Introduction

Nowadays, pollutant release has been expanded to environments entirely due to industry, urbanization, or uses related to excess agriculture modifications (Hasan et al. 2017; Wang et al. 2017). Heavy metal (HM) toxicity not only reduces plant growth but also degrades food quality. Although some heavy metals (HMs) are essential for plants, others are not. Essential heavy metals are needed by small quantities for plant activity, while high concentrations are toxic (Wuana and Okieimen 2011).

Copper (Cu) is somewhat toxic at higher levels than basic levels and causes an imbalance in plants (Gajewska and SkŁodowska 2010; Habiba et al. 2014). Consequently, excess Cu (ECu) has the possibility to disrupt cellular redox balance, ion transport, signal transmission and distort membrane integrity and permeability, enzyme activity, biosynthesis of photosynthesis machinery, and respiratory function, thus causing plant growth inhibition (Souza et al. 2014; Xu et al. 2015; Giannakoula et al. 2021).

Phytoremediation is a method in which plant is used to eliminate environmental pollutants (Anderson and Coats 1994) and considered a solution for environmental cleaning. Plants with metallic accumulation can extract, transfer, and concentrate minerals from the soil to their roots and transport them above the ground. However, the minerals’ extraction efficiency depends on the mineral concentrations in the harvested part and the plant biomass (Huang et al. 2018). There are many Cu-hyperaccumulator such as Elsholtzia splendens and Commelina communis, whereas they are ineffective for large-scale processing because of poor mass and slow growth. Maize (C4) has high biomass productivity and is an important crop in the universe (FAO, 2011). Moreover, the ability of maize to uptake high concentrations of heavy metals, such as Cd, Cr, Fe, Mn, Pb, and Zn from soils, has been observed (Aliyu and Adamu 2014). The ability to extract cadmium, copper, lead and zinc from the sewage sludge was also demonstrated (Xu et al. 2015). However, the exact mechanism for extracting Cu from soil or water contaminated with minerals is unknown in maize. The plant has several tolerance systems to maintain the suitable level of minerals such as Cu in the different active parts to avoid toxicity linked with the metals (Yruela 2005; 2009; Shahid et al. 2014). Kang et al. (2015) notified that copper is mainly associated with polar acidic compounds with the carboxyl and hydroxyl groups in Ricinus communis root cell walls. Antioxidant-related activity is also increased, which is the defense mechanism within plants against Cu toxicity (Adrees et al. 2015). Moreover, the accumulation of secondary metabolites may show resistance to Cu toxicity (Morales et al., 2012). However, there is no study to evaluate the effect of ECu on macromolecules in maize plants.

Therefore, the primary goal of this investigation was to explore the potency of maize to clean Cu from Cu-contaminated environments. This research addressed the growth rate and some physiological responses. Fourier infrared spectroscopy (FT-IR) was also used to detect changes in primary and secondary metabolites in shoots and roots.

Materials and methods

Plant growth

Maize (Zea mays L.) cultivar 321 is used in Egypt; caryopses were surface-sterilized in 5% NaClO solution for 10 min and rinsed with sterile distilled water (SDW) and then were cultivated in sterile-moistened sand for 3 days. After 3 days, three plants were transferred into a polyethylene container containing 1L of tap water, to avoid the shock of the high concentration of nutrients, and placed in the greenhouse of the Faculty of Science, Assiut University (27°11′00″N 31°10′00″E) with day/night temperature of 35–37/18–21 °C, relative humidity of 25–27%, and daily photon flux density about 673.98 µmol m−2 S−1. After 4 days of cultivation, water was changed by 1/4 N of Hoagland and Arnon (1950) solution, and after 2-d nutrient was increased to 1/2 N. After 8-d of growth in hydroponics, three levels of ECu, as CuSO4·5H2O (0, 5, and 10 μM), were added to the nutrient solution, and then, pH was adjusted to 5.7. Each container contained three plants, and each treatment was four duplicate containers. After 48 h, the seedlings, 13-day-old, were harvested for analysis. Leaves and roots were frozen in liquid nitrogen and stored at − 80 °C; other shoots and roots were dried at 70 °C to determine the dry weight (DW).

