**Insight into the Phytoremediation Capability of** *Brassica juncea* **(v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity**

**Arleta Małecka 1,\*, Agnieszka Konkolewska 2, Anetta Han´c 3, Danuta Barałkiewicz 3, Liliana Ciszewska 2, Ewelina Ratajczak 4, Aleksandra Maria Staszak 5, Hanna Kmita <sup>6</sup> and Wiesława Jarmuszkiewicz <sup>6</sup>**


Received: 13 August 2019; Accepted: 31 August 2019; Published: 5 September 2019

**Abstract:** Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. *Brassica* species are known to display hyperaccumulation capability. *Brassica juncea* (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 μM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all *B. juncea* plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of *B. juncea* in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.

**Keywords:** oxidative stress; antioxidative system; Brassicaceae family; heavy metals

### **1. Introduction**

Trace metal element contamination in soils is one of the world's major environmental problems, posing significant risks to human health, as well as to ecosystems ([1]). Metals such as zinc (Zn), iron (Fe), and copper (Cu) are essential micronutrients required for a wide range of physiological processes in all plant organs, and the processes are based on the activities of various metal-dependent enzymes and proteins. However, they can also be toxic at elevated levels. Metals such as arsenic (As), mercury (Hg), cadmium (Cd), and lead (Pd) are nonessential and potentially highly toxic [2]. Trace

metal element toxicity includes changes in the chlorophyll concentration in leaves and damage of the photosynthetic apparatus, inhibition of transpiration, and destruction of carbohydrate metabolism, as well as nutrition and oxidative stress, which collectively affect plant development and growth [3–7].

Biological organisms are incapable of degrading metals, so they persist in their body parts and environment, leading to health hazards [8]. Metal accumulation and other abiotic stresses cause excess reactive oxygen species (ROS) generation, leading to oxidative stress [7]. Plant cells are equipped with enzymatic mechanisms to eliminate or reduce oxidative damage that occurs under metal accumulation. The antioxidative defense system includes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which are regarded as responsible for maintaining the balance between ROS production and scavenging [9].

The Brassicaceae family includes many genera abundant in metallophytes, such as *Thlaspi*, *Brassica,* and *Arabidopsis*. They accumulate a wide range of heavy metals, especially Zn, Cd, nickel (Ni), thallium (Tl), chromium (Cr), and selenium (Se) [10]. The term hyperaccumulator is used for plants that accumulate 1000 mg per kg of dry matter of any above-ground tissue when grown in their natural habitat [11,12]. As of 2013, approximately 500 metal hyperaccumulator plant species were described [13,14], and the number is increasing. *B. juncea* exhibits some traits of a metal hyperaccumulator—this species can take up significant quantities of Pb, Cd [15,16], Cr, Cu, Ni, Pb, and Zn [10,17], although its translocation ability is not as efficient as shown for other known hyperaccumulators. Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Therefore, the objective of the present study was to estimate the contribution of the *B. juncea* (v. Malopolska) enzymatic antioxidant system to combating the oxidative stress induced by essential (Cu, Zn) and non-essential (Pb, Cd) metal elements to allow survival under adverse environmental conditions. The analysis included trace metal accumulation, level of stress parameters, and antioxidant enzyme activity, as well as estimation of encoding mRNA and enzyme protein levels.

### **2. Results**

### *2.1. Levels of Metal Accumulation*

Research using laser ablation combined with plasma mass spectrometry (LA-ICP-MS) made it possible to determine the levels of metal accumulation in *B. juncea* organs (Figure 1). The analyses were performed for roots, stems, and leaves. In the case of roots, Pb constituted approximately 60% of all accumulated metals. In addition, approximately 4 times higher levels of accumulated Cu and Zn, as well as more than 140 times higher levels of Cd, were found in roots compared to control plant seedlings. In the stems and leaves, high levels of Cu and Zn were observed to be approximately 20 times higher than in control plants. The data allowed for calculation of the amount of accumulated Cu, Cd, Zn, and Pb in the above-ground parts, which were 58%, 55%, 52%, and 38% higher, respectively, than the amount in the roots. The results indicate that *B. juncea* is a good accumulator of trace metals, especially Cd.

### *2.2. Biomass and Morphological Changes*

The metals used in the research did not dramatically increase *B. juncea* (v. Malopolska) seedling biomass (Figure 2). The highest inhibition of biomass growth was observed for seedlings exposed to Cu. After 96 h of treatment, the seedling biomass was approximately 34% lower than that of control plants. The weakest effect was observed for seedlings treated with Pb, as after 96 h of treatment, the seedlings were approximately 10% lighter compared to control plants. The metals used in the study also did not appreciably inhibit the increase in root length. The value of the index of tolerance (IT), based on average root length also did not change dramatically (Figure 2). After 96 h of treatment, we observed the lowest IT value for Pb (70%) and the highest IT value for Cd, i.e., 90,4%. We observed the occurrence of necrotic spots on leaves and the inhibition of leaf blade surface growth with respect to control

seedlings in the above-ground parts of seedlings. Moreover, in Cd-treated seedlings, leaves were slightly twisted, whereas Cu caused strong chlorosis and shortening of the end of leaves. The smallest morphological changes were observed for seedlings treated with Zn.

**Figure 1.** Accumulation of Pb, Cu, Cd, and Zn in the roots, stems, and leaves of *B. juncea* var. Malopolska seedlings grown in Hoagland's medium and treated with lead, cooper, cadmium, and zinc ions. Metal solutions Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 were applied at a 50 μM concentration. Mean values of three replicates (±SD).

**Figure 2.** Stress parameters in *B. juncea* seedlings treated with trace metals: Pb, Cu, Cd, and Zn. The results are expressed as the mean ± standard deviation (*n* = 3). Metal solutions Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 were applied at a 50 μM concentration. Mean values of three replicates (±SD).

### *2.3. Production and Localization of ROS*

The metal-treated seedlings increased O2 .- production at levels comparable for shoots and roots compared to control seedlings, but the fluctuation in the production observed for control plants was maintained (Figure 3). In the roots, the highest values were mainly observed in the first 72 h (over 30%), whereas in the above-ground parts, the highest values were observed for 48 h (over 30–40%). After 96 h, the levels of O2 .<sup>−</sup> decreased, which may indicate high activity of the SOD enzyme. The highest level of O2 .<sup>−</sup> in roots was observed for plants treated with Zn compared with shoots treated with Zn and Cd.

The profile of the changes in the H2O2 level was similar for control roots and shoots, but the levels were distinctly higher in roots. The highest H2O2 amount was observed in roots treated with Cu, Cd, and Zn. For metal-treated samples, a significant increase in H2O2 occurred between 48 and 72 h of treatment, and the observed profile of H2O2 changes was more homogenous for shoots. We noticed a large difference in the level of H2O2 in roots after 96 h of treatment, reaching approximately 20–50% higher compared to the control. As in the case of O2 .<sup>−</sup>, H2O2 levels were also confirmed by confocal microscopy (Figure 4). The most intensive fluorescence DHE, indicating the presence of O2 .−, was observed for the *B. juncea* roots treated for 24 h with 50 μM Cd and Zn. The highest amount of H2O2 generated was observed in roots treated with 50 μM Cu, Cd, and Zn (Figure 5).

**Figure 3.** Superoxide anion (A580 g−<sup>1</sup> FW) level and SOD (USOD mg-1 protein) activities in roots and above-ground parts of *B. juncea* var. Malopolska seedlings grown in Hoagland's medium and treated with lead, cooper, cadmium, and zinc ions. Metal solutions Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 were applied at a 50 μM concentration. Mean values of three replicates (±SD).

**Figure 4.** Hydrogen peroxide level (nMol H2O2 <sup>×</sup> min−<sup>1</sup> <sup>×</sup> mg protein<sup>−</sup>1), CAT (μMol min−<sup>1</sup> mg−<sup>1</sup> protein), and APX (μMol <sup>×</sup> min−<sup>1</sup> <sup>×</sup> mg protein<sup>−</sup>1) activities in roots and above-ground parts of *B. juncea* var. Malopolska seedlings grown in Hoagland's medium and treated with lead, cooper, cadmium, and zinc ions. Metal solutions Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 were applied at a 50 μM concentration. Mean values of three replicates (±SD).

**Figure 5.** Trace metals-induced O2 −· and H2O2 production in *B. juncea* var. Malopolska roots. Fluorescent images of *B. juncea* roots grown in Hoagland's medium in the presence of 50 μmol of Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 for 24 h and control roots of plants stained with DHE for 12 h (**A**) and DCFH-DA for4h(**B**). The bar indicates 1 μm.

### *2.4. Levels of Oxidized Proteins*

The levels of protein oxidative modification imposed by the metal treatment were 12–44% higher for roots and above-ground parts compared to control plants (Figure 1). The level of oxidized proteins reached a maximum after 48 h and was 3-fold higher than in the shoots of control plants.

### *2.5. Enzyme Antioxidant Activity*

SOD activities were 25–50% higher in the roots of plants treated with trace metals. In the above-ground parts, greater differences in SOD activity between research variants, ranging from 8 to 70%, were observed. However, the general activity of SOD was higher in roots and shoots compared to control seedlings (Figure 3) and changed differently for the seedling parts. In the case of roots, the activity level and profile were comparable to those of control seedlings, whereas for shoots, after the initial increase, the activity decreased significantly after 96 h. The generation of H2O2 caused a rapid increase in CAT activity within 24 h of cultivation, i.e., from 30% to 70% in the roots of plants treated with trace metals, especially in plants treated with Zn (Figure 4). In the next days, we observed a slight decrease (approximately 12–55%), but this decrease remained higher than that in control plants. The highest CAT activity was observed above ground in the first 48 h of cultivation (56%) in plants exposed to Cd. Activities of APX, a second enzyme involved in the dismutation of hydrogen peroxide, systematically increased in roots exposed to metals during the cultivation period, especially in plants grown in the presence of Cu and Zn, which had approximately 10–43% higher levels than those observed in the control (Figure 4). In the above-ground parts of *B. juncea* cultured in the presence of trace metals, we observed an increase in the intensity of APX during the first 48 h, reaching a maximum in plants treated with Cd for 48 h, approximately 62% higher than in the control, and then a slight decrease, but the activities were approximately 2-fold higher than those in control plants. The activity profiles of CAT and APX differed between the control roots and shoots (Figure 4). The metal treatment increased the activity of both enzymes, and the CAT activity profile appeared to be maintained in roots and shoots. However, the APX profile did not differ from that of the control plants with respect to treated shoots, whereas in treated roots, the APX activity profile was variable and metal-dependent, although comparable for Cu and Zn.

### *2.6. Levels of Gene Transcripts*

To estimate possible changes at the level of CuZnSOD and MnSOD encoding gene transcripts, we used an electrophoretic separation technique and the CpAtlas program (Figure 6). In the case of CuZnSOD, a decrease in the expression of the gene encoding CuZnSOD was observed in the roots of plants treated with trace metals after 4 h and 24 h of cultivation, with the exception of the roots of

*B. juncea*-treated Cu. Induction of the gene in the above-ground parts was visible, with an approximate 2-fold increase in the level of the transcript in plants after 8 h of copper treatment and an approximate 2-fold decrease in plants after 4 h of zinc treatment. The results indicate that the presence of cadmium ions had no significant effect on the induction of CuZnSOD gene expression because no significant changes in the level of the transcripts was observed in either the roots or above-ground parts of *B. juncea* plants.

**Figure 6.** Transcriptional levels of genes encoding antioxidative enzymes in roots and above-ground parts of *B. juncea* var. Malopolska seedlings grown in Hoagland's medium and treated with lead, cooper, cadmium, and zinc ions. Metal solutions Pb(NO3)2, CuSO4, CdCl2, and ZnSO4 were applied at a 50 μMol concentration. Enzymes chosen for the experiment were amplified using semi-quantitative RT-PCR with primers designed for *Arabidopsis thaliana* genes: *CSD1* for CuZnSOD and *MSD1* for MnSOD.

When analyzing changes in the expression of the gene encoding MnSOD, a decrease in the expression was observed in the roots and above-ground parts of plants after 4 h of treatment with lead ions; in the remaining research variants, there were no significant differences in transcript levels compared to control plants. An approximate 2-fold increase in the level of the transcript was found in plant roots after 24 h of Pb and Zn treatment in comparison to the control. The greatest decrease in expression was observed after 24 h in the above-ground parts of plants treated with Cu, which was almost 5-fold higher than that in the control (Figure 6).

