > cell wall > organelle ( Figure 3 ). This trend was also detected in the leaves. Plants absorb Cd and store most of it in the soluble fraction, followed by the cell wall. However, plant responses to Cd stress may vary among species and stress levels. The transgenic poplar plants had a higher Cd capacity than the WT controls. The accumulation of Cd in the cell wall and soluble fraction can effectively decrease Cd toxicity in plants. In the current study, the Cd content decreased in the cell walls of the transgenic roots and leaves, whereas it increased in the soluble fraction. Hence, in the SpCTP3-overexpressing transgenic poplar plants, Cd ions were separated and fixed in the cytoplasm in the form of relatively stable metal chelates/complexes. Compared with the WT plants, the overexpression of SpCTP3 in the transgenic plants increased the Cd content of the roots and leaves, while also altering the subcellular distribution of Cd by increasing and decreasing the Cd proportions in the soluble fraction and cell wall, respectively. These findings reflect the importance of SpCTP3 for minimizing the toxic effects of Cd in plants.

Under heavy metal stress conditions, multiple mechanisms may be activated in plants to prevent the excessive accumulation of metals in the cytosol and limit the toxicity of the metals. If heavy metals enter the soluble part of the cell, the internal heavy metal tolerance mechanism is activated, resulting in the production of metal chelators, including organic acids, amino acids, and binding proteins (Hall, 2002). Organic acids are an extremely important class of metal ligands that are crucial for the absorption, transport, storage, and detoxification of heavy metals (Long et al., 2003). The proportion of the Cd content in the cell wall of the roots and leaves of the transgenic poplar plants decreased, which was in contrast to the increase in the proportion of the Cd content in the soluble fraction, implying that more Cd ions in cytoplasm need to be immobilized in the form of stable metal chelates/complexes in the transgenic poplar plants than in the WT plants. Accordingly, poplar plants can avoid the harmful effects of Cd stress by decreasing the free Cd concentration in the cytoplasm. In response to the 30-day Cd treatment in this study, the titratable acid content was significantly higher in the transgenic poplar plants than in the WT plants. This indicates that SpCTP3 is critical for ameliorating the toxicity of Cd in plants.

The chemical form of Cd reflects the degree of migration and the toxicity of Cd (Kudo et al., 2021). Highly mobile inorganic and organic Cd compounds are usually more toxic than Cd_HAc, Cd_HCl, and Cd_NaCl (Wu et al., 2016). In our study, Cd_W was the dominant form in all transgenic lines. Additionally, the Cd_W content was lower in the roots of the transgenic lines than in the WT roots. The Cd_W form of Cd is converted to Cd_HAc and Cd_HCl. However, Cd_HAc is associated with Cd uptake, transport, and detoxification (Qiu et al., 2011). It is also indicative of the adaptive response of plants to an exposure to stress. Cd_W extracted as free inorganic Cd, and these free Cd²⁺ are not only easily localized in the organelles, but it is also transported to the above-ground tissues. Our analysis of leaves showed that Cd_W was more abundant in the transgenic lines than in the WT plants, indicating that the transgenic lines had considerably more free Cd²⁺ (Xin et al., 2017). The Cd content as well as the translocation factor were higher in the transgenic lines than in the WT plants, which is consistent with the results of a recent study (Lai et al., 2021). In the WT control, the proportion of Cd_NaCl increased, indicative of the increase in the binding between Cd and pectin or protein. Therefore, the leaf cell wall Cd content was higher for the WT plants than for the transgenic lines. Both Cd_HAc and Cd_HCl are mainly present in the cell wall and vesicles (Jiang et al., 2007). In the current study, Cd_HAc and Cd_HCl contents were higher in the transgenic lines than in the WT plants, which corresponds to the subcellular distribution of Cd. In summary, the conversion of Cd to Cd_HAc, Cd_HCl, and Cd_NaCl is an important mechanism related to the Cd uptake, translocation, and detoxification in plants.

Plants take up heavy metals from the soil primarily through their roots. Therefore, determining the Cd uptake and efflux from the roots is important for measuring plant Cd accumulation. Noninvasive micro-test techniques can measure real-time ion transport changes in living plant and animal tissues, cells, and internal/external environments. Our NMT analysis revealed that the Cd in-flow rate was significantly higher for the transgenic roots than for the WT roots. The overexpression of SpCTP3 promoted the influx of Cd in root cells and the accumulation of Cd, indicating that the roots of the transgenic lines had a greater Cd uptake capacity than the WT roots, which is consistent with the findings of an earlier study by Zhang et al. (2021).

