Introduction

While the demands for crop products continue to increase, global crop production has undergone sever variations in current years and it is severely restricted by different abiotic stresses such as drought, salinity, and heavy metals (Soares et al. 2019). Among these stresses, contamination of soils by heavy metals is a considerable environmental problem worldwide over the past several decades due to different human activities, including the use of fertilizers and pesticides, mining and smelting operations, industrial effluents, use of fossil fuels, and wastewater (Bokhari et al. 2016; Zhou et al. 2019). It may result in a critical threat to animal and human health as well as production capability and food quality (Jan et al. 2015). Although some heavy metals like copper (Cu), boron (B), zinc (Zn), molybdenum (Mo), manganese (Mn), and iron (Fe) are essential micronutrient, many other metals such as mercury (Hg), silver (Ag), cadmium (Cd), and lead (Pb) have no biological role and they are non-essential elements (Adrees et al. 2015). Essential micronutrients are needed in little amounts for healthy plant growth and development throughout life but are toxic to plants at higher levels (Dalcorso et al. 2014). Copper is an essential micronutrient used in several electron transport reactions in mitochondria and chloroplasts (Contreras et al. 2018). It also participates in hormone signaling, protein trafficking machinery, cell wall metabolism, iron mobilization, and transcription signaling (Ducic and Polle 2005). However, at high levels, copper is strongly phytotoxic and induce the increase of reactive oxygen species (ROS) levels within subcellular compartments and might, therefore, cause oxidative damage such as protein oxidation, lipid peroxidation, and nucleic acid cleavage (Martins and Mourato 2006). Several techniques are available to remedy soils that are contaminated by heavy metals (Nouri et al. 2009). Cleaning up of metal-contaminated soils using traditional remediation methods is not economical, and in some instances, they can degrade the biodiversity and result in deterioration of soil structure and fertility (Ghazaryan et al. 2019). Phytoremediation is an emerging sustainable, cost-effective, and eco-friendly (green) technique that uses the ability of hyperaccumulator species to immobilize, remove, or degrade pollutants from contaminated soils (Goswami and Das 2016). There are many techniques in phytoremediation such as phytoextraction, phytodesalination, rhizofiltration, phytovolatilization, phytostabilization, and phytofiltration (Palanivel et al. 2020). However, phytoextraction, phytostabilization, and rhizofiltration are commercially more viable than other methods (Thakur et al. 2016). The phytoextraction is the process of using hyperaccumulator plants to eradicate heavy metals from the surroundings by taking them from roots to shoots and consequently, eliminating the elements from the polluted environment (Reeves 2003). Hyperaccumulators can uptake high quantities of one or more heavy metals from the growth medium. Furthermore, the heavy metals are not maintained in the roots but are transferred to the shoots and gathered in the aerial parts, particularly leaves, at concentrations 100–1000-fold greater than those found in non-hyperaccumulators, without showing toxicity symptoms (Muszyńska and Hanus-Fajerska 2015). Generally, 5–30 mg/kg of Cu in crop tissues are considered as sufficient (Ballabio et al. 2018). (Hyper)accumulation of Cu was first defined as > 1000 mg/kg foliar Cu (Malaisse et al. 1978), but recently revised downwards to 300 mg/kg (van der Ent et al. 2013; Reeves et al. 2018). The fast and efficacious translocation of heavy metals from roots to shoots, high biomass yield, and capability to withstand heavy metal stress and metal toxicity is prominent characteristics of hyperaccumulators (van der Ent et al. 2013; Tiwari and Lata 2018). The excessive Cu can catalyze the generation of the ROS and free radicals in Fenton-type reactions, which induce oxidation of macromolecules like carbohydrates, proteins, lipids, and nucleic acids and causing oxidative stress in cells (Fatnassi et al. 2015). The detoxification of ROS compounds in hyperaccumulator plants is required to protect the plant cells against the deleterious consequence of ROS and free radicals (Rascio and Navari-Izzo 2011). The toxic effects resulting from the oxidative stress may be alleviated by enzymatic defense systems including superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), and nonenzymatic protection systems like proline, phenolic compounds, ascorbate, and glutathione (Flora 2009). Therefore, examining the antioxidant defense system (ADS) in such a plant may furnish valuable data about its phytoremediation ability (Goswami and Das 2016). The genus Verbascum L., relates to the family Scrophulariaceae and Iran, Pakistan, and Turkey, are the foremost centers of its diversity. It contains 360 species all over the world and 42 species in Iran (Karamian and Ghasemlou 2014). The V. thapsus (common mullein) is a biennial plant that is widely distributed throughout Asia, Europe, North Africa, and North America (Zhao et al. 2011). In Iran, it is grown mainly in the Northern, North-Western, and Central provinces (Morteza-Semnani et al. 2012). The species tolerates a wide range of environmental conditions and is adapted to the various habitats and different regions of Iran, including roadsides, open forests, rocky mountains, the margins of the rivers, and heavy metal polluted environments (Karamian and Ghasemlou 2014). However, very few studies have explored the potential of V. thapsus to remediate Cu-polluted environments (Morina et al. 2016). The objectives of the current study are to (1) observe the phytotoxicity of Cu on morphological and biochemical parameters of V. thapsus, (2) to determine the Cu accumulation efficiency in below and above ground biomass of V. thapsus growing on a Cu enriched medium, (3) to analyze the role of the antioxidant defense system in the Cu tolerance mechanism of V. thapsus, and (4) to investigate the possibility of using V. thapsus for Cu phytoremediation. Findings from the present study will add to our knowledge about the mechanism of Cu tolerance and accumulation in V. thapsus.