### *2.7. Identification of Enzyme Forms*

To distinguish between the enzyme forms, Western blot analysis was performed for protein extracts from roots and above-ground seedling parts in the absence and presence of the metal treatment (Figure 7). This allowed for the detection of MnSOD (25 kDa) and CuZnSOD (15 kDa and 20 kDa) subunits. The obtained signal was similar for both the treated and control seedlings. Thus, the metal presence likely did not change the levels of the CuZnSOD and MnSOD proteins.

**Figure 7.** Effects of 50 μM Pb, Cu, Cd, and Zn for 24 h on the CuZnSOD and MnSOD of roots and above-ground parts of *B. juncea* var. Malopolska seedlings. The protein content was evaluated by Western blot using specific antibodies.

### **3. Discussion**

Trace metals are one of the most important abiotic stress factors affecting the natural environment. As a result of anthropogenic activities, we can observe their increasing levels from year-to-year. Metal toxicity results in effects at physiological and cellular levels, leading to distorted metabolism, including plant metabolism [18]. Abiotic stresses, including the presence of trace metals in soil, are estimated to be the main cause of global crop yield reduction of ca. 70% and thus are considered a great constraint to crop production. This situation has worsened due to disturbed equilibrium between crop production and human population growth. Therefore, it is especially important to understand plant responses to such stress factors. This also applies to trace metals [12]. In the present study, this was clearly visible in the growth of plant biomass, which significantly decreased during the culture in the presence of heavy metals. Copper and zinc ions are essential for the normal growth and development of all organisms but can be toxic to plants at excessive levels. Lead and cadmium are nonessential elements and are toxic to plants even at low levels [8]. Essential and nonessential trace elements, when exceeding the threshold limits, can cause different physiological, morphological, and genetic plant anomalies, including reduced growth, mutations, and increased mortality [8]. Therefore, plants suitable for phytoremediation are, at present, of great importance.

In our study, we noticed that in the case of *B. juncea* v. Malopolska, all the mentioned metals used at 50 μM concentration displayed moderate phytotoxic properties. The biomass increments ranged between 96 mg for Pb-treated plants and 61 mg for Cu-treated plants, and the values were approximately 7% and 41% lower, respectively, than those in control plants. Several studies have shown that high concentrations of trace metals in the soil cause plant growth impairment [9,19]). In *Sesbania drummondii*, a reduction in seedling biomass was caused by Pb—21%, Cu—46.3%, Ni—31.5% and Zn—25.2% [20]. The inhibition of shoot growth by trace metals may be due to a decrease in photosynthesis, as trace metals disturb mineral nutrition and water balance, change hormonal status, and affect membrane structure and permeability [21]. Trace metals might cause an inhibition of root growth that alters water balance and nutrient absorption [12] and decrease calcium uptake in root tips, leading to a decrease in cell division or cell elongation [9,22,23]. According to Marshner [23], Cd-induced mineral stress can reduce plant dry weight accumulation. Other authors have shown a negative influence of Pb [24], Cu [25], Cd [26], and Zn [20]. Despite the inhibitory effect caused by trace metals on the growth of the biomass of *B. juncea,* we observed a high IT amounting to approximately 90% resistance of the plants to trace metals.

The bioaccumulation of trace metals is different for various plant species, reflected by their growth, reproduction, occurrence, and survival in metal-contaminated soil, because the mechanisms of elemental uptake by plants are not the same for all species. The capacity of plants to take up trace metals is different for different metals, and the same trace metal can be accumulated at different ratios in different plant species [27]. Metal bioavailability is also affected by the presence of organic compounds of that metal in plants [8]. The ICP-MS results we obtained indicate that the accumulation of trace metals was higher in above-ground parts than in roots, especially for cadmium, lead, and zinc. The metal concentrations followed an order of Pb > Cu > Zn > Cd in roots, Zn > Cu > Pb > Cd in

the stem, and Zn > Cu > Cd > Pb in leaves [28]. Based on the obtained results, it can be concluded that *B. juncea* is a hyperaccumulator of Cd, Zn, and Pb. Cherif and co-authors [29] reported that Zn induced a decrease in Cd uptake and a simultaneous increase in Zn accumulation, indicating a strong competition between these two metals for the same membrane transporters. In our earlier study [28] in *B. juncea* plants treated with a binary combination of metals, namely, PbCu, PbCd, PbZn, CuZn, CuCd, and ZnCd, at a concentration of 25 μM of each, a synergistic response between Zn and Pb was observed, resulting in an increased accumulation of the two metals. The accumulation results obtained for plants treated with Cu are different from those of other researchers. Purakayastha and others [30] showed that Cu is accumulated mainly in above-ground parts of *B. juncea*. This difference may result from different exposure durations of the plant to the metal, other metal concentrations, and different plant ages at the time of analysis of the collected metal. Quaritacci et al. [31] reported that *B. juncea* was identified as a species able to take up and accumulate metals in its above-ground parts, such as Cd, Cu, Ni, Zn, Pb, and Se. It has been observed that this species concentrated Cu, Pb, and Zn in its above-ground parts in amounts much higher than those detected in the metal soluble fractions present in a soil contaminated by acidic water and pyritic slurry [31].

The accumulation of trace metals in organs is dangerous for plants. In an earlier study [32], we confirmed that plants are not adequately protected by the detoxification system because trace metals penetrate in areas with high metabolic activity, such as the cytoplasm, mitochondria, or cell membrane.

The occurrence of oxidation stress conditions in *B. juncea* treated with the trace metals Pb, Cu, Cd, and Zn was confirmed by the increase in the level of oxidized proteins in the roots (approximately 7–12%) and above-ground parts (approximately 13%). Several metals, including Cd, Pb, and Hg, have been shown to cause protein oxidation by depletion of protein thiol groups [33]. ROS cause protein modifications through the formation of carbonyl groups at certain amino acid residues. Such modifications were caused by the presence of heavy metals, e.g., cadmium [34], mercury lead, aluminum, zinc, copper, cobalt, nickel, and chromium [35].

ROS also act as signaling molecules involved in the regulation of many key physiological processes, such as root hair growth, stomatal movement, cell growth, and cell differentiation, when finely tuned and regulated by an antioxidative defense system [12]. We showed an increase in the level of ROS compared to control plants in all plants treated with heavy metals. The O2 .<sup>−</sup> rate after 2 h of culture was 2 times higher than that observed in plants grown under control conditions. The high level of O2 .<sup>−</sup> was the highest between 24 to 72 h of the treatment depending on the research variant. The highest value of O2 <sup>−</sup> was measured in plants treated with Zn, while the highest H2O2 values were observed in plants treated with Cu and Cd. Similar results were obtained by other researchers. Markovska et al. [36] showed a 10-fold higher level of H2O2 in the leaves of *B. juncea* after 5 days of treatment with Cd ions at a concentration of 50 μM. Wang et al. [37] observed the highest levels of H2O2 in *B. juncea* roots treated with Cu ions for 4 days. In our research, the highest level of H2O2 was obtained after 4 days in plants treated with single metals. The reduction of O2 .<sup>−</sup> and the H2O2 content in roots and above-ground parts of plants treated with trace metals during the cultivation period suggests that some antioxidative enzymes would work effectively in the removal of ROS. To detect ROS in plant cells, we used incubation with fluorescent labels such as 2 7 -difluoroscein and dihydroethidium and imaging under confocal microscopy. We observed increased generation of O2 .<sup>−</sup> and H2O2 in the roots of *B. juncea* treated with trace metals—especially Cd and Zn for O2 .<sup>−</sup>, and Cu, Cd, and Zn for H2O2.

The increase in ROS production in metal-treated plants was precisely associated with changes in the activity of antioxidant enzymes. We always observed the induction of antioxidant enzyme activity in *B. juncea* roots and leaves, although there were no significant differences between the used metals. We observed increasing activity of antioxidant enzymes, i.e., 20–158% for SOD, 15–147% for CAT, and 6–68% for APX. The highest activity of SOD in both roots and shoots was observed in plants treated with Zn and Cu. The first line of defense against ROS-mediated toxicity is through SOD, which catalyzes the dismutation of superoxide anions to H2O2 and O2. The stimulation of SOD activity has also been reported in several plants exposed to Pb, Cu, Cd, Zn, Ni, and as ions [20,25,38,39]. We noticed that in the roots of *B. juncea,* the most induced activity of CAT was for Zn, compared with Cd in the above-ground parts. APX was definitely lower than catalase, especially in the above-ground parts, which means that this enzyme complements CAT catalytic activity. APX activity was significantly elevated in the metal-treated plants, which suggests its role in the detoxification of H2O2. Enhanced CAT and APX activity has been observed in various plant species after application of trace metals: Pb, Cu, Cd, Zn, Ni, and As [20,25,38–40]. APX may be responsible for controlling the levels of H2O2 as signal molecules, and the CAT function is to remove large amounts of oxygen during oxidative stress. APX may be responsible for controlling the levels of H2O2 as signal molecules, and the CAT function is to remove large amounts of oxygen during oxidative stress [41]. Mohamed et al. [42] showed in *B. juncea* that the higher activity of antioxidant enzymes offers a greater detoxification efficiency, which provides better plant resistance against trace metal-induced oxidative stress. Yadav and co-authors [25] reported increases in the activities of antioxidant enzymes: SOD by 16.2%, DHAR by 27–58%, GR by 35.74%, GST and GPX by 19.19%, and APX by 42.75% in *B. juncea* plants treated with 0.0005 M Cu. The authors indicated that brassinostereoids can regulate the activity of the antioxidant system and help in scavenging overproduced ROS, and can provide tolerance by inducing the expression of regulatory genes such as respiratory burst oxidase homologue, mitogen-activated protein kinase-1, and mitogen-activated protein kinase 3, as well as activating genes involved in antioxidative defense and responses [25]. Other authors [12] have noted that brassinosteroids are a group of hormones that regulate ion uptake in plant cells and reduce trace metal accumulation in plants. An exogenous application of brassinosteroids is widely used to improve crop yield, as well as stress tolerance, in various plant species.

We previously demonstrated an increase in the activity of the antioxidant system at the physiological and biochemical levels. The next step was to determine whether trace metals influence the transcription level of genes encoding suitable defense proteins. ROS concentration at an appropriate level can promote plant development and reinforce resistance to stressors by modulating the expression of a set of genes and redox signaling pathways [12]. In our research, we observed differences in the expression induction depending on the exposure time and the metal used. We observed an increase in the level of the gene coding for CuZnSOD in plants treated with copper, zinc, and lead. The highest level of expression was obtained after 4 h in roots and 8 h in above-ground parts. Romero-Puertas and co-authors [34] noted a drastic reduction in the expression of genes coding for CuZnSOD and no changes in MnSOD in *Pisum sativum* under conditions of stress caused by the presence of Cd. Their results showed a reduction in CuZnSOD levels in the presence of Cd, while in our study, we did not observe significant differences in the level of transcript for plants treated with this metal in relation to control plants. We observed the induction of gene expression encoding MnSOD in *B. juncea* roots after 8 h of exposure to Zn and Pb ions, compared with lead ions in above-ground parts. Other authors did not observe any changes or a low expression of genes coding for SOD, e.g., Fidlago et al. [43] showed no differences in MnSOD-related mRNA accumulation in leaves and roots, but CuZnSOD-related transcripts decreased in leaves but did not change in roots in Cd-treated *Solanum nigrum* L. Others authors [44] indicated that Cd stress induced an upregulated expression of FeSOD, MnSOD, Chl CuZnSOD, Cyt CuZnSOD, APX, GPX, GR, and POD at 4–24 h after treatment began for *Lolium perenne* L., and their results suggested that the gene transcript profile was related to the enzyme activity under Cd stress. Romero-Puertas et al. [34] indicated two groups of genes in pea plants treated with Cd. First, some elements of the signal transduction cascade accentuated or attenuated the Cd effect on CAT, MDHAR, and CuZnSOD mRNA expression. The second was formed by the genes MnSOD, APX, and GR that were not affected by these modulators during the Cd treatment because their expression was not modified compared to control plants.

The effect of Cd on the expression of CuZnSOD was reversed by a nitric oxide (NO). scavenger, indicating that NO. must be a key element in the regulation of this SOD, showing the existence of a relationship between an increase in ROS production and NO. NO-dependent downregulation was also observed for MnSOD, while the opposite effect was found for APX and GR. This suggests that protein

phosphorylation is involved in the response to Cd stress [34]. Bernard and co-authors [45] indicate that molecular analysis (gene expression) is the first level of integration of environmental stressors, and it is supposed to respond to stressors earlier than biochemical markers.

