Assessing the Correlations between Different Traits in Copper-Sensitive and Copper-Resistant Varieties of Jute (Corchorus capsularis L.)

The current study was conducted to explore the potential for phytoremediation in different varieties of jute grown under toxic concentrations of copper (Cu). For this purpose, a Petri dish experiment was conducted under controlled conditions using four varieties of jute, i.e., HongTieGuXuan, C-3, GuBaChangaJia, and ShangHuoMa, grown in double filter paper under 50 µmol L−1 of artificially spiked copper (Cu) using CuSO4.H2O. The results of the present study revealed that jute varieties C-3 and HongTieGuXuan were able to survive under high concentrations of Cu without a significant decrease in plant height, plant fresh and dry weights, total chlorophyll content, or seed germination, while varieties GuBaChangaJia and ShangHuoMa exhibited a significant reduction in their growth and biomass. Furthermore, high concentrations of Cu in the medium resulted in lipid peroxidation. This could be due to the oxidative damage induced in the roots and leaves of the jute varieties, which might be a result of by hydrogen peroxide (H2O2) and electrolyte leakage. Reactive oxygen species (ROS) generated due to Cu toxicity can be overcome by the increasing activity of antioxidants, and it was also noted that GuBaChangaJia and ShangHuoMa exhibited high Cu stress, while C-3 and HongTieGuXuan showed some resistance to Cu toxicity. Contrastingly, Cu accumulation and uptake was higher in C-3 and HongTieGuXuan, while a little Cu was accumulated in the roots and leaves of GuBaChangaJia and ShangHuoMa. On the basis of these findings, it can be suggested that C-3 and HongTieGuXuan have the potential to cope with Cu stress and can be considered Cu-resistant varieties, while GuBaChangaJia and ShangHuoMa are considered Cu-sensitive varieties. Moreover, C-3 and HongTieGuXuan have the potential to revoke large amounts of Cu, and can be cultivated as phytoremediation tools in Cu-contaminated soil.


Growth Conditions and Experimental Treatment
The seeds of different jute varieties were collected from Bast and Fiber Research Center of Huazhong Agricultural University, Hubei Province, China. The seeds of C-3, HongTieGuXuan, GuBaChangaJia, and ShangHuoMa (which are types of white jute) were used for the Petri dish experiment, and 40 seeds were planted in each Petri dish. Two filter papers (90 mm in diameter) were used in each Petri dish and 5 mL of 50 µmol L −1 Cu solution was added. Cu solutions were prepared with pure distilled water using copper sulfate (CuSO 4 .5H 2 O) (99% purity). After quantification of Cu, as percent availability in CuSO 4 .5H 2 O, 50 µmol L −1 doses of this compound were taken. Cu solution was applied every alternate day for the prevention of fungal infection and other contamination [29]. After washing carefully with distilled water, seeds were tested on filter paper (What-man No. 1). Seeds were surface sterilized with 0.1% HgCl 2 for the prevention of surface fungal/bacterial contamination [30]. The experiment was conducted in March 2018 at Huazhong Agricultural University, Wuhan China, in a growth chamber under white lights (100 W, Guangdong PHILIPS Co., Guangdong, China) with a day/night temperature of 25 ± 2 • C and day/night humidity of 80%. The nutrient solution was provided once in a week, replacing the Cu treatment for 24 h. The experiment was conducted in a complete randomized design with nine replications for each treatment. The seed germination was recorded at 4 days after sowing (DAS). The seeds were considered to have germinated when the shoots were more than 2 mm [10], and plant height, plant fresh and dry weight, chlorophyll content, and different antioxidants were analyzed at 14 DAS. Furthermore, the Cu concentration of roots and shoots were also measured in this study. All chemicals used were of analytical grade, procured from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Growth and Morphological Traits
Different morphological attributes were noted after the harvest of all plants. Plants in each treatment were harvested and separated into roots and shoots for growth and morphology traits. Total length was defined as the length of the plant from the surface growth medium line of the Petri dish to the tip of the uppermost shoot. Total fresh weight was measured by measuring the weight of roots and shoots with the help of a digital weighing balance. After that, plant samples were oven dried for 1 h at 105 • C, then 65 • C for 72 h until the weight become uniform and dry biomass was recorded. Roots were washed with distilled water and dipped in 20 mM Na 2 EDTA for 15-20 min, washed thrice with distilled water and finally with de-ionized water, and then oven dried for further analysis. Chlorophyll contents were determined following the method of Arnon [31] and are expressed as mg g −1 FW.