Photosynthetic pigments

Chlorophyll (Chl. a & b) and carotenoid (Carot.) concentrations were determined in ethanol (96%; v/v) extracts of the fresh leaf by the spectrophotometric method (Lichtenthaler 1987). The absorbance of chl. a + b and carot. was estimated at 665, 649, and 470 nm and calculated as mg g−1 fresh weight (FW).

Lipid peroxidation

Malondialdehyde concentration (MDA) was measured (Madhava Rao and Stresty 2000). The samples were extracted in trichloroacetic acid (TCA) and filtrated; then, TCA and thiobarbituric acid reagents were added. MDA concentration was calculated from the extinction coefficient of 155 mM cm−1 and expressed as µmol g−1 FW.

Proline and free amino acids

Proline was quantified using the technique of Bates et al. (1973). The sample was extracted in sulfosalicylic acid and filtrated; then, the acid-ninhydrin reagent was added. The chromophore of toluene was monitored at 520 nm. Proline content was measured from a standard curve of proline and calculated as mg g−1 FW.

Free amino acids (FAAs) were quantified (Moore and Stein 1948). The sample was extracted in potassium phosphate buffer (PPB, 100 mM; pH 7.8) and filtrated; then, the SnCl2 reagent was added. The absorbance was monitored at 570 nm. FAAs’ content was measured from a standard curve of glycine and calculated as mg g−1 FW.

Total soluble proteins (TSPs)

TSPs were quantified (Lowry et al. 1951) in samples extracted by PPB as described previously. The absorbance was monitored at 750 nm. TSPs’ content was measured from a standard curve of bovine serum albumin and calculated as mg g−1 FW.

Total soluble sugars (TSS)

TSS was measured (Fales 1951; Schlegel 1956) in samples extracted by PPB as described previously. The absorbance was monitored at 620 nm. TSS content was measured from a standard curve of glucose and calculated as mg g−1 FW.

Phenolic compounds

Free and bound phenolics were measured (Kofalvi and Nassuth 1995). Free phenolics were extracted in methanol (50%) and centrifuged, and then, the remaining pellets were saponified with NaOH (0.5 N) to quantify bound phenolics. The absorbance was monitored at 725 nm. Phenolic content was measured from a standard curve of gallic acid and calculated as µg g−1 FW.

Antioxidant enzymes

Frozen leaves (0.5 g) were homogenized in 5 mL PPB buffer (100 mM; pH 7.8) including ethylenediaminetetraacetic acid (EDTA; 0.1 mM) and polyvinylpyrrolidone (0.1 g). The supernatant was used to test the enzyme activity after centrifugation (4 °C, 18,000 rpm, 10 min). The activity was calculated as differences in the absorption wavelength (DA) unit protein−1 min−1 (Farghaly et al. 2020).

Ascorbate peroxidase (EC 1.11.1.11).

APX activity was checked (Nakano and Asada 1981). The assay medium consisted of enzyme aliquots (50 μL), PPB (0.05 M; pH 7), EDTA (0.0001 M), H2O2 (0.0012 M), and ascorbate (AsA; 0.0005 M); changes in absorbance were evaluated at 290 nm.

Guaiacol peroxidase (EC 1.11.1.7)

POD activity was checked (Zaharieva et al. 1999). The assay medium consisted of enzyme aliquots (100 μL), 30 mM PPB (0.05 M; pH 7), 6.5 mM H2O2, and guaiacol (1.5 mM), and changes in absorbance were measured at 470 nm.

Catalase (EC 1.11.1.6)

CAT activity was checked (Aebi 1984). The assay medium consisted of enzyme aliquots (20 μL), PPB (0.05 M; pH 7), and H2O2 (10 mM), and changes in absorbance were measured at 240 nm.