Our results from Western blotting indicate that the presence of trace metals does not increase the synthesis of the proteins CuZnSOD and MnSOD in the organs of *B. juncea* plants, but induces an increase in their activity.

### **4. Materials and Methods**

### *4.1. Plant Material*

*Brassica juncea* v. Malopolska seeds were grown in Petri dishes for 7 days under optimal conditions. Next, seedlings were cultivated hydroponically on Hoagland's medium for 7 days in a growth room with a 16/8 h photoperiod, day/night at room temperature and light intensity of 82 μmol m<sup>2</sup> s<sup>−</sup>1. Then, the applied medium was changed into 100×-diluted Hoagland's medium and a heavy metal solution in combination; Cu, Pb, Cd, and Zn ions at a concentration of 50 μM were applied. In the cultivation, a solution of Pb(NO3)2, CuSO4, CdCl2, Zn SO4 was used. The roots and shoots were cut off after 0, 24, 48, 72, and 96 h of cultivation. The roots were dipped sequentially in cold solutions of 10 mM CaCl2 and 10 mM EDTA for 5 min each to eliminate trace elements adsorbed at the root surface. Then, roots and shoots were rinsed three times with distilled water, frozen in liquid nitrogen, and stored at −80 ◦C until molecular analysis.

### *4.2. Phytotoxic Test*

The index of tolerance (IT) was calculated according to Wilkins [46]:

$$\text{IT} = \frac{\text{average length of roots in tested solution}}{\text{average length of roots in control}} \times 100\% \tag{1}$$

The changes in fresh biomass of control plants and plants treated with metals were measured on a Radwag scale after 0, 24 28, 72, and 96 h of cultivation.

### *4.3. Accumulation of Trace Metals*

The determination of trace metal accumulation was performed using inductively coupled plasma mass spectrometry (ICP-MS) model Elan DRC II, (Perkin Elmer Sciex, Concord, Ontario, Canada) connected with laser ablation (LA) model LSX-500 (CETAC Technologies, Omaha, NE, USA). Plant material (roots, stems, and leaves) was rinsed with distilled water, gently dried on blotting paper, weighed, and dried at 70 ± 2 ◦C. The dried samples were mineralized in an MDS-2000 microwave digestor oven (CEM Corporation Matthews, NC, USA). A three-stage dilution was conducted in a closed system using 5 mL of 65% HNO3. After mineralization, samples were transferred to 10 mL flasks filled with deionized water. An ICP-MS was used to determine the concentration of elements in the mineralized plant tissues.

Plant roots, stems, and leaves were collected after 72 h of treatment for the analysis of metal distribution. Samples were cut into 3 mm long pieces and ablated along the pre-defined line across the cross-sections. Laser performance was optimized according to a detailed scheme [47] using a single variable method.

### *4.4. Superoxide Anion Determination*

The superoxide anion content was determined according to Doke [48]. *B. juncea* roots (0.5 g) were placed in the test tubes that were filled with 7 mL of a mixture containing 50 mM phosphate buffer (pH 7.8), 0.05% NBT (nitro blue tetrazolium), and 10 mM of NaN3. Next, the test tubes were incubated in the dark for 5 min, and then 2 mL of the solution was taken from the tubes, heated at 85 ◦C for 10–15 min, cooled on ice for 5 min, and the absorbance was measured at 580 nm against the control.

### *4.5. Hydrogen Peroxide Content*

The hydrogen peroxide content was determined using the method described by Patterson et al. [49]. The decrease in absorbance was measured at 508 nm. The reaction mixture contained 50 mM phosphate buffer (pH 8.4) and reagents, 0.6 mM 4-(-2 pyridylazo)resorcinol, and 0.6 mM potassium-titanium oxalate (1:1). The corresponding concentration of H2O2 was determined against the standard curve of H2O2.

### *4.6. In Situ Detection of Superoxide Anion and Hydrogen Peroxide*

The roots and shoots from plants exposed to metals for 24 h were submerged for 12 h in 100 μM of CaCl2 containing 20 μM of dihydroethidium (DHE, pH 4.75; samples for superoxide anion radicals) or 4 μM dichlorodihydrofluorescein diacetate (DCFH-DA) (samples for hydrogen peroxide) in 5 mM dimethyl sulfoxide (DMSO). After rinsing with 100 μM of CaCl2 or 50 mM phosphate buffer (pH 7.4), the roots and shoots were observed with a confocal microscope (Zeiss LSM 510, Axiovert 200 M, Jena, Germany) equipped with no. 10 filter set (excitation 450–490 nm, emission 520 nm or more).

### *4.7. Estimation of Protein Oxidation*

For carbonyl quantification, the reaction with DNPH was used basically as described by Levine et al. [50]. For each determination, two replicates and their respective blanks were used. Roots and shoots (0.5 g) were incubated with isolation buffer containing 0.1 M Na-phosphate buffer, 0.2% (*v*/*v*) Triton X—100, 1 mM EDTA, and 1 mM PMSF. After centrifugation at 13,000× *g* for 15 min, supernatants (200 μL) were mixed with 300 μL of 10 mM DNPH in 2 M HCl. The blank was incubated in 2 M HCl. After 1 h incubation at room temperature, proteins were precipitated with 10% (*w*/*v*) trichloroacetic acid (TCA), and the pellets were washed three times with 500 μL of ethanol/ethylacetate (1:1). The pellets were finally dissolved in 6 m guanidine hydrochloride in 20 mM potassium phosphate buffer (pH 2.3), and the absorption was measured at 370 nm. Protein recovery was estimated for each sample by measuring the absorption at 280 nm. The carbonyl content was calculated using the molar absorption coefficient for aliphatic hydrazones, 22,000 m−<sup>1</sup> cm<sup>−</sup>1.

### *4.8. Determination of Antioxidant Enzyme Activities*

The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of NBT, adopting the method of Beauchamp and Fridovich [51]. The reaction mixture contained 13 μM riboflavin, 13 mM methionine, 63 mM NBT, and 50 mM potassium phosphate buffer (pH 7.8). Absorbance at 560 nm was then measured. One unit of SOD activity has been defined as the amount of enzyme that causes a 50% decrease in the inhibition of NBT reduction. The activity of CAT was determined by directly measuring the decomposition of H2O2 at 240 nm for 3 min as described by Aebi [52] in 50 mM phosphate buffer (pH 7.0) containing 5 mM H2O2 and enzyme extract). CAT activity was determined using the extinction coefficient of 36 mM−<sup>1</sup> cm−<sup>1</sup> for H2O2. The activity of APX was assayed using the method described by Nakano and Asada [53] by monitoring the rate of ascorbate oxidation at 290 nm (extinction coefficient of 2.9 mM−<sup>1</sup> cm<sup>−</sup>1) for 3 min. The reaction mixture consisted of 25–50 μL supernatant, 50 mM phosphate buffer (pH 7.0), 20 μM H2O2, 0.2 mM ascorbate, and 0.2 mM EDTA.

### *4.9. Isolation of Total RNA and RT-PCR*

Roots and above-ground parts (100 mg) of *B. juncea* plants in the presence of trace metals and under control conditions were collected for total RNA isolation. The RNA was isolated with TRIzol reagent and tested spectrophotometrically for purity at 260 and 280 nm. Then, RNA was reverse-transcribed with oligo (dT) primers using the RevertAid Reverse Transcriptase Kit (Thermo Science, Lithuania, European Union) after DNA was treated with DNase I (Thermo Science).

Primer pair sequences were as follows (forward/reverse, gene accession number): gtgattgcttgca gggtttt/cagaatacggaagcaaatgtca, X54844.1 (TUB1), ggagcaagtttggttccatt/aaggttattcggccagattg, U30841.1 (MnSOD), gaacaatggtgaaggctgtg/gtgaccacctttcccaagat M63003.1 (CuZnSOD).

As a reference gene, the gene encoding tubulin was used. PCRs were performed with 30 (BJMnSOD) and 34 (BjCuZnSOD) cycles of denaturation, 95 ◦C for 30 s; annealing primers, 53 ◦C for 30 s; and elongation, 72 ◦C for 30 s using a 1:100 diluted cDNA template and REDAllegro*Taq* DNA Polymerase (Novazym, Pozna ´n, Poland).

PCR products were separated by electrophoresis on a 1.3% agarose gel with ethidium bromide in TBE (445 mM Tris-HCL; 445 mM boric acid; 10 mM EDTA; pH 8.0), visualized under UV light and photographed using the Photo Print 215SD V.99 Vilber Lourmat Set. CP Atlas 2.0 were used for densitometric analysis of relative gene expression.

### *4.10. Western Blot*

RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) was used to lyse cells for protein extraction. The protein concentrations were determined using the Bradford method, and 20 μg of each extract was loaded onto a 12% SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gel. Separated proteins were transferred to polyvinylidene fluoride membrane (ImmobilonTM-P, Millipore, Burlington, MA, USA) at 350 mA for 1 h using the Mini Trans-BlotCell (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 1% BSA and incubated with an antibody against CuZnSOD at a final dilution of 1:2500. The secondary antibody, goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA), was used at a 1:3000 dilution to visualize protein bands by reaction with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) (Sigma-Aldrich, St Louis, MO, USA/CALBIOCHEMV.S. and Canada) as a substrate.

### *4.11. Protein Quantification*

Total soluble protein contents were determined according to the method of Bradford [54] using the Bio-Rad assay kit with bovine serum albumin as a calibration standard.

### *4.12. Statistical Analyses*

Each experiment was performed in three biological and technical replicates. The mean values ± SE are given in the tables and figures. The data were analyzed statistically using IBM SPSS Statistics (Version 22 for Windows). Significant differences among treatments were analyzed by one-way ANOVA, taking *p* < 0.05 as the significance threshold, and the b-Tukey post-hoc test was conducted for pairwise comparisons between treatments.

### **5. Conclusions**

This study was conducted to determine the interactive role of Pb, Cu, Cd, and Zn in metal uptake, plant growth, and the antioxidative system of *B. juncea*. Plants accumulated high amounts of trace metals, i.e., more than 40% in the roots, and in the above-ground parts, the values for Cu, Cd, Zn, and Pb were 58%, 55%, 52%, and 38%, respectively. The results suggest that *B. juncea* var. Malopolska is a good hyperaccumulator of trace metals, especially Cu, Cd, and Zn, and can be useful in phytoremediation. The presence of metals resulted in a considerable reduction in *B. juncea* biomass; the highest reduction was observed in plants treated with Cu and Cd. Despite the visible influence of trace metals on plant morphology, the IT coefficient was high and exceeded 90%, indicating the high resistance of *B. juncea* plants. Trace metals lead to the production of ROS, which causes an imbalance in the redox state in the plant cells and increases the level of oxidized proteins. We noticed that under the conditions of oxidative stress, the antioxidant system was activated: SOD, CAT, and APX. We observed that the presence of metals influenced the increase in the activity of antioxidant enzymes, while no significant differences were observed in the levels of CuZnSOD and MnSOD transcripts and proteins. The results obtained indicate that *B. juncea* var. Malopolska has efficient defense mechanisms to cope with different metals.

**Author Contributions:** A.M. conceived and designed the experiments, performed part of analysis, wrote-original draft, review and editing, A.K. performed the molecular experiments, A.H. performed analysis LA-ICP-MS and ICP-MS, analyzed the data from ICP-MS D.B. analyzed the data from LA-ICP-MS, L.C. performed biochemical tests, E.R. perfomed immunological research, A.M.S. graphical analysis of results, H.K. manuscripts edition, W.J. substantive care.

**Funding:** This work was partially supported by the National Science Centre no. N N305 381138. AH was supported by the National Science Center in Poland under the grant number 2017/01/X/ST4/00373

**Conflicts of Interest:** The authors declare no conflicts of interest.

### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Selenium Modulates the Level of Auxin to Alleviate the Toxicity of Cadmium in Tobacco**

**Yong Luo 1, Yuewei Wei 1, Shuguang Sun 2, Jian Wang 2, Weifeng Wang 3, Dan Han 1, Huifang Shao 1, Hongfang Jia 1,\* and Yunpeng Fu 1,\***


Received: 23 June 2019; Accepted: 30 July 2019; Published: 1 August 2019

**Abstract:** Cadmium (Cd) is an environmental pollutant that potentially threatens human health worldwide. Developing approaches for efficiently treating environmental Cd is a priority. Selenium (Se) plays important role in the protection of plants against various abiotic stresses, including heavy metals. Previous research has shown that Se can alleviate Cd toxicity, but the molecular mechanism is still not clear. In this study, we explore the function of auxin and phosphate (P) in tobacco (*Nicotiana tabacum*), with particular focus on their interaction with Se and Cd. Under Cd stress conditions, low Se (10 μM) significantly increased the biomass and antioxidant capacity of tobacco plants and reduced uptake of Cd. We also measured the auxin concentration and expression of auxin-relative genes in tobacco and found that plants treated with low Se (10 μM) had higher auxin concentrations at different Cd supply levels (0 μM, 20 μM, 50 μM) compared with no Se treatment, probably due to increased expression of auxin synthesis genes and auxin efflux carriers. Overexpression of a high affinity phosphate transporter NtPT2 enhanced the tolerance of tobacco to Cd stress, possibly by increasing the total P and Se content and decreasing Cd accumulation compared to that in the wild type (WT). Our results show that there is an interactive mechanism among P, Se, Cd, and auxin that affects plant growth and may provide a new approach for relieving Cd toxicity in plants.