Materials and methods

Plant material, growth condition, and treatment

The seeds of common mullein were prepared from the Pakan Bazr Company (Isfahan, Iran). The seeds were sterilized with 5% NaOCl for 5 min and then rinsed three times with ultrapure water. The seeds were placed on water-moistened filter paper in 9.5-Cm Petri dishes and germinated in the dark condition at 20 °C. After 10 days of germination, the seedlings were transferred to small plastic beakers (20 × 15 × 10 cm) filled with one-tenth (v/v)-strength Hoagland nutrient medium (600 mL) and transferred to a growth chamber (Noor Sanat Ferdows, Iran) with a 16-h light/8-h dark photoperiod and 15 °C/25 °C day/night temperature regime. Plants were irradiated by white fluorescent 36-W tubes that provided irradiance (400–700 nm) of ca. 80 μmol photons/m2/s1. The nutrient medium was renewed every second day, and the concentration of nutrients was increased by 10% every week. After 8 weeks, the uniform plants bearing 8-leaves were selected and exposed to different levels of Cu (0 (control), 125, 250, 375, and 500 mg/L) in 80% Hoagland nutrient solution. Copper was supplied as CuSO4. 5H2O (Merck, KG, Darmstadt, Germany). Plants were harvested after 14 days of Cu treatment without damaging the roots. Roots were washed twice in 10 mM Na2EDTA (pH 7.0) and subsequently rinsed with distilled water to remove adsorbed metal ions. The plant separated into shoots and roots and stored at − 70 °C after freezing in liquid nitrogen for further analysis.

Biomass determination

The dry biomass of shoots and roots was determined 14 days after the initiation of the experiment. The plant samples were then dried in an oven for 48 h at 80 °C, and their dry biomass was measured (Morina et al. 2016).

The capacity of copper uptake in plant organs

Copper concentration in common mullein shoots and roots was analyzed after digesting the dried samples in an acidic mixture of nitric and perchloric acid (HNO3: HClO4), according to the APHA method (1992) using an Atomic Absorption Spectrometer (Vario6, Analytik Jena, Germany). The phytoremediation ability of V. thapsus plants was evaluated by the translocation factor (TF) and the extraction coefficient (EC) using the following formula (Goswami and Das 2015):

$$ {\displaystyle \begin{array}{c}\mathrm{TF}=\frac{\mathrm{copper}\ \mathrm{in}\ \mathrm{shoots}}{\mathrm{copper}\ \mathrm{in}\ \mathrm{roots}}\\ {}\mathrm{EC}=\frac{\mathrm{copper}\ \mathrm{concentration}\ \mathrm{in}\ \mathrm{aerial}\ \mathrm{part}\ \left(\mathrm{shoots}\right)}{\mathrm{copper}\ \mathrm{concentration}\ \mathrm{in}\ \mathrm{solution}}\end{array}} $$

Measurement of the photosynthetic pigments

For the estimation of the photosynthetic pigments, 0.25 g of shoot samples were extracted with 80% acetone. The extract was then centrifuged at 3000 g, and the absorbances of the extract were taken at 646.8, 663.2, and 470 nm with a UV/Vis spectrophotometer (Specord 200 Plus, Analytik Jena, Germany) based on the Lichtenthaler (1987) method.