Copper and potassium content

The dry samples were digested with perchloric acid (60%), concentrated nitric acid, and sulfuric acid in a 1: 3: 1 (V: V) ratio (Farghaly et al. 2020). Potassium and copper were examined in digested samples using an atomic absorption spectrometer (PAC 210 Vgp model, USA) and calculated as mg g−1 DW. Translocation factor (translocation; TF), uptake, and bioaccumulation factor (BF) were measured to determine the degree of element accumulation in plants and were calculated using equations (Ekvall and Greger 2003; Usman et al. 2019):

Element uptake = Element taken up in whole plant/DW root.

Translocation factor % (TF%) = Concentration of element in plant shoot/Concentration of element in plant root ×100.

Bioaccumulation factor shoot (BF) = Concentration of element in shoot/Concentration of element in the medium.

Bioaccumulation factor root (BF) = Concentration of element in root/Concentration of element in the medium.

Fourier transform infrared (FT-IR) analysis

Changes in macromolecules were studied using FT-IR (Nicolet IS 10 FT-IR) spectroscopy. Samples from the dried plant fractions were ground into a fine powder (~ 100 μg) and converted into pellets with KBr by pressing. Special care was taken for preparing pellets of identical thickness using the same sample amount and pressure. Waves ranging from 4000 to 400 cm−1 infrared transmittance data were collected. All samples were analyzed with blank KBr pellets. Spectral data were compared with a reference to determine the functional groups within the sample.

Statistical analysis

The values obtained (± standard deviation) were averages of four biological replicates of three technical measurements. One-way analysis of variance (ANOVA) was performed and followed by Tukey’s test for multiple comparisons (P ≤ 0.05). Pearson correlation was performed to obtain the relation between the mean rates of different parameters under CuSO4 treatments. Asterisks indicate a significant relationship (* and ** at 5 and 1%, respectively).

Results

Growth and pigments

Seedlings were treated with various levels of ECu for 2 days to investigate the excess Cu influence on growth (Fig. 1A). The successive increase in Cu concentration in the nutrient medium inhibited the RDW of the seedlings. The decrease in RDW of seedlings occurred at the highest CuSO4. Compared to controls, the highest reduction in RDW was 14.68% and 27.85% for shoots and roots, respectively, for plants exposed to 10 μM CuSO4. Seedlings treated with the low ECu level (5 μM) showed a lower reduction in RDW compared to controls, where decreases for shoots and roots were 6.33% and 25.98%, respectively. The results manifested that the negative effect of ECu on shoots growth was less than roots. Moreover, our data revealed that shoot RDW (− 0.79*) and root RDW (− 0.87*) displayed a strong negative association with Cu contents.

Fig. 1
figure 1

Relative dry weight of shoots and roots (A), photosynthetic pigments (B), and MDA content (C) in leaves of 13-day-old maize plants exposed for 48 h to different concentrations of CuSO4. The data are means ± SD (n = 4). Different letters, capital for shoots; chl. a & b and small for roots; carot, indicate statistically significant differences according to one-way ANOVA; Tukey HSD- post hoc

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Chlorophyll a & b and carot. (being an antioxidant) are the primary types of pigments, and differences in their content reflect the degree of photosynthesis (Fig. 1B). The concentrations of CuSO4 used had inhibitory effects on the photosynthetic pigments of seedlings. Under ECu, a significant decrease was observed in concentrations of chlorophyll a & b and compared to the control was 15.51% and 31.12%, respectively. The data in Fig. 1B showed 5 μM of ECu did not significantly reduce carotenoid concentration, while 10 μM of ECu reduced its concentration by 29.07% over the control. In addition, chlorophyll a & b (− 0.78*) and carot. (− 0.79*) had a strong negative association with the Cu content in shoots.

Membrane integration

To verify whether ECu treatments affected the lipid oxidation induction of plant membranes, we assessed lipid oxidation as the MDA content in leaves (Fig. 1C). Treatments with ECu increased MDA content by 17.28% and 52.30%, respectively, over control. Moreover, the data revealed that MDA content was not significantly correlated to Cu content in shoots.