**Keywords:** selenium; cadmium stress; auxin; root architecture; phosphate transporter; *Nicotiana tabacum*

### **1. Introduction**

Cadmium (Cd) is a highly toxic heavy metal, which is widely distributed in the environment [1]. In recent years, industrial and agricultural production have discharged Cd to varying degrees, and it has become one of the most widely distributed agricultural pollutants [2]. Cd is not needed for plant growth and development but is more likely to accumulate in plants than other heavy metals [3]. Evidence suggests that a high concentration of Cd in soil affects the growth and development of plants through physiological and biochemical processes, including inhibition of plant enzyme and membrane activity [4], decreased cell division [5], reduced growth rate [6], damaged photosynthesis [7], inhibition of stomatal opening [8], and promotion of lipid peroxidation [9]. Cd bioaccumulates and in humans can cause diseases such as osteoporosis, anemia, hypertension, and kidney damage. Soil pollution by Cd has become a serious threat to the safety of agricultural produce. To address this issue, it is important to develop a comprehensive understanding of the mechanism of Cd uptake in plants. Selenium (Se) is not essential for plant nutrition, but it can play a beneficial role in plant health. A suitable dose of Se can

enhance antioxidant capacity, delay aging, increase photosynthesis, boost auxin content, and promote plant growth [10,11]. By contrast, a high dose of Se can damage plants through reactive oxygen species (ROS) accumulation and inhibit plant growth [12]. Plants are able to utilize soil Se in its inorganic Se (IV) and Se (VI) forms (selenite and selenate, respectively). Studies have shown that plants absorb selenate through sulfate transporters and selenite through phosphate channels [13,14]. A previous study has suggested that Se (IV) uptake is mediated by Pi (inorganic phosphate) transporters [15]. Recent research suggests that auxin is involved in the interaction between Pi and Se in tobacco, which provides convincing evidence for understanding the molecular mechanism of how Se regulates plant growth [16].

Se inhibits Cd uptake, which can relieve the toxic effects of Cd in plants. Cary et al. (1981) first reported that Se fertilizer reduced the absorption of Cd in wheat and lettuce [17]. Increasing numbers of studies have shown that there is antagonism between Cd and Se in plants [18–20]. Cd stress has also been shown to inhibit root growth and leaf photosynthesis in winter wheat, although adding some doses of Se can alleviate the Cd toxicity by enhancing root growth [21]. Despite studies investigating the antagonistic relationship between Se and Cd, this relationship—especially the molecular mechanism of Se remission of toxic Cd effects—is still not clear.

Tobacco is an important economic and model crop. In this study, we aimed to clarify the molecular mechanism of Se remission of toxic Cd effects in tobacco. We chose *DR5::GUS* and *NtPT2* overexpressed transgenic tobacco as the test material. We studied the influence of Cd on growth; the antioxidant system; the auxin distribution; and the Se, Cd uptake of tobacco under various Se and Cd treatments. We aimed to (1) clarify the function of auxin in tobacco growth, under different doses of Se and Cd; (2) determine the mechanism by which Se enhances tolerance to Cd stress; and (3) reveal the function of phosphate transporter (*NtPT2*) in the interaction of Se and Cd in plants.

### **2. Results**

### *2.1. E*ff*ects of Se on Tobacco Phenotype and Biomass under Cd Stress*

To determine whether Se affects the growth of tobacco under different Cd treatments, we checked the characterization of tobacco plants. We found that Cd treatment had a substantial toxic effect on tobacco seedlings, with greater yellow leaf area and more poorly developed root architecture in Cd-treated seedlings than those in the Cd0 treatment (Figure 1A and Figure S1). Interestingly, we also found that low Se (Se10) could promote the growth of tobacco under Cd stress (Figure 1A); under Cd20 and Cd50 stress conditions, the biomass of shoots with Se10 treatments increased by 16.3% and 20.8%, respectively, compared to those with no Se added (Figure 1B), and the biomass of roots with Se10 treatments increased by 24.2% and 30.2%, respectively (Figure 1C). By contrast, high Se (Se50) levels suppressed tobacco growth under different Cd (Cd0, Cd20 and Cd50) treatments (Figure 1). These results show that Se has a dual effect on Cd stress, and the appropriate concentration of Se can alleviate Cd toxicity in tobacco.

**Figure 1.** Characterization of tobacco under different Se and Cd concentration supply conditions for 21 days. (**A**) The phenotype of shoot, third young leaf and root under Se (IV) and Cd (II) treatments in tobacco; (**B**) The biomass of shoot under different Se (IV) and Cd (II) concentration supply conditions; (**C**) The biomass of root under different Se (IV) and Cd (II) concentration supply conditions. 14-days-old seedlings (wild-type, Yunyan 87) were grown in pots with sand under different Se (0, 10, 50 μM) and Cd (0, 20, 50 μM) concentrations for 21 days. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0:** no Cd; **Cd20**: Cd, 20 μM; **Cd50**: 50 μM. Shown are mean ± standard deviation (SD) from five biological replicates. DW, dry weight. Different letters indicate significant differences (*p* < 0.05).

### *2.2. E*ff*ects of Se and Cd Interactions on Tobacco Antioxidant Capacity*

Previous studies showed that Se can enhance the enzymatic and non-enzymatic anti-oxidation systems and improve plant resistance to abiotic stresses in plants [4–7]. To determine the function of Se under Cd stress conditions, we measured the antioxidant capacity and chlorophyll content of tobacco. Firstly, we determined the accumulation of H2O2 by nitroblue tetrazolium (NBT) staining. Low Se (Se10) obviously reduced the accumulation of H2O2 (Figure 2A), which was consistent with the results of Malondialdehyde (MDA) content under Cd stress conditions (Figure 2B). We also checked the chlorophyll content, and found that Se had notable effects on chlorophyll content in the leaves (Figure 2C). At no Se (Se0) and high Se (Se50) levels, increased Cd levels significantly reduced tobacco chlorophyll content. Notably, the chlorophyll content of the low Se (Se10) treatment showed a remarkable increase under different Cd levels, which implies that low Se can promote tobacco growth under Cd stress by improving the anti-oxidation activity of tobacco plants.

**Figure 2.** Effects of Se and Cd treatments on antioxidant capacity of tobacco. (**A**) nitroblue tetrazolium (NBT) staining of tobacco seedlings under different Se (IV) and Cd (II) concentration supply conditions; (**B**) the content of MDA of tobacco seedlings under different Se (IV) and Cd (II) concentration supply conditions; (**C**) the SPAD of the third young leaf of tobacco seedlings under different Se (IV) and Cd (II) concentration supply conditions; 14-days-old seedlings (wild-type, Yunyan 87) were grown in pot with sand under different Se (0, 10, 50 μM) and Cd (0, 20, 50 μM) concentrations for 21 days. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0**: no Cd; **Cd20**: Cd, 20 μM; **Cd50**: Cd, 50 μM. Shown are mean ± SD from five biological replicates. Different letters indicate significant differences (*p* < 0.05).

### *2.3. Accumulation of Se and Cd in Tobacco*

To investigate the accumulation of Cd in tobacco under low Se (Se10) and high Se (Se50) conditions, we monitored the Se and Cd content in the roots and shoots of tobacco seedlings (Figure 3). Under high Se and low Se conditions, the Se content of the shoots and roots increased in Cd20 and Cd50 treatments compared with the Cd0 treatment (Figure 3A,B). Under Cd stress (Cd20, Cd50) conditions, the low Se treatment significantly reduced the Cd content of tobacco shoots and roots, especially under the Cd20 treatment, where the root Cd content was 37.3% lower than that observed with the Se0 treatment (Figure 3C,D). We also found that high Se did not reduce the Cd content of tobacco plants, suggesting that variation in Se content can affect the uptake of Cd in tobacco.

**Figure 3.** Content of Se and Cd in tobacco under different Se and Cd concentration supply conditions for 21 days. (**A**,**B**) Se content of the shoots and roots under different Se (IV) and Cd (II) concentration supply conditions; (**C**,**D**) Cd content of the shoots and roots under different Se (IV) and Cd (II) concentration supply conditions. 14-days-old seedlings (wild-type, Yunyan 87) were grown in pot with sand under different Se (0, 10, 50 μM) and Cd (0, 20, 50 μM) concentration for 21 days. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0**: no Cd; **Cd20**: Cd, 20 μM; **Cd50**: Cd, 50 μM. Shown are mean ± SD from five biological replicates. Different letters indicate significant differences (*p* < 0.05).

### *2.4. E*ff*ects of Se on Auxin and Expression of Auxin-Related Genes in Tobacco under Cd Stress*

To investigate whether auxin is involved in the growth of tobacco roots under Se and Cd treatment, we used *DR5::GUS* transgenic tobacco, which could reflect the distribution of auxin in the plant [22–24]. We detected *GUS* expression in the root tip under Se and Cd treatments (Figure 4A). Under Cd stress (Cd20, Cd50) conditions, the *GUS* expression in the root tip of plants was much lower than observed in the Cd0 treatment. Under low Se (Se10), the expression of *GUS* in the root tip was much greater than that in Se0 tobacco. Low Se could increase the expression of *GUS* under Cd stress. We also checked the auxin content of shoots and roots, which was consistent with the *GUS* expression results (Figure 4B,C).

**Figure 4.** Histochemical localization of *DR5::GUS* and indole-3-acetic acid (IAA) contents of tobacco under different Se and Cd concentration supply conditions. (**A**) Histochemical localization of *DR5::GUS* in root tips of tobacco under different Se (IV) and Cd (II) concentration supply conditions; (**B**,**C**) IAA content of the shoots and roots under different Se (IV) and Cd (II) concentration supply conditions. 14-days-old seedlings (*DR5::GUS* transgenic tobacco) were grown in pot with sand under different Se (0, 10, 50 μM) and Cd (0, 20, 50 μM) concentrations for 21 days. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0:** no Cd; **Cd20:** Cd, 20 μM; **Cd50**: Cd, 50 μM. Shown are mean ± SD from five biological replicates. Different letters indicate significant differences (*p* < 0.05).

Local auxin levels are determined by biosynthesis and intercellular transport in plant roots [25]. To detect whether Se and Cd treatments affect the auxin-signal pathway, we analyzed the expression of *YUCCAs* and *PINs* family genes, which are involved in auxin biosynthesis and transport in tobacco under different Se and Cd treatments. Under Cd stress conditions, the expression of *NtYUCCA 6*, *8*, and *9*, and *NtPIN 1a*, *1c*, and *4* was substantially higher under low Se treatments, which was consistent with the auxin content results (Figure 5A–F). All these results suggest that auxin may play a key role in growth under Se and Cd treatment conditions.

**Figure 5.** Expression of auxin-relative gene in tobacco under different Se and Cd concentration supply conditions. (**A**–C) Expression of three members (*YUCCA6, 8, 9*) of the tobacco *YUCCAs* family genes in shoots under different Se and Cd concentration supply conditions.; (**D**–**F**) Expression of three members (*PIN1a, 1c, 4*) of the tobacco *PINs* family genes in roots under different Se and Cd concentration supply conditions. 14-days-old seedlings (*DR5::GUS* transgenic tobacco) were grown in pot with sand under different Se (0, 10, 50 μM) and Cd (0, 20, 50 μM) concentrations for 21 days. The tobacco housekeeping gene *L25* was used as an internal control. The relative expression levels are shown compared with the expression under Cd0 and Se0 (Cd0 + Se0) conditions as 1 expression. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0**: no Cd; **Cd20**: Cd, 20 μM; **Cd50**: Cd, 50 μM. Shown are mean ± SD from five biological replicates. Different letters indicate significant differences (*p* < 0.05).