Assessment of lipid peroxidation and electrolyte leakage

The level of lipid peroxidation was estimated using the measurement of the malondialdehyde (MDA) content based on the Sun et al. (2010). The fresh sample (0.5 g) was homogenized in 10% trichloroacetic acid (TCA) and centrifuged at 12,000 g for 10 min. Then, 2 mL of extract was added to 2 mL of reaction solution containing 10% TCA and 0.5% thiobarbituric acid (TBA). The mixture was heated in boiled water for 30 min and then cooled immediately on an ice bath. The mixture was centrifuged at 10,000 g for 15 min, and the absorbance was read at 450, 532, and 600 nm. The concentration of MDA was measured as 6.45 (A532-A600) − 0.56 A450.

Electrolyte leakage was determined according to Dionisio-Sese and Tobita (1998). Root sample (100 mg) was cut into 5-mm length and placed in closed tubes containing 10 mL of deionized water. The tubes were covered with rubber caps and put in a water bath maintained at 32 °C. After 2 h, the initial electrical conductivity (EC1) of the medium was determined using a conductivity meter (SG3-FK2 SevenGoTM, Mettler-Toledo, USA). The samples were then autoclaved at 120 °C for 30 min, cooled down to 25 °C, and their final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was calculated from EL = (EC1/EC2) × 100 (%). The experiment was performed with three replicates for each treatment.

Antioxidant enzyme extraction and assays

Shoot and root samples (0.1 g) were extracted in 2 mL of 50 mM sodium phosphate buffer (pH 7.0) including 1 mM EDTANa2 and 0.5% polyvinylpyrrolidone. The homogenate was centrifuged for 30 min at 13,000 g, and the extract was applied for enzymatic assays. Total protein content was determined according to Bradford (1976) assay with the use of bovine serum albumin as the standard solution. The SOD activity was measured spectrophotometrically based on its ability to inhibit the photochemical reduction of nitroblue tetrazolium, adopting the method of Dhindsa et al. (1981). The CAT activity was determined based on the method of Beers and Sizer (1952), by measuring the H2O2 absorbance reduction at 240 nm. The APX activity was assayed following the ascorbate oxidation by the decrement in absorption at 290 nm according to the method of Nakano and Asada (1981).

Statistical analysis

In all experiments (except for the ion leakage experiment which was performed in three replicates), five replicates were considered for each treatment. The data analysis was performed with one-way ANOVA followed by Duncan’s multiple range tests (p = 0.05) for the comparison of mean values using SAS 9.1.

Results and discussion

Influence of Cu on biomass and photosynthetic pigments

The presence of Cu in the growth medium resulted in a gradual reduction in the shoot and root biomass of V. thapsus plants, and the highest growth retardation was found in plants treated with 500 mg/L Cu. Data on the biomass production indicated that V. thapsus plants could tolerate 125 mg/L Cu for 14 days without a significant decrease in shoot and root biomass compared with control. With increasing Cu levels in the nutrient medium, the growth of V. thapsus plants slowed. For 250, 375, and 500 mg/L Cu levels, the shoot biomass decreased by 17%, 33%, and 45%, and the root biomass declined by 30%, 47%, and 63%, respectively, compared with the control (Table 1). Moreover, excess copper was progressively resulted in significant increase of leaf chlorosis and a concomitant decrease in the percentage of healthy green leaves (Fig. 1).

Table 1 Effects of different Cu concentrations (mg/L) in the nutrient solution for 14 days on the biomass of the shoots and roots of V. thapsus plants
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Fig. 1
figure 1

Effects of different Cu concentrations (mg/L) in the nutrient solution for 14 days on the aerial parts of the V. thapsus plant

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The results presented in Table 2 designated that in comparison with control, no significant variation was found in terms of chlorophylls (Chlorophyll a, Chlorophyll b, and total chlorophyll) at 125 mg/L Cu level. Increasing the Cu concentration in the nutrient solution significantly reduced the chlorophylls in the leaves of V. thapsus plants. A considerable decrement in total chlorophyll contents (15%, 40.7%, and 54.1% for 250, 375, and 500 mg/L, respectively, compared with the control) was registered. The chlorophyll a/b ratio was slightly decreased with elevating the Cu concentration. However, there were significant differences only at 375 and 500 mg/L Cu levels. Other results indicated that Cu had no punctual impact on carotenoid content in V. thapsus leaves up to 250 mg/L. Indeed, although the content of carotenoid had a decreasing trend with raising Cu concentration, the influence was not statistically significant (p < 0.05). However, when the Cu level in the nutrient solution was exceeded 250 mg/L, the carotenoid content significantly declined, and at 500 mg/L, Cu was 41% lower than the control.