Some metabolic compounds

Free amino acids

To study whether seedling treatments with ECu induced effects on FAAs, which affect many physiological and support processes in resistance to stress, we evaluated FAAs in roots and leaves. Based on the results elucidated in Fig. 2A, it can be revealed that ECu had a significant catalytic effect on FAAs accumulation within the leaf. However, CuSO4 levels failed to induce any significant modification in FAAs accumulation of roots. Additionally, FAAs accumulation was significantly related to Cu concentration (0.80**) in leaves; however, this relation was not significant in roots.

Fig. 2
figure 2

Proline (A), other free amino acids (B), soluble proteins (C), and soluble sugar (D) of leaves and roots, phenolic compounds (E), and antioxidant enzyme [ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT)] (F) in leaves of 13-day-old maize plants exposed for 48 h to different concentrations of CuSO4. The data are means ± SD (n = 4). Different letters, capital for leaves, free phenolics and small for roots, bound phenolics indicate statistically significant differences according to one-way ANOVA; Tukey HSD- post hoc

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Soluble proteins and sugars

TSPs and TSS increase in response to (a)biotic stresses; therefore, it was interesting to examine the influence of ECu treatments on TSPs and TSS (Fig. 2B and C). ECu applications nonsignificantly stimulated TSPs’ content in leaves and roots; only ECu at the high level stimulated their content in leaves (76.04%). Moreover, in leaves treated with ECu, the relationship between TSPs and Cu content in leaves was significant (0.732*), while this correlation was insignificant in roots.

Similar to TSPs, ECu induced insignificant changes in TSS accumulation in leaves and roots; only roots recorded an increase (152.56%) at 10 μM. In contrast, in leaves treated with ECu, the relation between TSS and Cu content was not significant, while this relationship was significant in roots (0.784*).

Proline

It is known that proline accumulation is an adaptation to (a)biotic stress and plays a significant role in its management, so we studied the effect of ECu on proline synthesis (Fig. 2D). Increasing Cu levels within nutrient medium significantly increased proline content within leaves and roots of seedlings. The highest level of CuSO4 showed an increase in proline concentration of 51.30% and 39.75% in leaves and roots, respectively, compared to the controls. In addition, our data appeared that the relation between proline and Cu within leaves was insignificant, while this relationship was significant in roots (0.745*).

Phenolic compounds

Phenolic compounds, which show significant roles in plant defense, were tested in leaves exposed to ECu levels (Fig. 2E). Increasing CuSO4 concentrations significantly increased free and bound phenolic content within leaves. Compared with control plants, the highest increase in free (46.09%) and bound (46.05%) phenolic was observed after subjecting seedlings to the high ECu level. Furthermore, free phenolic displayed a significant association with Cu content within shoots (0.83**), while the relation between bound phenolic and Cu content was nonsignificant.

Antioxidant enzymes

Under different levels of ECu, the activity of some antioxidant enzymes was estimated (Fig. 2F). CAT activity increased significantly with increasing levels of CuSO4 in the nutrient medium, compared to controls elevation was 76.02% and 145.50% in treatments 5 and 10 μM, respectively. CAT activity was also considerably correlated with Cu content in seedling shoots (0.76*).

Concerning APX activity, compared to controls, CuSO4 levels (5 and 10 μM) significantly reduced its activity by 50.18% and 80.46%, respectively. In addition, APX activity showed a negative association with the copper content in shoots (− 0.86**).

For POD, low ECu level slightly reduced its activity, while high level (10 µM) significantly reduced its activity. Moreover, POD activity had a strong negative correlation with the Cu content in shoots (− 0.74*).

Potassium accumulation

K+ was estimated during this research because it plays many vital regulatory functions in plant growth (Fig. 3A). Treatments of the tested plant with 5 and 10 µM CuSO4 considerably increased potassium content in shoots by 41.57% and 92.13%, respectively, over the controls. However, CuSO4 treatments did not considerably catalyze K+ accumulation in the roots. As expected, K+ content in shoots showed a strong positive association with Cu content in shoots (0.85**), while this association was weak in the roots.