Determination of Oxidative Stress
The method used to describe the concentration of lipid peroxidation was presented by Heath and Packer [32] and expressed as µmoles g −1 FW.
For the estimation of H 2 O 2 content, an H 2 O 2 Assay Kit (Suzhou Comin Biotechnology Co., Ltd.) was used.
The electrolyte leakage (EL) was measured according to the standard procedure of Dionisio-Sese and Tobita [33].

Analysis of Antioxidant Enzyme Activities
The activity of SOD was measured by the method of Chen and Pan [34] and expressed in Ug −1 FW. The activity of POD was measured by the method of Sakharov and Aridilla [35] and expressed in Ug −1 FW.
The enzymatic activity of CAT was measured by the method of Aebi [36] and expressed in Ug −1 FW. Ascorbate peroxidase activity was measured according to Nakano and Asada [37] and expressed in Ug −1 FW.

Determination of Cu Concentration
The dried samples were ground into powdered form using stainless steel and 0.1 g of root and shoot samples was taken for digestion in HNO 3 /HClO 4 (4:1) solution. Final readings were taken from an atomic absorption spectrophotometer (AAS) model Agilent 240 FS-AA [7].
Bioaccumulation factor (BAF) was measured as the proportion of Cu concentration in plant tissues and Cu concentration in medium using the following formula: Cu concentration in plant tissues Cu concentration in the medium (1) Translocation factor (TF) was evaluated as the proportion of Cu concentration in shoots with respect to the roots: Cu concentration in shoots Cu concentration in the roots (2)

Statistical Analysis
The data recorded were statistically analyzed using Statistix 8.1 (Analytical Software, Tallahassee, FL, USA). Testing showed that all the data were approximately normally distributed. Thus, the differences between treatments were determined using analysis of variance, and the least significant difference test (p ≤ 0.05) was used for multiple comparisons between treatment means. Graphical representation was carried out using SigmaPlot 12 and R studio.

Effects of Cu Toxicity on Seed Germination, Growth, and Chlorophyll Content
Jute varieties showed various germination behaviors in response to Cu exposure (Table 1). Germination rate ranging from 75-100% in different varieties of jute under Cu toxicity. Based on the observations of seed germination, HongTieGuXuan and C-3 showed a better germination rate than GuBaChangaJia and ShangHuoMa. Maximum germination percentage was observed in HongTieGuXuan (100%) and C-3 (100%), followed by GuBaChangaJia (77.5%) and ShangHuoMa (75%) under toxic concentrations of Cu in the medium. Table 1. Effect of Cu stress on plant height (cm), plant fresh weight (g), plant dry weight (g), total chlorophyll content (mg g −1 FW), and seed germination (%) in different varieties of jute. Values in the table are from one harvest ± SD (n = 5). Different letters within a column indicate significant difference between the treatments (p < 0.05).