### *2.5. Overexpression of NtPT2 Could Enhance the Tolerance of Cd Stress under Low Se Conditions*

Phosphate transporters are not only involved in Pi uptake, but also in selenite uptake [14,26]. Recently, we reported that the expression of a high-affinity phosphate transporter (*NtPT2*) involved in Se uptake in tobacco is induced in the roots and shoots under low Pi conditions [16]. In this study, we used *NtPT2* overexpression (*NtPT2-Oe*, two independent transgenic lines: Oe1 and Oe2) in transgenic tobacco to clarify the molecular mechanism by which Se can alleviate Cd toxicity. We found that *NtPT2* overexpression in plants enhanced their tolerance to Cd stress, with plants exhibiting better roots and shoots than the WT under Cd stress conditions. This was consistent with the auxin content differences between the *NtPT2-Oe* plant and the WT (Figure 6A,B and Figure S2). We also checked the P, Se, and Cd content in both *NtPT2-Oe* and WT plants; the total P and Se content of *NtPT2-Oe* plants was higher

than that of the WT plants under Cd stress conditions (Figure 6C,D), suggesting that *NtPT2* is involved in P and Se uptake in tobacco. We also found that Cd content was significantly reduced in *NtPT2-Oe* plants under Cd stress conditions (Figure 6E), confirming that overexpression of *NtPT2* could enhance the tolerance of tobacco plants to Cd stress.

**Figure 6.** Effects of Cd on *NtPT2-Oe* transgenic tobacco under different Se and Cd concentration supply conditions. (**A**,**B**) The phenotype and IAA content of *NtPT2-Oe* transgenic tobacco seedlings in Se (10 μM) and different Cd (0, 20, 50 μM) concentrations supply conditions; (**C**–**E**) Total P, Se and Cd content of the whole transgenic plant. Tobacco seeds were grown in 1/2MS culture under Se (10 μM) and different Cd (0, 20, 50 μM) concentrations for 14 days. **Se0**: no Se; **Se10**: Se, 10 μM; **Se50**: Se, 50 μM; **Cd0**: no Cd; **Cd20**: Cd, 20 μM; **Cd50**: Cd, 50 μM. Shown are mean ± SD from five biological replicates. Different letters indicate significant differences (*p* < 0.05).

### **3. Discussion**

### *3.1. Se A*ff*ects the Growth of Tobacco Roots by Changing Auxin Concentration under Cd Stress*

Cd has high levels of biological toxicity and can inhibit the growth and development of plants [27]. Biomass is an important indicator of plant growth and development. In this study, we showed that under Cd stress conditions, the biomass of tobacco, especially in the roots, was significantly lower than

plants not exposed to Cd stresses. This indicates that the roots and shoots were damaged by Cd stress, which is consistent with the results of Li et al. [28]. We also found that low Se levels stimulated growth in tobacco and could effectively alleviate the toxic effects of Cd stress (Figure 1), which is consistent with the results of previous studies [21,29]. Previous studies have shown that low Se enhances the antioxidation of enzymatic and non-enzymatic systems, changes root growth, and promotes absorption of nutrients [30,31], thus, improving the ability of plants to resist abiotic stresses. Low Se increased anti-oxidation activity (Figure 2), reduced the content of Cd (Figure 3) and changed root development by increasing auxin concentration in the roots (Figure 4), which may have increased growth. Further analyses on auxin-related genes showed that the expression levels of *YUCCAs* and *NtPINs* family genes were markedly higher under low Se conditions (Figure 5), indicating that low Se affects the growth of roots by changing auxin concentration under Cd stress.

Se often exerts a dual effect on plant growth. High levels of Se cause toxicity in plants, such as accumulation of ROS and inhibition of plant development [12,30,31]. A recent study showed that high Se levels inhibit root growth [16]. In this study, we also found high Se significantly decreased the biomass and auxin content of roots. Notably, under high Se conditions, the Cd treatments significantly decreased the biomass and auxin content in the shoots and roots. To investigate whether auxin is a major regulator of plant growth and development under Cd stress conditions, we also checked the characterization of root in *DR5::GUS* transgenic tobacco under different Se and Cd concentration supply conditions by adding IAA (100 nM). The results showed that exogenous IAA could increase the root biomass and the length of primary root, which implies that IAA plays a key roles in regulating the root architecture under Cd stress conditions (Figure S3).

Altogether, our data suggest that, under Cd stress conditions, Se might affect the growth and development of plants by changing auxin concentration. Not only the auxin pathway, but also other endogenous hormones (e.g., cytokinin, ethylene and gibberellin) may have an effect on plant root development. Recent studies showed that Se increases primary root length through alteration of the auxin and ethylene balance in rice, growth inhibition in Se-treated *Arabidopsis* is associated with an incomplete mobilization of starch, and high concentrations of selenite-induced enhancement of ethylene biosynthesis may result in plant cell death [32–34]. Therefore, the function of cytokinin, ethylene, gibberellin and other hormones in the development of tobacco roots under the treatment of Se and Cd need further study.

### *3.2.* NtPT2 *Might be a Potential Candidate Gene for Breeding Cd-Tolerant Plants*

A large number of genes encoding Pi transporters have been identified in different plant species. Pi transporters are generally classified into the *Pht1*, *Pht2*, *Pht3*, and PT gene families [35,36]. Most of the high-affinity Pi transporter (*Pht1*) family genes had induced expression under low Pi stress conditions, suggesting that *Pht1* plays a crucial role in both Pi uptake and translocation under Pi deficiency [37,38]. Recent studies have suggested that Se and Pi share similar uptake mechanisms and that Pi transporters are involved in Se uptake in plants, which might occur via a Se-H<sup>+</sup> symport process in the plant cell membrane [14,16,26].

Although a large number of *Pht1* family genes have been studied in rice and *Arabidopsis*, the function of key Pi transporters in plants need further research. For instance, recently, two key phosphate transporter genes *OsPT2* and *OsPT8* were found to play a key role in As and Se uptake and translation in rice, implying that some phosphate transporters are involved in the uptake of heavy metals or trace elements [39]. At present, studies on the *Pht1* family gene in tobacco are few. In our previous work, we showed that *NtPT2* is the most closely related to *OsPT2,* and that *NtPT2* has similar expression patterns to *OsPT2* (low Pi-induced expression in roots) [16]. In this study, we used *NtPT2* overexpressing transgenic tobacco to further clarify whether Se could alleviate the toxicity of Cd. Our results showed that when 10 μM Se was supplied, the *NtPT2* overexpression significantly increased biomass, total P, axuin, and Se content. In contrast, the Cd content in transgenic tobacco obviously reduced under Cd

treatments compared with WT (Figure 6), implying that a suitable level of Se modulates the level of auxin, enhancing the tolerance of tobacco to Cd stress.

With the improvement of health awareness, how to reduce the Cd content in crop production and food chain has become a research hotspot in recent years. It has a potential impact on reducing the accumulation of Cd in tissues such as liver, kidney, lungs and bones, and avoiding the induction of various diseases such as lung cancer, hypertension and cardiomyopathy [40,41]. Our results suggest that *NtPT2* might be a potential candidate gene for breeding Cd-tolerant plants, which also need further research.

Based on this study, we propose a possible model for revealing the mechanism controlling the Se, Cd stress, P, and auxin response in tobacco (Figure 7). Under Cd stress conditions, (1) plant growth is inhibited. (2) Under low Se and Cd stress conditions, the Pi transporter is involved in the uptake of Se (IV) and P in the root, and low Se (accumulation of a small amount Se in plant) changes the auxin-related gene expression, which increases auxin content to promote plant growth. (3) Low Se not only increases the biomass of the root but also enhances the antioxidant capacity. We conclude that low Se alleviates Cd toxicity in tobacco. However, the function of other hormones in the development of tobacco roots under the treatment of Se and Cd needs further study.

**Figure 7.** The model for the interaction mechanism between Se, Cd stress, *p* and auxin response in tobacco. The model is based on the results presented here. + indicates positive regulations and – indicates negative regulations. ? indicates that requires further research.

### **4. Materials and Methods**

### *4.1. Plant Material and Experimental Conditions*

The tested materials were the wild-type (*Nicotiana tabacum* cv,Yunyan 87), *DR5::GUS* and *NtPT2-Oe* transgenic seeds (T2 generation) (Figure S4). The generation of transgenic tobacco material and the construction of *pDR5::GUS* had been detailed in the previous studies [16,26,42].

Tobacco seeds were sterilized in solution of 75% (*v*/*v*) ethanol for 30 s and 10% (*v*/*v*) sodium hypochlorite for 7 min, then followed by washing 6 times with sterile distilled water. The seeds were then transferred to seedling tray (3 days) kept in the culture room at 28 ◦C in dark for proper germination. Germinated seedlings were placed in greenhouse for 10 days. The culture during the first five days used one-fourth-strength Hoagland's nutrient solution, and half-strength nutrient solution was used in the second five days. The tobacco seedlings were transferred in pot with sand and half-strength nutrient solution was used for 4 days in seedling recovering stage. Then they were exposed to Se (Na2SeO3) and Cd (CdCl2·2.5H2O) for 21 days. The experiment of three Se levels, i.e., 0, 10 and 50 μM, and three Cd levels, i.e., 0, 20 and 50 μM were designed. There was a total of nine treatments: Cd0+Se0, Cd0+Se10, Cd0+Se50, Cd20+Se0, Cd20+Se10, Cd20+Se50, Cd50+Se0, Cd50+Se10, and Cd0+Se50. Se and Cd were added into the nutrient solution in the forms of Na2SeO3 and CdCl2·2.5H2O, respectively.

The tobacco seedings were harvested after 21 days of treatment. Shoots and roots were washed with deionized water for further analysis. (1) We observed and recorded the phenotype of tobacco plants. (2) Some seedings were used to measure the IAA, chlorophyll and MDA content. (3) Some of the leaves and roots were used for NBT and *DR5::GUS* staining. (4) The other tobacco seedings were oven-dried and used to measure the contents of Se and Cd. (5) Some seedings were used for detecting the gene expression and enzyme activity.

For IAA (Indole-3-acetic acid, dissolved in 1 M NaOH) treatments, 100 nM IAA was added to the nutrient solution under different Se and Cd treatments, respectively. The nine treatment plants, namely Cd0+Se0+IAA, Cd0+Se10+IAA, Cd0+Se50+IAA, Cd20+Se0+IAA, Cd20+Se10+IAA, Cd20+Se50+IAA, Cd50+Se0+IAA, Cd50+Se10+IAA and Cd50+Se50+IAA. The tobacco seedings were grown in a growth chamber for 7 days. The plants were then harvested for next stage of analysis. (1) To observe the phenphtype of tobacco plants. (2) To record the histochemical localization of GUS.

### *4.2. GUS Staining and Nitroblue Tetrazolium (NBT) Staining of Plant Tissues*

Plants were stained for GUS activity for 24 h at 37 ◦C, and then seedlings were immersed in 95% ethanol to eliminate chlorophyll pigmentation. Plants were stained in NBT solution for 5 h at 30 ◦C, and 95% ethanol was used until decolorization was complete. The stereo microscope (Olympus SZX16, Olympus, Tokyo, Japan) equipped with a colorcharge coupled device (CCD) camera were used to photograph stained plant tissues.

### *4.3. Chlorophyll and MDA Measurement*

The relative amount of chlorophyll in plants was determined by a SPAD-502 chlorophyll meter (Konica, Tokyo, Japan) [43]. Five sites of one leaf were measured, and the results were averaged. The content of MDA was determined by the thio-barbituric acid method (TBA) which was calculated by using the difference in absorbance of the extract at 532 nm and 600 nm [44,45].

### *4.4. IAA Measurement*

The IAA contents of shoots and roots in tobacco seedlings were measured as described by Sun et al. (2014) and Jia et al. (2018) [16,46]. The samples were grinded with appropriate amount of the antioxidant butyleret hydroxytoluen (BHT) and 80% pre-cooled methanol for 12–16 h. We collected and concentrated the extracted fluid by a rotary evaporator to 10 mL at 40 mL, and then the fluid was extracted with petroleum ether of the same volume. Under a layer liquid it was adjusted to pH 8.5 and added 0.2 g polyvinylpyrrolidone (PVP) then vibrated for 30 min, and then filtered through a 0.45 μm filter over an OASIS HLB (St. Louis, Mo, USA), and chromatographic conditions were described by: Waters 600–2487; Hibar column RT 250 × 4.6 mm; Purospher STARRP-18 (5 μm); column temperature 45 ◦C; fluid phase: methanol:1% acetic acid (*v*/*v*, 40/60), isocratic elution; fluid rate: 0.6 mL min−1; ultraviolet (UV) detector, *l* = 269 nm; injection volume 20 μL. A 0.22 μm filter was used for filtration of both the buffer and the samples before high-performance liquid chromatography (HPLC) analysis.