Fig. 3
figure 3

Concentration of Cu (A) and K (C) of roots and shoots, roots Cu uptake and translocation (B) and roots K uptake and translocation (D) of 13-day-old maize plants exposed for 48 h to different concentrations of CuSO4. The data are means ± SD (n = 4). Different letters, capital for shoots, Cu and K translocation and small for roots, Cu and K uptake, indicate statistically significant differences according to one-way ANOVA; Tukey HSD- post hoc

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Copper accumulation, translocation, and uptake

Cu uptake, translocation, and accumulation in maize tissues were estimated under ECu to find out the ability of maize to accumulate it. The Cu absorption was relied on the Cu level within the medium, and a gradual increase in Cu treatments increased copper accumulation in plant organs (Fig. 3B). Under ECu (5 and 10 µM), roots had higher Cu concentrations (65.62%, 87.79%, respectively) than shoots (338.49%, 555.15%, respectively). These results were confirmed by a low Cu translocation from roots to shoots (54.22%, 63.87%, at 5 and 10 μM CuSO4, respectively), while root uptake was 11.11-fold in 10 μM CuSO4 compared to control (Fig. 3C).

The BF is an important parameter used for studying the feasibility of phytoremediation potential (Usman et al. 2019). In controls (without ECu conditions), roots (1.80 g kg−1) had a higher BF than shoots (0.72 g kg−1) (Fig. 3D). The results also showed that increasing CuSO4 levels in the nutrient solution reduced the BF in shoots and roots. Compared with Cu-unstressed seedlings, CuSO4 (5–10 µM) treatments reduced the BF by 97.55%, 98.60% in shoots, and 93.52%, 95.12% in roots, respectively.

FT-IR analysis

FT-IR was applied to distinguish the alterations in macromolecules of maize organs under different ECu levels (Figs. 4, 5, and 6). In shoots, ECu treatments (5 and 10 µM) showed a decrease in the transmittance of the peaks at 1656.70 and 1250.12 cm−1 by 13.34, 5.77, 3.13, and 6.1 cm−1, respectively, compared to the controls (Fig. 4E, 5E). However, low ECu level elevated the transmittance of 1053.95 cm−1 peaks by 7.48 cm−1 compared to control (Fig. 6C). Moreover, CuSO4 treatments significantly failed to shift the transmittance of other peaks (3388.5, 2918.76, 2850.15, 1514.73, 1377.71, 1159.89, 608.7 cm−1). Interestingly, the low ECu level decreased the intensity of all bands, while the high level induced different effects compared to the control. Regarding roots, the high ECu level (10 μM) increased broadband transmittance as indicated by 12.36 cm−1 compared to the control (3395.82 cm−1) (Fig. 4B). In comparison with control, the highest shift in transmittance of bands was 52.67 and 53.61 cm−1, respectively, in the small peak 557.21 cm−1, when plants were exposed to 5 and 10 µM ECu stress (Fig. 6F). Moreover, there was no apparent peak shift in other FT-IR spectra (2921.23, 1651.67, 1515.65, 1378.01, 1250.93, 1159.23, 1050.96 cm−1) for maize roots treated with ECu. Compared to control, the intensity of all peaks was increased under the low ECu treatment, except the band at 609.88 cm−1 decreased. The high ECu level increased the intensity of 3408.18, 2923.24, 1646.87, 1159.32, and 1050.49 cm−1 bands while decreasing the 1515.85 cm−1 intensity.