Table 2 Effects of different Cu concentrations (mg/L) in the nutrient solution for 14 days on shoot pigment contents of V. thapsus plants
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Cu stress significantly declined V. thapsus biomass in a dose-dependent manner. However, at 125 mg/L Cu, no significant changes in shoot and root biomass were found. The reduction of biomass and yield is a noticeable effect of exposure to excess Cu in plants, which was also detected in this study (Adrees et al. 2015). Various studies have demonstrated the adverse impact of excessive amounts of Cu on the biomass and yield of plants grown in soil conditions and hydroponics (Fidalgo et al. 2013; Li et al. 2018; Saleem et al. 2020a). An overall reduction of plant biomass and photosynthesis rate, inhibition of root growth, altered nutrient uptake, membrane disruption, chlorosis, and necrosis are the typical reported symptoms of a Cu excess (Lange et al. 2017; Morina et al. 2016). Photosynthesis is one of the principal constituents determining plant biomass (Aggarwal et al. 2011). Chlorophylls are the essential photosynthetic pigments and can be regarded as a fundamental biochemical index of the photosynthetic potential of the plant in response to different constraints (Ashraf and Harris 2013). The reduction of photosynthetic pigments is amongst the common detrimental effects of excessive Cu concentration in the plant's culture medium (Llagostera et al. 2016). It has been suggested that Cu at toxic doses blocks the enzymes acting in chlorophyll biosynthesis and stimulates the chlorophyll degradation by chlorophyllase activity (Shiyab 2018). High Cu concentrations can also induce degradation of the internal and structural constituents of chloroplasts and may even replace the Mg ion needed for chlorophyll biosynthesis, which impairs its synthesis (Marques et al. 2018). Our results indicated that Cu stress significantly dwindled the photosynthetic pigment in plants, which eventually caused a reduction in plant biomass. Diminution of chlorophyll content owing to higher doses of Cu was also reported in Chinese cabbage (Shahbaz et al. 2010), sunflower (Kolbas et al. 2014), rapeseed and Indian mustard (Feigl et al. 2015), Calendula officinalis L. (Goswami and Das 2016), and ramie (Boehmeria nivea L.) (Rehman et al. 2019). An overall reduction in carotenoid content was observed with increasing Cu concentrations; however, no significant changes were observed in leaf carotenoid contents for 125 and 250 mg/L Cu levels. Carotenoids play a crucial role in the quenching of singlet oxygen and detoxifying of other ROS compounds that are generated upon exposure to various constraints, including heavy metals (Bose et al. 2014). In addition to their activity as subsidiary pigments, carotenoids exhibit antioxidant characteristics (Strzalka et al. 2003). Therefore, the lack of significant changes in the carotenoid contents of the V. thapsus at 125 and 250 mg/L Cu levels maybe due to this role. However, when the Cu level was raised, a significant reduction in leaf carotenoid contents was observed. Diminution in carotenoid contents in higher Cu concentrations might be a result of Cu-induced oxidative stress, which manifested as MDA accumulation. This indicates that carotenoids as antioxidants are not capable of inhibiting the interference of Cu ions and the production of the ROS compounds that damage photosynthetic pigments.