In our study, SpCTP3 was induced to be expressed by the Cd treatment and Cd accumulation was increased in the transgenic lines. Then ROS levels were significantly elevated, which induced the up-regulated expression of SpCTP3, thereby promoting ROS detoxification. This is one of the reasons for the increased antioxidant enzyme activities in the SpCTP3-overexpressing transgenic lines. In addition, Cd induced the accumulation of ROS in the roots and leaves, ultimately resulting in oxidative stress, which is in accordance with the findings of recent studies involving other plant materials (Bari et al., 2019; Zhang et al., 2020a). In the current study, Cd stress induced the excessive accumulation of O 2 · − in the roots and leaves. Moreover, the H₂O₂ content increased as the treatment duration increased. To protect against excessive ROS, plants employ a complex array of antioxidant defense mechanisms to prevent or mitigate the oxidative damage caused by abiotic stresses (Shafi et al., 2022). Because they are signaling compounds, the accumulation of H₂O₂ and O 2 · − may increase the activities of CAT, SOD, POD, and other enzymes during plant responses to Cd stress (Wrzaczek et al., 2013). Superoxide dismutase activity is positively correlated with the intracellular ROS level within a certain range (Zhou et al., 2021). Under stress conditions, an increase in the ROS content increases the activities of SOD and other antioxidant enzymes (Gupta et al., 2017). In addition, SOD converts superoxide radicals into less toxic substances in reactions that produce H₂O₂ (Fujita and Hasanuzzaman, 2022). This may explain the observed accumulation of H₂O₂ in the transgenic poplar lines. Subsequently, CAT and POD are activated as H₂O₂ scavengers to maintain plant redox homeostasis (Tadjouri et al., 2022). In our study, the SpCTP3-overexpressing transgenic lines had significantly elevated CAT and POD activities after the Cd treatment, and the CAT activities were significantly higher in the Cd-treated transgenic lines than in the WT plants ( Figure 6 ). Similarly, Cd stress reportedly causes the SOD, POD, and CAT activities in the above-ground parts of rice plants to increase to some extent to limit the toxic effects of Cd (Afzal et al., 2018). An exposure to Cd stress can significantly increase SOD, CAT, APX, and POD activities in marigold (Calendula calypso) by 150%, 79%, 97%, and 60%, respectively, leading to the conversion of H₂O₂ to non-toxic oxygen and water (Farooq et al., 2020).

The physiological process leading to the accumulation of Cd in above-ground plant parts is divided into the following three stages: root cell absorption; ectoplastic pathway-, symplast pathway-, and apoplast pathway-mediated transport; and above-ground accumulation (Uraguchi et al., 2009). Cadmium is transported to the above-ground tissues via numerous transporter proteins. In plants, the NRAMP proteins form one of the heavy metal transporter families in monocotyledons and dicotyledons (Ishida and Corcino, 2022). Their functions have been demonstrated in rice (Chang et al., 2020), poplar (Chen et al., 2019), and other plants (Li et al., 2021).

The ability of roots to absorb Cd is the decisive factor influencing the accumulation of Cd in plant shoots (Chaney, 2015). The overexpression of the S. alfredii SaNRAMP1H gene significantly increases the Cd contents of tobacco plants (above-ground and below-ground parts), implying that SaNRAMP1H promotes Cd uptake and accumulation (Zhang et al., 2020). The ZIP family proteins also function as heavy metal transporters. A previous study showed that Cd²⁺ can enter plant root cells through the ZIP transporter, which regulates Cd uptake, sequestration, and translocation (Ma et al., 2014). In addition, the overexpression of the Vicia sativa VsIRT1 gene in A. thaliana enhances the transport and accumulation of Cd (Zhang et al., 2020b). In poplar, NRAMP1.3, ZIP2, and ZIP6.2 encode plasma membrane proteins that control the entry of Cd²⁺ into root cells. In the current study, the Cd treatment up-regulated the NRAMP1, ZIP2, and ZIP6.2 expression levels and increased the cytoplasmic Cd content in the transgenic lines; the Cd content was higher in the transgenic lines than in the WT plants ( Figure 8 ).

Cytosolic Cd²⁺ may bind to phytochelatins (PCs), which are synthesized from glutathione in a reaction catalyzed by PC synthase (PCS) (Liu et al., 2021). The generated PC–Cd complex can be transported to vesicles via ABC transporters (e.g., ABCC1 and ABCC2) (Feng et al., 2020). Cytoplasmic Cd²⁺ may also be transported into vesicles via MTP1, which is located in the vesicle membrane (Singh et al., 2021). In poplar, PCS, ABCC1, and MTP1 are differentially expressed in response to Cd²⁺. In the present study, PCS, ABCC1, and MTP1 expression levels increased 3.7-fold, 7.4-fold, and 16.4-fold, respectively, in the Cd-treated transgenic poplar plants. Earlier research confirmed HMA4 is localized to the plasma membrane, wherein it helps export divalent cations (e.g., Cd²⁺/Zn²⁺) from the cytoplasm and facilitates their transport into the xylem (He et al., 2015; Wang et al., 2020). A recent study indicated HMA4 is responsible for the transport of Cd²⁺ through the central cylinder and xylem (Wiyono et al., 2021). In the root system, HMA4 expression increased significantly in the transgenic lines exposed to Cd stress, which increased the transport of Cd from the roots to the above-ground parts. Moreover, HMA4 expression was up-regulated in the roots, but not in the leaves. These results suggest that the substantial accumulation of Cd in the transgenic lines may have been due to enhanced heavy metal transport.