Fig. 4
figure 4

FT-IR spectra of shoots and roots of 13-days-old maize plants exposed for 48 h to 0, 5 and 10 μM CuSO4. Spectral region associated with carbohydrates, proteins, alcohols, phenolic compounds (A, B), fatty acids (C, D) and amide I (E, F)

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Fig. 5
figure 5

FT-IR spectra of shoots and roots of 13-day-old maize plants exposed for 48 h to 0, 5 and 10 μM CuSO4. Spectral region associated with lignin (A, B), cell wall polysaccharides, lipids and proteins (C, D) and pectic substances (E, F)

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Fig. 6
figure 6

FT-IR spectra of shoots and roots of 13-day-old maize plants exposed for 48 h to 0, 5 and 10 μM CuSO4. Spectral region associated with cell wall polysaccharides (A, B), cellulose and hemicellulose (C, D) and C-alkyl chloride (E, F)

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Discussion

The various applications of copper raise concerns about its potential actions on human health and thus ecosystems. Therefore, the ability of maize plants to remediate the ECu toxicity was estimated in this study. Bioaccumulation mass is the main variable indicating the enhanced remediation capacity of plants growing in contaminated soils (Yu et al. 2015). In this investigation, maize growth decreased with exposure of seedlings to ECu, and this reduction did not reach 15% in shoots and 28% in roots at the high level, which indicates the ability of this plant to withstand ECu. Root growth was less than shoot because the accumulation of copper in roots (6.6-fold) was more than shoots (1.9-fold). This finding is in line with the previous study, which indicates that Cu tends to accumulate within the root (Shiyab 2018). The decrease in plant growth due to HM toxicity is associated with oxidative stress causing physiological changes (Liu et al. 2014; Abdel-Wahab et al. 2019).

Inhibition of maize growth due to ECu could be associated with a reduction within the synthesis of photosynthetic pigments. In this investigation, ECu reduced chlorophyll synthesis due to membrane oxidation, and the evidence for this is MDA accumulation. These results are consistent with Chen et al. (2015) who reported that ECu leads to suppression of synthesis of essential enzymes involved in chlorophyll synthesis, disruption of thylakoid fatty acid formation, and membrane integrity. Carotenoids protect the chlorophyll from oxidative damage by quenching reactive oxygen species (ROS) (Behera et al. 2002). Our results revealed that the high ECu level inhibited the synthesis of carotenoids, which indicates stimulated ROS generation. This finding is consistent with the previous study (Choudhary et al. 2012).

The main target related to the metal injury is cell membranes, and thus, membrane injury is a marker of lipid peroxidation (Wang et al. 2008). Our data indicated that MDA content, a product of lipid peroxide, was significantly increased under the high ECu level, indicating a decrease in the effectiveness of its antioxidant systems at this level. Various studies notified that MDA output significantly increased in response to ECu exposure and that the cell membrane was the first target of copper toxicity (Chen et al. 2015; Huang et al. 2018; Abdel-Wahab et al. 2019). This result indicates that ECu treatments may lead to significant damage to the structure and performance of cell membranes. In plants, cytoplasmic chelation is a known intracellular mechanism for maintaining low concentrations and detoxifying free HMs (Anjum et al. 2015). In the current study, stimulation of FAAs synthesis and their positive association with Cu content in ECu-stressed plants indicated an improved leaf growth. Our results are consistent with Manara (2012) who concluded that FAAs and their derivatives may confer resistance to plants through the possibility of binding HMs. This result could indicate that seedling tolerance was related to FAA fixation in roots. However, under ECu toxicity, FAA functions are not yet clear and require additional investigations.

In plants, protein synthesis can be a mechanism affected by HMs. In this investigation, at the high ECu level, increased TSPs in the leaves could increase the Cu sequestration to diminish the toxicity. It may also result from the output of metallothioneins and phytochelatins to detoxify Cu ions. The vacuole plays a major role in Cu tolerance, and it was the most accumulating organelle (Chen et al. 2015). Additionally, the increase in proteins may be similar to stress proteins that can include various antioxidant enzymes (Lamhamdi et al. 2010). In roots, insignificant changes in TSPs were agreed with Mocquot et al. (1996). Soluble sugars are known to act as signaling molecules against reactive oxygen species, and they are a source of energy (Gautam et al. 2019). Within this study, ECu modified TSS content and distribution in maize plants. In leaves, insignificant changes in TSS suggested that a possible cause of plant resistance to ECu stress may be related to unchanged TSS. Also, Contreras et al. (2018) found that exposure to ECu did not cause differences in total reducing sugars in Colobanthus quitensis plants compared to controls. In roots, under the high ECu level, the enhancement of TSS accumulation could be related to their role in stress management and osmolyte needed for growth. Previous reports have notified that sugar acts as a nutrient, retains cells from mineral toxicity (Li et al. 2013), and acts as signal molecules (Wu et al. 2013).