Influence of Cu on lipid peroxidation, electrolyte leakage, and protein contents

In our study, The MDA levels, which portend oxidative damage, were increased in the shoots and roots of V. thapsus plants at all the Cu levels compared with the control (Table 3). In 125, 250, 375, and 500 mg/L Cu levels, the shoot MDA contents increased by 2.79-, 4.56-, 8.2-, and 12.59-fold, whereas the root MDA contents increased by 2.2-, 3.64-, 7.07-, and 10.72-fold, respectively, compared with the control. Electrolyte leakage tended to increase significantly in the roots of V. thapsus plants with increasing Cu concentration. At higher Cu concentration (500 mg/L), the electrolyte leakage increased more than 3 times compared with the control (Table 3). Although Cu as a metal cofactor participates in the structure of the SOD enzyme, it can directly induce the production of ROS compounds via Fenton and Haber–Weiss reactions (Küpper et al. 2009). It has been revealed that additional Cu stimulates the production of free radicals and the ROS compounds and can induce lipid peroxidation in plants (Fatnassi et al. 2015). Lipid peroxidation due to excess copper interrupts the membrane organization, which may inactivate membrane-bound enzymes and receptors, increasing the permeability of the cell membranes and promote the leakage of electrolyte and subsequent loss of cell turgor pressure (Panda 2008). The MDA is the end product of membrane lipid peroxidation and accumulates in plants after exposure to oxidative stress. Electrolyte leakage showed the same trend with MDA contents in the root of V. thapsus plants. This finding suggests that Cu-induced MDA accumulation could cause damage to the cell membranes, resulting in increased electrolyte leakage. Similar results were reported in Hibiscus cannabinus L. (Saleem et al. 2020b), Verbascum olympicum Boiss. (Akpinar et al. 2016), Alternanthera bettzickiana L. (Khalid et al. 2020), etc. Accordingly, the level of MDA and electrolyte leakage are commonly applied as an indicator to assess the sensibility of the plant to oxidative stress under different constraints including heavy metal stress (Juknys et al. 2012).

Table 3 Effects of different Cu concentrations (mg/L) in the nutrient solution for 14 days on MDA contents, electrolyte leakage, and soluble protein of V. thapsus shoots
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The total soluble protein content of shoots was progressively enhanced with the elevated concentrations of Cu, reach its highest in 375 mg/L Cu level, and after that decreased at 500 mg/L Cu concentration. However, no statistical differences were found among 125, 250, and 375 mg/L Cu Levels. Alteration in the protein metabolism of plants is one of the harmful effects of heavy metal-induced oxidative stress. Accordingly, the content of soluble protein is employed as an essential index of the physiological status of plants (Doğanlar 2013). The increment in total soluble protein content of V. thapsus plants under Cu stress may be due to inducing the biosynthesis of stress-related proteins, including metal chelator polypeptides (metallothionein and phytochelatin) and other proteins involved in metal detoxification or homeostasis for adaptation to excess Cu level (Parmar et al. 2013). The increase in total soluble protein content in response to heavy metals stress was expressed by other researchers (Heiss et al. 2003; Rolli et al. 2010; Doğanlar 2013; Zoufan et al. 2018). With increasing the Cu concentration in the nutrient solution, the content of total soluble protein remarkably declined. The reduction in protein content under the highest Cu level can be related to inhibition of protein synthesis or an increase in protein degradation due to ROS accumulation (Hasan et al. 2017).

Different letters indicate significant differences at p < 0.05 according to the Duncan’s test