Varieties
In the present study, growth in terms of plant height, plant fresh weight, and plant dry weight was also measured in different varieties of jute when cultivated under high concentrations of Cu in the medium. Results regarding growth and biomass of different varieties of jute are presented in Table 1. Maximum plant height and fresh and dry biomass were observed in HongTieGuXuan and C-3 under toxic levels of Cu in the medium. Moreover, the minimum plant height (0.61 ± 0.13), plant fresh weight (0.101 ± 0.003), and plant dry weight (0.040 ± 0.02) was observed in ShangHuoMa, while maximum plant height (3.73 ± 0.85), plant fresh weight (0.30 ± 0.01), and plant dry weight (0.100 ± 0.01) were observed in C-3 under toxic concentrations of Cu in the medium.
In the present study, total chlorophyll contents of the leaves of different varieties of jute are presented in Table 1. Different chlorophyll contents were observed in different varieties to jute after exposure of high concentrations of Cu in the medium. Maximum contents of chlorophyll were observed in HongTieGuXuan and C-3. The maximum contents of chlorophyll were observed in C-3 (2.82 ± 0.03 mg g −1 FW) followed by HongTieGuXuan (2.64 ± 0.09 mg g −1 FW), while minimum contents of chlorophyll were observed in ShangHuoMa (1.47 ± 0.08 mg g −1 FW) followed by GuBaChangaJia (1.51 ± 0.03 mg g −1 FW).

Effect of Cu Toxicity on Oxidative Stress
In this study, the effects of Cu toxicity on different varieties of jute on malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), and electrolyte leakage (EL) from the roots and shoots were also investigated ( Figure 1). Exposure of Cu concentration to different varieties of jute significantly increased the MDA, H 2 O 2 , and EL in the roots and leaves of different varieties of jute ( Figure 1). The contents of MDA, H 2 O 2 , and EL were higher in the roots when compared to the above-ground parts of the plant. According to the results, maximum contents of MDA (17.7 ± 0.8 µmoles g −1 ), H 2 O 2 (539 ± 6 µmoles g −1 ), and EL (70 ± 2%) were observed in the roots of ShangHuoMa. Similarly, in the shoots, the maximum contents of MDA (11.9 ± 0.2 µmoles g −1 ), H 2 O 2 (445 ± 5 µmoles g −1 ), and EL (56 ± 2%) were also observed in of ShangHuoMa compared to other varieties of jute.

Cu Uptake and Bioaccumulation in the Roots and Shoots
In this study, the concentrations of Cu from different parts (roots and shoots) of jute varieties were also determined under toxic concentrations of Cu in the medium. The concentrations of Cu in the roots and shoots of different varieties of jute are presented in Table 2. These results suggested that Cu concentration was higher in the varieties that exhibited significantly better growth than the varieties that were more affected by Cu stress. The concentration of Cu in the roots ranged from 37 to 60 mg kg −1 , while in the shoots, Cu concentration ranged from 38 to 61 mg kg −1 ( Table 2). The maximum Cu was accumulated in the roots of C-3 (60 ± 0.8 mg kg −1 ), followed by HongTieGuXuan (56 ± 1.4 mg kg −1 ) and GuBaChangaJia (40 ± 0.8 mg kg −1 ). Similarly, in the shoots, maximum Cu concentration was accumulated in C-3 (61 ± 0.9 mg kg −1 ), followed by HongTieGuXuan (57 ± 1.1 mg kg −1 ) and GuBaChangaJia (41 ± 0.6 mg kg −1 ). Table 2. Accumulation of Cu in roots (mg kg −1 ) and shoots (mg kg −1 ) of different varieties of jute. In this study, bioaccumulation factor (BAF) and translocation factor (TF) were also calculated ( Figure 3). It was noticed that the values of BAF and TF of HongTieGuXuan and C-3 were greater than 1 while the values of BAF and TF of GuBaChangaJia and HongTieGuXuan were less than 1 (Figure 3). The maximum BAF value was shown by C-3 in the roots (1.19) and shoots (1.20), compared to other varieties of jute. Contrastingly, the minimum BAF value was shown by HongTieGuXuan in the roots (0.74) and shoots (0.74), compared to other varieties of jute. Similarly, the maximum value of TF was also shown by C-3 (1.01), followed by HongTieGuXuan (1.01) and GuBaChangaJia (0.99).