Proline accumulation acts as an osmoprotectant and anti-oxidant to save the plant from various abiotic stressors (Dar et al. 2016). Our results showed an induction of proline content in maize organs by raising copper levels in the nutrient medium. This increase indicates that proline could be beneficial to counteract ECu toxicity. In this context, De Knecht et al. (1994) stated that proline accumulation under HM stress causes the synthesis of phytochelatin that chelates HMs. Cao et al. (2018) manifested that increased proline within tolerant willow species may increase the osmotic ability of the cell and maintain normal intercellular metabolism, which improves the Cu tolerance of plants. Additionally, Dar et al. (2016) established that proline plays a defensive role in mitochondria.

Secondary metabolites with phenolic groups are essential for stress management, as they act as powerful non-enzymatic antioxidants within the cell. Our data disclosed that ECu stimulated the phenolic synthesis in leaves, and these metabolites could be significant in protecting against oxidative. The strong negative relationship between free phenolic and Cu in shoots confirms previous reports of Jung et al. (2003) who reported that phenolic groups help bind HMs such as Cu. Zhao et al. (2016) also added that phenolics can increase antioxidant activity.

ECu toxicity stimulates the production of hydroxyl radicals that cause alterations in antioxidant pathways (DalCorso et al. 2013; Giannakoula et al. 2021). CAT, POD, and APX are important enzymes in breaking down H2O2. Antioxidant enzymes revealed that ECu treatments stimulated CAT activity while decreasing POD and APX activity. These finds suggested that the CAT enzyme was associated with the response of plants to ECu stress, and the role of POD and APX in eliminating H2O2 can be overcome by increased CAT activity. The strong negative relation between shoots Cu, POD, and APX activity, and the strong positive correlation of CAT confirmed the possibility of overcoming APX and POD roles in H2O2 elimination by boosting CAT activity. Likewise, ECu toxicity stimulated CAT activity in annual capsicum (Islek and Unal 2015). Also, the adverse effects of ECu toxicity on POD and APX activity were reported in Moso bamboo and Arabidopsis thaliana (Drążkiewicz et al. 2003; Chen et al. 2015).

Potassium plays a protective function against (a)biotic stresses, where it reduces the activity of nicotinamide adenine dinucleotide phosphate oxidase, maintains photoelectron transport, and thus reduces ROS synthesis (Cakmak 2005). In this study, potassium accumulation in shoots increased significantly under different concentrations of CuSO4, whereas there was an insignificant change in roots. Strong and weak relations between K+ and Cu contents within shoots and roots, respectively, showed that maintaining elevated K+ levels in shoots may also be necessary to reduce ROS and enhance plant resistance against ECu stress. In shoots, potassium can accumulate in guard cells, promote photosynthesis, and thus increase plant growth (Wang et al. 2014). In this context, Laohavisit et al. (2012) showed that OH− induces K+-permeability behavior in Arabidopsis. Nonetheless, Zhao et al. (2016) reported that the elevated Cu level may have synthesized the OH− that induced K+ imbalance.

Cu is a necessary micronutrient for plants. The toxicity limit of copper varies widely between plant species and affects tissues that depend differently on metabolic needs (Burkhead et al. 2009). In our study, the ECu treatments caused a considerable increase in Cu content in plant organs and mainly accumulated within roots (about 90%) compared to shoots confirmed by the decrease of TF, which could confirm the tolerance of maize to ECu toxicity. Consistent with our findings, Fernandez and Henriquez (1991) suggested that Cu-tolerant plants might prevent copper from reaching the aerial organs by keeping it in their roots.