Effect of Cu on antioxidant enzymes activity

The antioxidant enzymes activity in shoots and roots of V. thapsus plants exposed to Cu for 14 days were shown in Fig. 2. The SOD activity in both the shoots and roots was progressively enhanced proportionally to the increasing Cu levels in the growth medium. The highest SOD activity was determined in the shoots and roots of plants exposed to 375 mg/L Cu. Further application of Cu at 500 mg/L slightly decreased the enzyme activity in both of the shoots and roots. However, the difference was not statistically significant (Fig. 2a). In plants exposed to Cu, the CAT activity in both the shoots and roots gradually increased and the highest value for CAT activity was observed at Cu Level of 375 mg/L and then was declined significantly at 500 mg/L Cu treatment (Fig. 2b). Our results demonstrated that 125 mg/L Cu had no significant effect on the APX activity of the roots and shoots. With enhancing Cu levels, the activity of APX was significantly increased. Similar to SOD and CAT enzymes activity, the highest APX activity was observed in the shoots and roots of plants exposed to 375 mg/L Cu for 14 days and then was significantly diminished at 500 mg/L Cu level (Fig. 2c). Different heavy metals, including copper, stimulate oxidative stress due to the overproduction of the ROS compounds in plant cells (Shahid et al. 2014). To refrain from the detrimental effects of the ROS on cellular components, plants have formed a highly organized and powerful antioxidant system that comprises enzymatic and non-enzymatic antioxidants that are ordinarily effective in limiting adverse effects of the ROS (Das and Roychoudhury 2014). Antioxidant enzymes are substantial constituents in hindering the oxidative stress in plants by detoxifying free radicals (Caverzan et al. 2016). Many investigations have stated that increases in activity of the antioxidant enzymes in plants are correlated to the level of tolerance against Cu toxicity (Thounaojam et al. 2013; Fatnassi et al. 2015; Goswami and Das 2016; Liu et al. 2018). In this study, the efficiency of enzymatic antioxidants was assessed to specify more accurately the strategies of Cu tolerance in V. thapsus plants. The SOD is a metalloenzyme and most potent antioxidant that constitutes the front line of protection versus ROS by accelerating the dismutation of superoxide (O2•–) to hydrogen peroxide (H2O2) in the cytosol, chloroplast, and mitochondria of plant cells (Gupta et al. 2018). Because SOD functions as a first-line enzyme in defense against oxidative stress, the alterations found in its activity confirm that exposure to excess Cu, produce superoxide radicals in the shoots and roots of V. thapsus plants, and induce the stress responses. The hydrogen peroxide, which is formed by the SOD activity, is yet toxic and requires to be eliminated in the succeeding reactions by conversion to H2O (Das and Roychoudhury 2014). Different enzymes are responsible for the elimination and adjusting of the intracellular levels of H2O2 in higher plants, but the most prominently characterized enzymes are CAT and APX (Caverzan et al. 2016). The CAT is one of the most vital antioxidant enzymes implicated in the elimination of toxic peroxides by decomposing H2O2 into water and molecular oxygen (Sharma and Ahmad 2014). The CAT activity was raised in all Cu concentrations except 500 mg/L, which can be regarded as inferred evidence for the role of CAT in scavenging of H2O2 that was induced under Cu stress in V. thapsus plants. The ascorbate-glutathione cycle is another significant hydrogen peroxide scavenging system in plant cells, in which APXs perform a pivotal function by catalyzing the transformation of H2O2 into H2O, applying ascorbate as a particular electron donor (Caverzan et al. 2012). These results indicate that the ascorbate-glutathione cycle probably has a significant role in the ROS detoxification in V. thapsus exposed to copper. Our result showed that high Cu concentration is detrimental for the antioxidative ability of V. thapsus and resulted in antioxidant enzyme activities reduction, which is in agreement with the finding in Vicia faba L. (Fatnassi et al. 2015), watercress (Nasturtium officinale R. Br.) (Ercan et al. 2018), Verbascum olympicum Boiss. (Akpinar et al. 2016), and B. nivea (Rehman et al. 2019). Detoxification of ROS can be attained by antioxidant enzymes such as SOD, CAT, and APX. Thus, the remarkable increase in activity of these enzymes, as found in our study, may be regarded as evidence for the capability of the plant to retain an equilibrium between the creation and detoxification of ROS. The elevated biosynthesis of important antioxidant enzymes in hyperaccumulator plants functions as a mechanism to augment the antioxidant defense system of the cell and confront the hazards of ROS increment as a result of heavy metal stress (Goswami and Das 2016). Therefore, it can be concluded that the production of SOD, CAT, and APX can serve as valuable biomarkers for Cu tolerance in V. thapsus.

Fig. 2
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The activity of antioxidant enzymes in shoots and roots of V. thapsus plants exposed to various Cu concentrations (mg/L) for 14 days. The values are represented as mean ± SD of five replicates. Different letters indicate significant differences at p < 0.05 according to the Duncan’s test

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The copper concentration in shoots and roots and indexes