### *4.5. Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis*

Trizol reagent was used to prepared Total RNAs from the roots and shoots of tobacco seeding. DNase I-treated total RNAs were used for RT by Superscript II. Triplicate quantitative assays were performed with SYBR Premix Ex Taq™ II (Perfect Real Time) kit (TaKaRa Biotechnology, Dalian, China) on the Step One Plus RealTime PCR Systems (Applied Biosystems, Bio-Rad, Berkeley, CA USA). The gene-specific primers for *YUCCAs* and *PINs* family genes of tobacco were used to perform reverse

transcription polymerase chain reaction (qRT-PCR) analysis. The primers were shown in Supplemental Table S1. The analysis of relative expression levels used *NtL25* (L18908.1) as internal reference gene and presented as 2−ΔΔ*C*<sup>t</sup> .

### *4.6. Determination of Total P in Plant*

Dry samples of about 0.05 g were digested with 5 mL of 98% H2SO4 and 3 mL of 30% hydrogen peroxide. Then, total P content was analyzed by the molybdate blue method [47].

### *4.7. Measurement of Se and Cd Contents*

The comminuted tobacco samples were digested with concentrated HNO3 and HClO4 (*v*/*v*, 4:1) [48]. Se and Cd contents were determined by inductively coupled plasma mass spectrometry (ICP-MS 7500A, Agilent, Palo Alto, CA, USA). The accuracy of elemental analysis was verified using standard reference materials from the China Standard Reference Center.

### *4.8. Statistical Analysis*

Two-way analysis of variance (ANOVA) and Tukey's multi-comparisons test (*p* ≤ 0.05) were applied to all data. The results were expressed as the means and the corresponding standard errors. All statistical analyses were completed using the Origin2018 (Origin Lab, Northampton, MA, USA) software.

### **5. Conclusions**

This study showed that proper Se supply effectively alleviates the toxicity of Cd in tobacco. Selenium affected the growth of tobacco in the Se–Cd interaction by regulating the expression of the auxin-related genes and enhancing the tolerance to Cd stress by increasing the content of auxin in tobacco. Overexpression of a high-affinity phosphate transporter *NtPT2* increased the content of P and Se and decreased the accumulation of Cd. This study reveals the interaction mechanism of P and auxin in plant growth under the action of Se and Cd and provides new ideas for the safe cultivation of crops in Cd-contaminated soil.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/15/ 3772/s1.

**Author Contributions:** H.J. and Y.F. conceived the research project. Y.L. performed most experiments and wrote the manuscript. Y.W. and W.W. conducted the transgenic tobacco. S.S. and J.W. checked the content of Se and Cd; D.H. and H.S. revised the manuscript. All authors saw and commented on the manuscript.

**Funding:** This work was supported by the grants from National Natural Science Foundation of China (grant no.31301837) and the Foundation of Henan Educational Committee (grant no.15A210029).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Ectopic Expression of Poplar ABC Transporter PtoABCG36 Confers Cd Tolerance in** *Arabidopsis thaliana*

**Huihong Wang** †**, Yuanyuan Liu** †**, Zaihui Peng, Jianchun Li, Weipeng Huang, Yan Liu, Xuening Wang, Shengli Xie, Liping Sun, Erqin Han, Nengbiao Wu, Keming Luo and Bangjun Wang \***

Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), College of Life Sciences, Southwest University, Chongqing 400715, China

**\*** Correspondence: bangjunwang@swu.edu.cn; Tel.: +86-23-6825-3235

† These authors contributed equally to this work.

Received: 17 May 2019; Accepted: 1 July 2019; Published: 4 July 2019

**Abstract:** Cadmium (Cd) is one of the most toxic heavy metals for plant growth in soil. ATP-binding cassette (ABC) transporters play important roles in biotic and abiotic stresses. However, few ABC transporters have been characterized in poplar. In this study, we isolated an ABC transporter gene *PtoABCG36* from *Populus tomentosa*. The *PtoABCG36* transcript can be detected in leaves, stems and roots, and the expression in the root was 3.8 and 2 times that in stems and leaves, respectively. The *PtoABCG36* expression was induced and peaked at 12 h after exposure to Cd stress. Transient expression of *PtoABCG36* in tobacco showed that PtoABCG36 is localized at the plasma membrane. When overexpressed in yeast and Arabidopsis, PtoABCG36 could decrease Cd accumulation and confer higher Cd tolerance in transgenic lines than in wild-type (WT) lines. Net Cd2<sup>+</sup> efflux measurements showed a decreasing Cd uptake in transgenic Arabidopsis roots than WT. These results demonstrated that PtoABCG36 functions as a cadmium extrusion pump participating in enhancing tolerance to Cd through decreasing Cd content in plants, which provides a promising way for making heavy metal tolerant poplar by manipulating ABC transporters in cadmium polluted areas.

**Keywords:** Cd; *PtoABCG36*; tolerance; poplar; accumulation; efflux

### **1. Introduction**

Cadmium (Cd) is a highly toxic pollutant in the environment. Cadmium is nephrotoxic, and it can lead to serious human diseases, including kidney disorders, bone damage and neurotoxicity [1]. For example, high environmental exposure in Japan resulting from a stable diet of cadmium contaminated rice caused itai-itai disease [2]. Cadmium can inactivate or denature proteins by binding to the sulfhydryl groups, leading to cellular damage by displacing co-factors from a variety of proteins including transcription factors and enzymes, and by indirectly generating reactive oxygen species [3,4]. Heavy metal pollution in agricultural soils has become a serious problem. Therefore, it is essential to prevent cadmium from getting into the food chain and make the best use of cadmium contaminated soil.

Plants are able to tolerate heavy metal stress to a certain extent, with the participation of some transporters. These transporters can enhance heavy metal tolerance by pumping heavy metals into vacuoles or out of cells. Previous studies showed that two type 1(B) heavy metal-transporting subfamily of the P-type ATPases AtHMA2 and AtHMA4 are localized at the plasma membrane and can transport excessive zinc and cadmium to the outside of the cell in *Arabidopsis thaliana*, which are important players in the plant detoxification process [5]. The members of the cation diffusion facilitator (CDF) family, natural resistance-associated macrophage protein (Nramp) and Zrt/IRT-like protein (ZIP) families of transporters are also involved in the transport of heavy metals in a variety of organisms [6,7]. In addition, ATP-binding cassette (ABC) transporters are essential for plant growth and development. ABC transporters are driven by ATP hydrolysis and can act as exporters as well as importers. The Arabidopsis nuclear genome encodes for more than 100 ABC transporters, which are divided into eight subfamilies (ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG and ABCI), largely exceeding that of animal. Most plant ABC transporters are present in cell membranes and are involved in detoxification processes, organ growth, plant nutrition, plant development and response to abiotic and biotic stresses [8]. Some ABC transporters are closely related to the detoxification of heavy metals. In *Saccharomyces cerevisiae*, an ABCC-like heavy metal transporter ScYCF1 (yeast cadmium factor 1) has been found to contribute to detoxifying cadmium by pumping it into vacuoles [9], and overexpression of *ScYCF1* in Arabidopsis can improve cadmium tolerance [10]. Similarly, a half-size ABC transporter HMT1 (heavy metal tolerance 1) from *Schizosaccharomyces pombe* can transport phytochelatin–Cd complexes into the vacuole, which is considered to be the first transporter to transport heavy metal-phytochelatin complexes [11]. AtABCB25/AtATM3, a close homolog of SpHMT1, contributes to Cd resistance and can transport glutamine synthetase conjugated Cd (II) across the mitochondrial membrane [12]. Full-size ABC transporters AtABCC1 and AtABCC2 have been demonstrated to be major vacuolar phytochelatins (PCs) transporters to participate in arsenic (As), mercury (Hg) and Cd resistance in Arabidopsis [13], and their homologous rice ABCC transporter OsABCC1 is involved in the As detoxification and reduces As accumulation in the rice grains [14]. Another homologous ABCC transporter PtABCC1 can enhance tolerance to Cd in poplar [15]. It has reported that AtABCC3 can complement the Cd sensitive phenotype of the *ycf1* mutant in *Saccharomyces cerevisiae* [16]. The level of expression of *AtABCC6*/*AtMRP6* can be up-regulated in response to cadmium (Cd) treatment [17]. Furthermore, some ABCG subfamily transporters are also involved in heavy metal resistance. AtABCG36/PDR8, localized at the plasma membrane, plays an important role in Cd extrusion from root cells [18]. The transcription of cucumber genes *CsPDR8*/*CsABCG36* can be up-regulated under Cd stress [19]. The rice OsABCG43/PDR5 is a Cd inducible-transporter and confers high Cd resistance in yeast cells [20].

Poplar is a woody plant with established genetic transformation system and abundant biomass. In China, there is a very large number of *Populus tomentosa*, taking up very large land resources. The previous research on poplar mainly focuses on insect resistance, herbicide resistance, biomass traits, stress tolerance, disease resistance, hormone modification, flowering modification and phytoremediation [21]. Recently, a study found that exogenous abscisic acid (ABA) stimulated the expression level of poplar ABCG40 transporter involved in lead (Pb) uptake, transport and detoxification [22]. The two multidrug and toxic compound extrusion (MATE) family genes *PtrMATE1* and *PtrMATE2* from poplar induced by aluminum (Al) can enhance aluminum resistance in acidic soils [23]. The *YCF1*-expressing transgenic poplar plants exhibited enhanced growth, reduced toxicity symptoms, and increased Cd content in the aerial tissue compared to the non-transgenic plants [24]. However, there are few studies on poplar ABCG transporters involved in Cd resistance. Therefore, the engineering of *Populus tomentosa* by manipulating ABC transporters is a significant step towards the effective utilization of Cd contaminated soil.

In the present study, we cloned a novel ABC transporter gene *PtoABCG36* (GenBank accession: MH660448) by BLAST search in the poplar database using *AtABCG36* as a query sequence. Yeast and Arabidopsis overexpressing *PtoABCG36* were measured in terms of their Cd tolerance and Cd content after Cd treatment. The results showed that overexpressing *PtoABCG36* is effective in enhancing Cd tolerance through decreasing Cd content in plants, indicating that PtoABCG36 transporter functions as a cadmium extrusion pump to participate in Cd stress in plants, which provides a reasonable way to make heavy metal tolerant poplar by manipulating ABC transporters in the areas with cadmium pollution.

### **2. Results**

### *2.1. Structural and Phylogenetic Analysis of PtoABCG36*

*PtoABCG36* was isolated from full-length cDNA of leaves of six-month-old *Populus tomentosa* and submitted to GenBank (accession number: MH660448). The sequence encoded 1478 amino acid residues and contained two putative transmembrane domains (TMD) and two putative nucleotide-binding domains (NBD) (Figure 1A). Each NBD domain has about 200 amino acid residues, and it contains a Walker A motif (GXXGXGKS/T), a Walker B motif (hhhhD) and an ABC signature motif (LSGGQQ/R/KQR) [25]. Some ABCG subfamily transporters have been identified in many plant species, including *Arabidopsis thaliana*, *Glycine Max*, *Ricinus conmunis*, *Vitis vinifera*, *Gossypium arboretum* and *Oryza sativa*. The two NBD domains are highly conserved (Figure 1A).

**Figure 1.** Amino acid sequence alignment and phylogenetic analysis. (**A**) Structure analysis and amino acid multi-alignment of the nucleotide-binding domains (NBD) of ABCG proteins from different plant species. ABCG domains are marked as two green and orange blocks. TMD, transmembrane domain; Walker A, ATP-binding cassette (ABC) signature; Walker B, NBD associated motifs. Blue indicates identical amino acids; pink indicates similar amino acids. (**B**) Phylogenetic analysis of ABCG proteins from *Populus trichocarpa* (PtoABCG36, MH660448); *Arabidopsis thaliana* (AtABCG36, NP\_176196); *Oryza sativa* (OsABCG36, XP\_015648358; OsABCG37, XP\_015648329; OsABCG43, XP\_015646575; OsABCG44, XP\_015650488); *Glycine Max* (GmABCG36, XP\_006585572); *Ricinus conmunis* (RcABCG36, XP\_002515970); and *Vitis vinifera* (VvABCG29, XP\_010654625); *Gossypium arboretum* (GaABCG36, XP\_017606959). The numbers beside the branches represent bootstrap values based on 1000 replications.