Correlation between Growth, Biomass, Total Chlorophyll Content, and Cu Uptake
A Pearson's correlation analysis was carried out to quantify the relationship between growth, biomass, total chlorophyll content, and Cu uptake in the roots and shoots of different varieties of jute ( Figure 4). Cu concentration in the roots was positively correlated with Cu concentration in the shoots, and also positively correlated with growth parameters and chlorophyll content. In the same way, Cu concentration in the shoots was also strongly positively correlated with plant height, fresh and dry biomass, and chlorophyll content. This correlation reflected the close connection between Cu uptake and growth in different varieties of jute.

Discussions
In the last few decades, soil concentrations of Cu have surpassed toxic levels (>30 mg kg −1 ) due to overpopulation and industrialization [5,6,15,38]. It has been observed that a reduction in plant growth and biomass in a common response of plants to Cu stress [4,8,39]. For the successful production of jute when cultivated in the Cu-contaminated soil of China, development and selection of tolerant varieties through screening is crucial. For better growth and development of jute for fiber extraction and natural resources, it is necessary to cultivate the most suitable variety of jute [25,26]. However, the resistance or tolerance mechanism of a plant depend upon its specific physiological and biochemical activities [14,40,41]. Therefore, a preliminary experiment was conducted to expose Cu-sensitive varieties and Cu-resistant varieties to toxic concentrations of Cu in the medium.
The germination assay is a basic method to observe the effects of heavy metal stress on plant seedlings. Moreover, seed germination is one of the most important biological parameters in the life cycle of a plant [10,16,42,43]. Seed germination of different varieties of jute exhibited different responses to exposure to Cu stress via the medium (Table 1). It was also noticed that HongTieGuXuan and C-3 showed the maximum germination percentage, while GuBaChangaJia and ShangHuoMa showed the minimum germination percentage, which might be due to high toxicity caused by the Cu in the medium. Previously, it has also been suggested that germination percentage at seedling stage can be affected by the phytotoxicity of Cu, which might be due to the accumulation of carbon partitioning in the tissues of the plant [10,16]. Furthermore, the minimum germination rate in GuBaChangaJia and ShangHuoMa might be due to water deficiency due to excess Cu in the medium inhibiting cell expansion and reducing carbon assimilation [10,44]. These results coincided with Nizam et al. [42], who found that high concentrations of As reduced the germination percentage of some varieties, while others showed a better germination rate compared to the control.
High concentrations of Cu are extremely toxic for growth and biomass of plants. It was noticed that dominant plants collected from Cu mining sites showed more resistance than normal plants [8,39]. In the present study, HongTieGuXuan and C-3 showed better growth in terms of plant height and plant fresh and dry biomass compared to GuBaChangaJia and ShangHuoMa ( Table 1). The poor growth and composition in GuBaChangaJia and ShangHuoMa under high concentrations of Cu in the medium can be attributed to insufficient uptake of nutrients, low availability of water, perturbed root architecture, and poor stomata regulation of plant metabolic processes [45]. Furthermore, nutrient acquisition, stimulation of the defense system (antioxidants), structural integrity of metabolites, and considerable water use efficiency are positively associated with heavy metal stress tolerance in different plant species [10,46,47]. Similar findings were shown by Uddin et al. [48] when they studied different varieties of jute and noticed that different varieties exhibited different responses to exposure to high concentrations of Pb in their soil.
Copper is a micronutrient that provides support to the shaping and function of chloroplasts in plant cells [4], but excess Cu affects chloroplast structure and ultimately reduces the photosynthetic pigment of the plants [49,50]. In the present study, excess Cu in the medium caused a drastic reduction in chlorophyll content in Cu-sensitive varieties ( Table 1). The reduction of chlorophyll content in GuBaChangaJia and ShangHuoMa under toxic concentration of Cu in the medium might be due to the inhibited activities of various enzymes associated with chlorophyll biosynthesis [11,50]. Furthermore, the accumulation of Cu concentrations in the tissues of GuBaChangaJia and ShangHuoMa also caused low chlorophyll content in the leaves of these jute varieties.