The copper concentration in shoots and roots of V. thapsus plants exposed to various treatments of Cu are given in Table 4. The Cu concentration progressively was elevated in both tissues up to 375 mg/L Cu, but at higher doses, the copper accumulation began to decline. Excluding for the highest concentrations of copper (500 mg/L), the accumulation of Cu in shoots was higher than in roots. The results indicate that at 500 mg/L Cu, as an exclusion strategy, the plant limited the transport of Cu to the aerial parts to avoid phytotoxicity. In this research, V. thapsus plants gathered Cu in the range 92.5–492.8 mg/kg, that is greatly exceeding from the standard (5–25 mg/kg) and noxious (20–100 mg/kg) levels of copper in plants (Ballabio et al. 2018). Arslan et al. (2010) reported that Verbascum bombyciferum Boiss. has the capability of Cd2+, Cr3+, Cu2+, Ni2+, Pb2+, and Zn2+ accumulation, especially in aboveground parts. Morina et al. (2016) showed that metal tolerance can occur in Verbascum populations after a relatively short time of exposure to metal-contaminated soil. Plants Cu tolerance varies with species and cultivar, but in general, plants are Cu-excluders and uncommon are Cu accumulator plants (Baker and Brooks 1989; Reeves 2003). However, the phytoextraction of copper by some plant species including Calendula officinalis L. (Goswami and Das 2016), Cyamopsis tetragonoloba L. (Amin et al. 2018), B. nivea (Rehman et al. 2019), and Indian mustard (Napoli et al. 2019) have been reported. The heavy metal uptake by the plant includes the movement of metal ions through the plasma membrane (PM) of the root cells, xylem loading, and translocation to shoots and, ultimately detoxification, and sequestration of metal ions at the whole plant, cellular, and subcellular levels (Cheng 2003). A characteristic feature of a suitable hyperaccumulator is its ability to gather metal ions principally in their shoots at different concentrations of exogenous metal ions (Peer et al. 2005). The prerequisite for the accumulation of metal in the plant is metal tolerance (Muszyńska and Hanus-Fajerska 2015). To depreciate the harmful effects of heavy metal exposure and their accumulation, plants have developed a variety of detoxification mechanisms. Such mechanisms principally relied on metal chelation and compartmentalization within the vacuole (Yruela 2009; Yadav 2010). Plants employ the conserved CTR/COPT family of high-affinity Cu transport proteins at the PM to facilitate cellular Cu acquisition (Sanz et al. 2018). Copper enters the cytoplasm in Cu+ form. The Cu ions are bound by chaperons and chelators. Chelators, including phytochelatins, metallothioneins, amino acids, and organic acids, are involved in the detoxification of metals, while chaperons mainly distribute metal ions to metal-requiring protein and organelles (Clemens 2001; Ducic and Polle 2005). The plant cells store amazing kinds of toxic compounds in their vacuoles, and there is plentiful evidence for vacuolar sequestration of metal ions in plant species (Clemens 2001). The vacuolar compartmentalization relies on two vacuolar pumps (V-PPase and V-ATPase) and some transporters on tonoplast, which are directly driven by proton motive force, and primal ATP-dependent pumps (Sharma et al. 2016). In the current research, the Cu concentrations in shoots were higher than in roots, suggesting the effective transfer of Cu from roots to shoots. The highest accumulation of Cu was 492.8 ± 20.143 mg/kg DW which was seen in the shoots at 375 mg/L treatment. The Phytoremediation efficiency for V. thapsus was quantified by evaluating the extraction coefficient (EC) and translocation factor (TF) values (Table 4). There was an increment in the EC index with raising the Cu levels. The maximum EC was 0.9856 at 375 mg/L. As the concentration of copper in the medium increased, the EC index declined. The TF is the index that shows the potential of the plant to translocate the Cu from roots to shoots. Except at 500 mg/L, which demonstrated TF of 0.869, the translocation of Cu from root to shoot at other concentrations, was > 1, which is the specified limit for an optimum hyperaccumulator (Muszyńska and Hanus-Fajerska 2015). The decrement in the TF index with raising Cu dose implicates an exclusion mechanism (Goswami and Das 2016). Lower shoot metal concentration and higher root metal concentration represent the confinement of Cu translocation from the roots towards upper parts through the xylem sap stream at the higher levels (phytostabilization).

Table 4 Effects of different Cu concentrations (mg/L) in the nutrient solution for 14 days on the copper concentrations (mg/kg DW) in shoots and roots, the extraction coefficient (EC), and the translocation factor (TF) of V. thapsus plants
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Conclusions

The current research demonstrated that V. thapsus showed excellent Cu tolerance and can accumulate phytotoxic quantities of Cu in its shoots. The species can also be recommended for its capacity for Cu phytoremediation objectives due to its extensive root system for metal uptake, and elevated production of biomass in the presence of a high level of heavy metal. With inducible and efficient antioxidant enzymes, high accumulation of metal in organs, EC close to 1, and TF > 1, V. thapsus can be suggested for broad-scale utilization to amend Cu polluted soils. Although the results of the current research are promising, additional investigation is required to appraise the usefulness of V. thapsus under field conditions for remediation purposes and in the amendment of soils polluted with various metals.