FT-IR analysis was conducted on plant organs treated with ECu to determine changes within the biochemical composition. The peak area (3388.5 cm−1) assigned to OH and NH for carbohydrates, proteins, alcohols, and phenolics (Türker-Kaya and Huck 2017) was somewhat unchanged in shoots under ECu treatments, whereas the change was high in roots at the high Cu level. The change in the root peak indicated an alteration in carbohydrates, proteins, alcohols, and phenolics for chelating ECu. Further, increased peak intensity could be attributed to the additional OH groups to chelate ECu. These results are consistent with increased TSS, TPs, and phenolics.

Fatty acids appear distinct transmittance in regions of wavenumbers 2950–2845 cm−1 (Gupta et al. 2015). Obtained data showed that ECu treatments only affected the intensity of fatty acid peaks, indicating that ECu may alter the fatty acid composition. Abdel-Wahab et al. (2019) found no changes in this peak under CuO nanoparticles, suggesting no chemical change in lipids. Moreover, the 2850.15 cm−1 peak was also intended for lipids (Dokken and Davis 2007) and did not change in shoots under ECu treatments, while it disappeared at roots. The disappearance of the absorption peak was associated with an increase in the previous peak (2921.23 cm−1); thus, the amplitude of the lipid peak indicates changes in root membranes under ECu. The variation in the wavenumbers of peaks and intensity of these peaks may indicate an increase in the number of gauche conformations leading to less rigid membranes (Türker-Kaya et al. 2016).

The peaks around 1656.70–1651.67 cm−1 are attributed to the C=O stretching, mainly associated with the -NH deformation mode and may be attributed to the amide I peak (α-helix structure) (Hlihor et al. 2013). Excess copper treatments reduced the transmittance of these peaks, which may provide information about changes in protein structure in maize organs to chelate copper, and this suggestion may confirm Abdel-Wahab et al. (2019).

Under ECu treatments, peaks, located around 1514.73, 1377.71–1378.01, and 1159.89–1159.23 cm−1, which were described as lignin, cell wall polysaccharides, lipids, and proteins (Türker-Kaya and Huck 2017), did not register significant changes, and only the intensity changes; this could link with binding of Cu with cell wall sugars. Otherwise, decrease the transmittance of peaks at 1250.12–1250.93 cm−1 (pectic substances peaks; Zuverza-Mena et al. 2016) of ECu-stressed plants that may be attributed to cell wall changes. Also, ECu did not change peaks (1050.96 cm−1), which were assigned for cellulose and hemicellulose (Rico et al. 2015) in roots, while changed them in shoots. In addition, the high peak intensity under ECu may associate with the participation of cell wall groups in Cu chelation. Under ECu, metamorphism within weak peaks at 557.21 cm−1 (C-alkyl chloride; Yedurkar et al. 2016) at roots (9.62%) may contribute to copper chelation. Increased peak intensity at most root peaks under ECu could indicate further bond formation for Cu chelation. This result indicated that the high peak intensity shows a significant role in linking with excess Cu and reduces the transfer of copper to shoots. In line with the published literature, improved peak intensity may indicate the interaction of the amino, carboxyl, hydroxyl, thiol, and phosphate groups with HMs (Abdel-Wahab et al. 2019).

In the present research, we attempted to study the process of phytoaccumulation of copper-containing water using maize cultivar 231. Increased Cu levels in the nutrients led to increased accumulation in maize tissues and most of the copper in roots and not in shoots. Also, copper content in roots exceeded the criterion 220 g kg−1 DW, and growth decreased by about 28% at the high ECu level. Increased CAT activity, amino acids, soluble proteins, soluble sugars, phenolics, and K could be a sign of reduced oxidative stress for Ecu. Moreover, ECu increased the C-alkyl chloride peak and increased the intensity of macromolecule peaks at roots, which may contribute to the chelation of Cu and prevent its transfer to shoots. These results give new insight into removing copper from aqueous environments via maize roots (rhizofiltration).