Heavy metal stress can alter the equilibrium of reactive oxygen species (ROS) production, which promotes membrane lipid peroxidation and ROS accumulation, and disturbs the function and structure of the cell membrane [20,23,39]. ROS have also been shown to play a role in ABA mediated root growth in Arabidopsis [50,51]. Moreover, the generation of ROS under high concentrations of Cu in the soil is enhanced by cuprous and cupric Cu ions, which induce oxidative damage in plant cells/tissues [9,18,52]. MDA is an oxidized product of membrane lipids and is supported by leakage of plasma membrane and cell turgor loss [39,53]. The production of ROS in plant cells/tissues is very dangerous, and plants have evolved special defense systems such as SOD, POD, CAT, and APX to scavenge ROS [7,8,14]. Plant responses to oxidative stress depend on the plant species and variety [47,54]. It was noticed that Cu-sensitive (GuBaChangaJia and ShangHuoMa) varieties underwent high oxidative stress, as shown by high contents of MDA, H 2 O 2 , and EL ( Figure 1). Similar results were suggested by Khan et al. [47]: that tolerant varieties undergo less oxidative stress when compared to sensitive varieties. Furthermore, it was also observed that to overcome oxidative stress, plants have a special defense system to scavenge ROS generation (Figure 2). GuBaChangaJia and ShangHuoMa showed more antioxidant activity than HongTieGuXuan and C-3, which might be due to more oxidative stress in these species. The difference in antioxidant activities might be due to species-specific biochemical responses, as shown by Akram et al. [45].
Metal accumulation in different parts of a plant depends upon plant species, growth stage, fertilizer application, and growth conditions [4,7]. Based on tolerance mechanisms, plant species can been divided into two types: (1) Metal excluders accumulate heavy metals from the substrate in their roots, but restrict their transport and entry into their aerial parts; (2). Hyperaccumulators are able to accumulate large amounts of metals in their above-ground parts rather than in belowground parts [55]. Furthermore, bioaccumulation factor (BAF) and translocation factor (TF) are important in screening hyperaccumulators for phytoremediation of heavy metals. Screening of hyperaccumulators depends upon BAF and TF (both should be greater than 1) for evaluation and selection of plants for phytoremediation [8,25,[56][57][58]. In many previous studies, jute has been considered as a hyperaccumulator species for different heavy metals; for example, Ahmed and Salima [25] studied jute under different heavy metal exposures and noticed that is a hyperaccumulator for different heavy metals including Cd, Cu, Cr, and Pb. Our results suggested that HongTieGuXuan and C-3 are able to accumulate large amounts of Cu in their above-ground parts and can be considered hyperaccumulator varieties, while GuBaChangaJia and ShangHuoMa are able to accumulate low Cu concentrations in their above-ground parts and can be considered Cu-excluder species. Furthermore, the values of BAF and TF of HongTieGuXuan and C-3 were greater than 1, while the values of BAF and TF for GuBaChangaJia and ShangHuoMa were less than 1. Phytoremediation potential of different varieties of jute has already been studied by Uddin et al. [48] under lead-contaminated soil, and it was found the similar results that Pb-tolerant varieties were good hyperaccumulator species (showed values of BAF and TF greater than 1) and were able to accumulate large amount of Pb in their above-ground parts rather than in the below-ground parts of the plant.

Conclusions
Overall, Cu stress significantly decreased growth and development (seed germination, plant height, fresh and dry biomass, chlorophyll content) in all varieties of jute, but Cu tolerance index was higher in HongTieGuXuan and C-3 than in GuBaChangaJia and ShangHuoMa. However, jute has an active antioxidative defense system to scavenge the ROS produced due to high concentrations of Cu in the medium. The results suggested that HongTieGuXuan and C-3 are Cu-tolerant varieties, while GuBaChangaJia and ShangHuoMa are Cu-sensitive varieties. Furthermore, the accumulation of Cu in different parts (roots and shoots) of the plants also indicated that HongTieGuXuan and C-3 can be considered Cu hyperaccumulator species, while GuBaChangaJia and ShangHuoMa can be considered Cu excluder species. However, future research is needed to study the effects of Cu stress on different varieties of jute for fiber extract in field areas.