To investigate the homology between PtoABCG36 and other plant species, ten plant ABCG transporters were analyzed. PtoABCG36 had 81.9%, 81.4%, 78.4%, 78.2% and 74.1% amino acid sequence similarity to GaABCG36 (XP\_017606959), VvABCG29 (XP\_010654625), GmABCG36 (XP\_006585572), RcABCG36 (XP\_002515970) and AtABCG36 (NP\_176196), respectively. Phylogenetic analysis also revealed that PtoABCG36 was homologous with the ABCG proteins from dicotyledons such as *Vitis vinifera*, *Gossypium arboretum*, *Ricinus conmunis*, *Glycine Max* and *Arabidopsis thaliana*, as well as monocotyledons such as *Oryza sativa* (Figure 1B).

### *2.2. The PtoABCG36 Gene Is Highly Expressed in Response to Cd Stress in Poplar*

To confirm the function of the PtoABCG36 transporter, we measured its gene expression level. *PtoABCG36* transcript can be detected in leaves, stems and roots, and the expression in the root was 3.8 and 2 times that of the stems and leaves, respectively. The higher expression level in the roots indicated that PtoABCG36 mainly functioned in the roots (Figure 2A). In addition, to confirm the function of PtoABCG36 in response to Cd stress, we performed induced expression using quantitative real-time PCR after the six-month-old poplars were immersed in woody plant medium (WPM) supplemented with different concentrations of CdCl2 for 12 h. Poplar gene-specific primers were used for qRT-PCR analysis of *PtoABCG36*. The results showed that the expression of *PtoABCG36* was significantly increased in roots with increasing cadmium concentration and reached the highest level when treated with 100 μM CdCl2 for 12 h. *PtoABCG36* expression was also significantly increased in stems and leaves but not as highly as that in roots. However, when treated with 150 or 200 μM CdCl2, the expression of the *PtoABCG36* gradually declined, but it could still be induced in roots, stems and leaves (Figure 2B). Furthermore, temporal spatial expression analysis upon treatment with 100 μM CdCl2 for 24 h showed that *PtoABCG36* transcript increased overtime and peaked at 12 h, with a level seven times that of the control, then gradually decreased (Figure 2C). These results further determined that *PtoABCG36* could be induced and participate in resisting Cd stress.

**Figure 2.** Expression analysis of *PtoABCG36* gene. (**A**) Relative expression level of *PtoABCG36* gene in roots, stems, leaves of *Populus tomentosa*. (**B**) Expression of *PtoABCG36* in poplar roots, stems and leaves under different concentrations of Cd2<sup>+</sup> for12 h. (**C**) Time course of *PtoABCG36* expression in poplar roots, stems and leaves in response to 100 μM Cd2<sup>+</sup> treatment. The results are shown as the mean expression ± standard deviation (SD) of three independent experiments. Poplar ubiquitin (*UBQ*) expression was used as a control and gene-specific primers were used for qRT-PCR analysis of *PtoABCG36* gene. Student's t-test, \* *p* < 0.05, \*\* *p* < 0.01.

### *2.3. The PtoABCG36 Transporter is Localized at the Plasma Membrane*

In order to determine the subcellular localization of PtoABCG36, the 35S:*PtoABCG36*-GFP construct, in which the *PtoABCG36*-GFP fusion gene was driven by the CaMV 35S promoter, was transiently expressed in the leaves of three-week-old *Nicotiana benthamiana*. Compared with the control where GFP was observed at the plasma membrane (PM), endoplasmic reticulum (ER) and nucleus (NU) in the epidermal cells (Figure 3A–D), the PtoABCG36 signal was observed only at the plasma membrane (Figure 3E–H), indicating that PtoABCG36 is localized at the plasma membrane to function as transporter, consistent with the localization pattern of AtABCG36 in *Arabidopsis thaliana*.

**Figure 3.** Subcellular localization of PtoABCG36 in epidermal cells of *Nicotiana benthamiana*. The fluorescence of green fluorescent protein (GFP) or PtoABCG36-GFP signal in tobacco leaf cell (**A**,**E**). Chlorophyll autofluorescence (**B**,**F**). Bright field (**C**,**G**). The overlap images of bright field and fluorescence images (**D**,**H**). Scale bars = 20 μm. NU, nucleus; PM, plasma membrane; ER, endoplasmic reticulum.

### *2.4. Heterologous Expression of PtoABCG36 Confers Cd Tolerance in Yeast*

To investigate whether PtoABCG36 is involved in Cd tolerance, pDR-*PtoABCG36* was produced and transformed into the yeast Cd sensitive mutant strain Δ*yap1* and wild-type strain Y252. We found that on the SD-Ura medium, growth was similar between the yeast cells carrying the empty vector and those expressing *PtoABCG36*. However, on the SD-Ura medium containing 100 μM or 200 μM CdCl2, the Δ*yap1* or Y252 with pDR-*PtoABCG36* exhibited stronger Cd tolerance than mutants or wild-type with the empty vector (Figure 4A). Yeast growth in liquid SD-Ura medium containing 40 μM CdCl2 was analyzed overtime. In the absence of Cd, there was no growth difference between the *PtoABCG36*-carrying yeast and the control (Figure 4B). However, upon CdCl2 exposure, the growth of *PtoABCG36*-carrying Δ*yap1* and Y252 were better than the yeast cells carrying the empty vector. Additionally, complementary strains partially restored their tolerance to Cd (Figure 4C), further confirming heterologous expression of *PtoABCG36* could confer Cd tolerance in yeast.

**Figure 4.** PtoABCG36 enhances cadmium tolerance in yeasts. (**A**) Δ*yap1* and the wild-type Y252 were transformed with EV (pDR196 empty vector) and pDR196-*PtoABCG36*, and grown on SD plates with indicated concentrations of CdCl2 for 7 d. (**B**,**C**) Growth curves of yeast cells Δ*yap1*-EV (square), Δ*yap1- PtoABCG36* (circle), Y252- EV (up-triangle) and Y252- *PtoABCG36* (down-triangle) under control (**B**) and 40 μM CdCl2 condition (**C**) for indicated time. (**D**) Accumulation of cadmium in Δ*yap1*-EV (navy blue), Δ*yap1-PtoABCG36* (purple), Y252-EV (dark cyan) and Y252-*PtoABCG36* (orange) yeasts. Yeast cells (1 <sup>×</sup> 107) were exposed to 40 <sup>μ</sup>M Cd treatment for 6, 12, 18 or 24 h at 30 ◦C. Cd concentrations in the yeast cells were measured by ICP-OES. Error bars indicate standard deviation (*n* = 3). Different letters indicated significant differences (*p* < 0.05).

Previous studies have shown that yeast could resist cadmium by transporting it into the vacuoles or out of the cells. We tested Cd concentration in the yeast cells culturing in liquid SD-Ura medium containing 40 μM CdCl2. As shown in Figure 4D, after 24 h of treatment, the accumulation of Cd in *PtoABCG36*-carrying Δ*yap1* and Y252 was significantly less (52.5% and 20.3% less, respectively) than that in mutant and wild-type. These results indicated that PtoABCG36 can contribute to Cd resistance by transporting it out of the yeast cells.

### *2.5. Overexpression of PtoABCG36 Increases Tolerance to Cd and Decreases Cd Accumulation in Plants*

In order to investigate the function of PtoABCG36 in plants, the construct 35S:*PtoABCG36* was introduced into Arabidopsis. The *PtoABCG36* transcript levels were detected by qRT-PCR for further analysis (Supplementary Figure S2). Arabidopsis transgenic plants T4, wild-type and mutant seeds were analyzed after treatment without Cd and with 20 μM, 40 μM or 60 μM CdCl2 for 2 weeks. There was no growth difference among these lines in the absence of Cd, while the growth of Arabidopsis was significantly inhibited when grown on half MS agar plates containing 20 μM, 40 μM and 60 μM CdCl2. The *abcg36* mutants displayed shortest roots. However, the transgenic plants had longer roots and grew better than wild-type plants (Figure 5A,B), indicating that PtoABCG36 was also involved in mediating tolerance to Cd in plants. Quantitative analysis showed that the roots of overexpression lines (OX-2 and OX-3) were significantly longer than those of wild-type plants in the presence of 40 μM CdCl2 (44% and 48% longer, respectively) and 60 μM CdCl2 (116.7% and 112.5% longer, respectively). These results further indicated that PtoABCG36 enhanced tolerance to Cd in plants.

**Figure 5.** PtoABCG36 enhances cadmium tolerance in Arabidopsis. Arabidopsis seeds were grown on half-strength MS medium containing 0, 40 or 60 μM CdCl2 for two weeks (**A**) and primary root length (**B**) were analyzed. (**C**) Accumulation of cadmium in plants after treatment with the half MS liquid medium containing 100 μM CdCl2 for 24 h. For root lengths, *n* = 120–124 from three independent experiments. WT, wild-type; *abcg36*, *abcg36* mutant SALK\_1422526; OX-1 and OX-2, OX-3 *PtoABCG36*-overexpressing Arabidopsis lines. White bars = 15 mm. Cd concentrations in plants were measured by ICP-OES. Error bars indicate standard deviation. Different letters indicated significant differences (*p* < 0.05). Student's t-test, \* *p* < 0.05, \*\* *p* < 0.01.

To explain the detoxification mechanism of PtoABCG36 in plants, we tested the cadmium content in the mutants, wild-type and transgenic plants after treatment with the half MS liquid medium containing 100 μM CdCl2 for 24 h. We found that transgenic plants OX-1, OX-2 and XO-3 had lower Cd content than wild-type in the shoots (46.68%, 57% and 42.91% lower, respectively) and roots (37.42%, 22.3% and 27.37% lower, respectively). In contrast, the mutant *abcg36* had higher Cd content than the wild-type in the roots (61.65% higher) and shoots (59.24% higher). More importantly, the levels of cadmium reduction in the roots of transgenic plants were much greater than those in the shoots (Figure 5C), suggesting that PtoABCG36 contributed to Cd tolerance by pumping it out of the plants and reducing Cd toxicity in plant roots.

To further determine the function of PtoABCG36 in plant roots, we investigated the Cd2<sup>+</sup> uptake in root tips of *abcg36* mutants, WT and plants overexpressing *PtoABCG36* through a non-invasive micro-test (NMT) technique. In the presence of 50 μM CdCl2, the net Cd<sup>2</sup><sup>+</sup> influxes of OX-1, OX-2 and OX-3 lines were lower than WT plants (62.39%, 54.50% and 53.30% lower, respectively) (Figure 6). In contrast, the mutant *abcg36* had higher Cd net Cd2<sup>+</sup> influx than the WT plants. These results

indicated that a decreasing Cd uptake capacity existed in lines overexpressing *PtoABCG36* than the WT plants.

**Figure 6.** Net Cd2<sup>+</sup> fluxes. Net Cd2<sup>+</sup> fluxes in the roots of WT, *abcg36* mutant and transgenic plants (OX-1, OX-2, and OX-3) treated with CdCl2 stress (**A**). The average 180 s net Cd2<sup>+</sup> fluxes are illustrated to highlight the trend differences (**B**). Bars indicate means ± SD. Student's t-test, \* *p* <0.05, \*\* *p* < 0.01.

### **3. Discussion**

To date, how to effectively use soil containing cadmium has become a worldwide problem. Previous studies have showed that several transporters, including the P-type ATPases AtHMA2 and AtHMA4, the CDF, Nramp and ZIP families of transporters and ABC transporters could be involved in the heavy metal tolerance [5–7,9–11].

In this study, we identified the ABC transporter ABCG36 of *Populus tomentosa.* Protein sequence analysis showed that it contained conserved Walker A, Walker B, and ABC signal (Figure 1A). In previous studies, Walker A, Walker B, ABC signal of NBD were demonstrated to function as ABC transporters motifs [26]. Phylogenetic tree analysis showed that PtoABCG36 in poplar is an ortholog of Arabidopsis AtABCG36, which acts as transporter involved in biotic or abiotic stress [18,27] (Figure 1B). Expression pattern showed that the accumulation of *PtoABCG36* transcript was mainly detected in the roots (Figure 2A). In line with our results (Figure 2B,C), it has been also reported that transcript levels of ABCG transporters were induced rapidly by biotic or abiotic stress [28–32]. Interestingly, *PtoABCG36* expression was induced by Cd, peaking at 12 h after Cd treatment. Additionally, the expression of *PtoABCG36* was significantly higher in poplar roots than that in shoots under Cd treatment, which is different from its ortholog in other species (Figure 2C).

It is important for plants to cope with heavy metal stress. In this study, first, we found that ectopic expression of *PtoABCG36* in yeast and Arabidopsis all significantly increased Cd tolerance (Figures 3 and 5). Interestingly, our data showed that the growth of *PtoABCG36*-carrying Δ*yap1* yeast stain, which has a lower level of Cd, was not better than that of the wild-type Y252 (Figure 4C,D). It is known that Yap1 increased cellular tolerance to cadmium by activating the expression of *ScYCF1* as a transcription factor. Yeast wild-type Y252 can resist Cd stress through ABC transporter ScYCF1 localized at vacuolar membrane and plasma membrane pumping Cd into vacuoles or out from the cells [10]. The expression of *YCF1* in Y252-*PtoABCG36* could pump Cd into vacuoles, while inhibition of *YCF1* in Δ*yap1*-*PtoABCG36* could decrease the transport of heavy metals to vacuoles. Therefore, Y252-*PtoABCG36* has higher accumulation of Cd compared to Δ*yap1*-*PtoABCG36* (Figure 4D). In addition, our data indicated that Arabidopsis *PtoABCG36*-overexpressing lines could enhance Cd tolerance (Figure 5). The *abcg36* plants are sensitive to Cd, whereas the *PtoABCG36*-overexpressing plants are tolerant (Figure 5). *PtoABCG36*-overexpressing plants have reduced cadmium content in their shoots and roots, but *abcg36* plants were the opposite. The wild-type plants accumulate 1.2 to 1.5 times as much Cd in roots and shoots as the transgenic plants (Figure 5C), suggesting that the overexpression of *PtoABCG36* could expel heavy metals from plants. Non-invasive micro-test (NMT)

technique showed that overexpressing *PtoABCG36* can decrease Cd uptake capacity in plants (Figure 6). The detoxification mechanism of PtoABCG36 might be similar to that of its homologous AtABCG36 located at the plasma membrane, which can transport Cd out from the cells.

Taken together, our study provided the evidence for the biological functions of PtoABCG36 as a transporter in regulating Cd resistance in plants. Additionally, it plays a crucial role in reducing Cd accumulation in plants, providing a theoretical basis to make heavy metal tolerant poplar by manipulating ABC transporters in cadmium polluted areas. The present study has also provided insight on the roles of ABCG transporters in economic forest cultivation.

### **4. Materials and Methods**

### *4.1. Materials and Growth Conditions*

Arabidopsis seeds of wild-type (ecotype Columbia-0), *abcg36* (a loss-of-function mutant of *AtABCG36*, SALK\_1422526) [18], and transgenic plants OX-1, OX-2, OX-3 were vernalized in the dark at 4 ◦C for 2 days, and then grew on half-strength MS agar medium plates containing 1.5% sucrose in a controlled environment with a 16 h light with 120 μmol m−<sup>2</sup> s−<sup>1</sup> light intensity and 8 h dark at 22 ◦C/18 ◦C for the indicated duration.

*P. tomentosa* Carr. (clone 741) (Chinese white poplar), kindly provided by Institute of Resources Botany, Southwest University, and transgenic poplars were cultivated in a greenhouse at 24 ◦C under a 14 h/10 h light/dark cycle with 45 μmol m−<sup>2</sup> s−<sup>1</sup> of light and maintained in sterile woody plant medium (WPM) containing 0.8% (w/v) agar. Gene expression patterns were analyzed in leaves, roots and stems from 6-month-old plants.

### *4.2. Gene Cloning, Expression Vector Construction, Structural and Phylogenetic Analysis of PtoABCG36*

Total RNA was extracted from the leaves of 6-month-old *P. tomentosa Carr*. by using the Trizol Reagent (Tiangen, China), then revers transcribed to cDNA by using the RT-AMV transcriptase Kit (TaKaRa, Dalian, China). The *PtoABCG36* specific fragment was amplified by PCR using specific primers (Supplementary Table S1). Cycling conditions were: 98 ◦C for 3 min followed by 34 cycles of 98 ◦C for 30 s, 56.6 ◦C for 30 s and 72 ◦C for 2 min 58 s, adding a final prolongation step at 72 ◦C for 10 min. The amplification products were cloned into the *Bam*HI site of the plant binary vector pCAMBIA-1300-GFP [33] as well as the *Spe*I and *Xma*I sites of the yeast vector pDR196 [34], to construct pCAMBIA-1300-*PtoABCG36* and pDR196-*PtoABCG36*.

Prediction and analysis of the structure of PtoABCG36 protein was performed with the Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de). The homologous amino acid sequences of PtoABCG36 in other species were downloaded from NCBI (http://www.ncbi. nlm.nih.gov), and aligned with DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA). The phylogenetic analysis of amino acid sequences was carried out with MEGA 5.0 software by using neighbor-joining (NJ).

### *4.3. Transformation and Selection for Yeast and Arabidopsis*

The yeast expression vectors pDR196 and pDR196-*PtoABCG36* were transformed into the Cd sensitive-yeast mutant Δ*yap1* (*MATa ura3 lys2 ade2 trp1 leu2 yap1::leu2*) and the wild-type Y252 (*MATa ura3 lys2 ade2 trp1 leu2*) [35], kindly provided by Ji-Ming Gong (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, shanghai, China) for metal sensitivity assay, as described [36]. Yap1 is a transcription factor that increases the tolerance of cells to cadmium by activating *YCF1* expression [37].

pCAMBIA-1300-*PtoABCG36* was transformed into the *Agrobacterium tumefaciens* strain GV3101, then transformed into wild-type Arabidopsis by the floral dip method [38]. The selection of putative transgenic plants was performed on half MS medium with 40 mg/L hygromycin and 200 mg/L

cefotaxime, and further confirmed by PCR analysis (Supplementary Figure S1) and qRT-PCR analysis (Supplementary Figure S2).

### *4.4. The Metal Assay of Yeast Cells and Plants*

For phenotypic analysis, yeast cells were cultured in SD-Ura liquid medium to log phase and diluted to the corresponding concentration after collection, then spotted onto SD-Ura plates containing 100 μM and 200 μM CdCl2. Plates were kept at 30 ◦C for 7 days before being photographed. Yeast cells were also cultured in liquid medium containing 40 μM CdCl2 for 12 h and OD600 was measured at indicated time [35].

For phenotypic analysis, Arabidopsis transgenic plants T4, wild-type and mutant seeds were grown on half MS agar plates in the absence or presence of 20, 40 and 60 μM CdCl2 for 2 weeks before being photographed and the averages of root lengths were measured in different experiments. Four untreated seedlings, each with a distinctive genotype, were grown in the half MS liquid medium with 100 μM CdCl2 for 24 h, and were used for determination of cadmium content. Three technical replicates were performed.

For induced expression experiment, 6-month-old poplars were immersed in WPM medium supplemented with different concentrations of CdCl2 for 12 h. Meanwhile, poplars treated with WPM medium without Cd were used as control. For the temporal spatial expression analysis, 6-month-old poplars were immersed in WPM medium supplemented with 100 μM CdCl2. Roots, stems and leaves were collected every 3 h for real-time quantitative PCR. Three technical replicates were performed.

### *4.5. Subcellular Localization of PtoABCG36*

*PtoABCG36* was ligated into pCAMBIA1300-GFP vector to produce 35S:*PtoABCG36*-GFP, which was transiently expressed in the leaves of 3-week-old *Nicotiana benthamiana* to examine the subcellular localization of PtoABCG36 after 72 h of infiltration. The 35S:*PtoABCG36*-GFP construct was transformed into GV3101 cells. The cells were grown at 28 ◦C to OD600 of 0.8, resuspended in infiltration buffer (10 mM MES, pH=5.7, 10 mM MgCl2, and 100 μM acetosyringone) to adjust the OD600 to 0.6 and infiltrated into 3-week-old *Nicotiana benthamiana* leaves. Analysis was carried out with a confocal microscope (Olympus FV1200, Tokyo, Japan). Conditions for imaging were set as 488-nm excitation, collecting bandwidth at 500 to 552 nm for GFP, 633-nm excitation, collecting bandwidth at 650 to 750 nm for chlorophyll autofluorescence.

### *4.6. Quantitative Real-Time PCR Analysis*

Total RNA was extracted from different plant tissues by using the RNA RNeasy Plant Mini Kit (Qiagen, Duesseldorf, Germany). First-strand cDNA synthesis was performed using the PrimeScript™ RT reagent kit (Perfect Real Time; Takara, Dalian, China). qRT-PCR was performed to detect the transcript of *PtoABCG36* in Arabidopsis and poplar by using the SYBR Green-based qPCR Master Mix (Promega, Madison, WI, USA). The gene-specific primers for qRT-PCR are listed in the supplementary Table S1. The poplar reference gene *UBQ* (FJ438462) was used as an internal control to normalize the expression data. The PCR conditions and relative gene expression calculations were conducted as previously described [14]. Three biological replicates and three technical replicates were performed.

### *4.7. Determination of Cadmium Content in Yeasts and Plants*

Cells of each line (1 <sup>×</sup> 107) were added to 30 mL of liquid SD-Ura medium containing 40 <sup>μ</sup>M CdCl2 and then cultured for different durations (6, 12, 18 or 24 h) at 30 ◦C. The cells were then collected and washed twice with distilled water and digested with HNO3 and H2O2 (3:1) at 140 ◦C for 10 min, 200 ◦C for 20 min and 140 ◦C for 10 min. The 2-week-old plants were immersed in half MS medium supplemented with 100 μM CdCl2 for 24 h. Then, shoots and roots were digested with HNO3 and H2O2 (3:1) at 140 ◦C for 10 min, 200 ◦C for 20 min and 140 ◦C for 10 min [39]. All of samples were

analyzed for total Cd detection by using Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES; ThermoFisher ICAP 6300, Waltham, MA, USA). All analyses were repeated three times.

### *4.8. Net Cd2*<sup>+</sup> *E*ffl*ux Measurements*

Fifteen-day-old seedlings were treated with 50 μM CdCl2 for 24 h and soaked in testing buffer (0.1 mM KCl, 0.1 mM CaCl2, 0.05 mM CdCl2, 0.3 mM 2-(*N*-morpholino) ethane sulfonic acid, pH 5.8) for 15 min. Roots were immobilized on the bottom of a measuring dish in fresh testing buffer. The measuring site was 800 μm from the root apex, and the net flux of Cd2<sup>+</sup> was detected using a non-invasive micro-test technique (NMT; BIO-001A, Younger United States Science and Technology Corp, Beijing, China). The ion flux of Cd2<sup>+</sup> was calculated according to Fick's law of diffusion, *<sup>J</sup>*<sup>0</sup> <sup>=</sup> <sup>−</sup>*<sup>D</sup>* <sup>×</sup> (*dC*/*dX*), where *J*<sup>0</sup> is the net ion flux (in <sup>μ</sup>mol·cm−<sup>2</sup> per second), *D* is the self-diffusion coefficient for the ion (in cm2·s<sup>−</sup>1), *dC* is the difference in the ion concentrations between the two positions, and *dX* is the 10 μm excursion over which the electrode moved in these experiments.

### *4.9. Statistical Analysis*

The experimental data related to roots length, Cd content, OD600 of yeast, and quantitative RT-PCR were analyzed by the statistical software SPSS 9.0. One-way analysis of variance (ANOVA) with Duncan's multiple range tests was considered as significance test. Different letters represented significant differences (*p* < 0.05). Values represented means ± standard deviation. Quantitative difference between two groups of data for comparison in each experiment was found to be statistically significant (\* *p* < 0.05; \*\* *p* < 0.01).

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/13/ 3293/s1.

**Author Contributions:** Data curation, H.W., Y.L., X.W., S.X., L.S. and E.H.; Investigation, Y.L.; Methodology, B.W.; Project administration, J.L., Y.L., Z.P. and W.H.; Supervision, N.W. and B.W.; Validation, K.L.; Writing–original draft, H.W.; Writing–review & editing, B.W. All authors reviewed the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (Grant No. 31571584 and 31370317), the Ministry of Science and Technology of China (Grant No. 2016YFD0100504), the Natural Science Foundation of Chongqing (Grant No. cstc2013jcyjA80016 and cstc2016shmszx20008), and Fundamental Research Funds for the Central Universities (XDJK2013B032), the National Undergraduate Training Programs for Innovation and entrepreneurship of China (Grant No. 201810635034).

**Acknowledgments:** We thank Ye Ning and Dexin Zhou for fruitful discussions and critical reading of the manuscript. We thank Ji-Ming Gong for kindly providing the yeast strains.

**Conflicts of Interest:** The authors declare no conflicts of interest.

### **References**


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