Integrative analysis of maize and sunflower responses to copper stress reveals species-specific phytoremediation strategies

Abstract

Background

Copper (Cu) contamination of arable land threatens crop yields, productivity, and environmental sustainability. While EDTA and IAA can enhance phytoremediation, understanding how different plant species exploit these amendments remains limited. This study tested the hypothesis that maize (Zea mays) and sunflower (Helianthus annuus) employ distinct physiological and Cu-accumulation strategies under Cu-induced stress.

Methods

In a controlled pot experiment with 25, 50, and 75 mg kg⁻¹ Cu, applied alone or in combination with soil-applied EDTA (5 mmol kg⁻¹) or with both EDTA and foliar-applied IAA (5 µmol L⁻¹), growth, physiology, antioxidants, and Cu partitioning/distribution of the selected plants were analyzed.

Results

Significant inhibition occurred in growth, biomass (40% in sunflower; 25% in maize), and photosynthetic pigments (30–35%), along with elevated oxidative stress responses in terms of enzymatic (CAT, APX) and non-enzymatic (flavonoids, phenolics, proline). EDTA alleviated stress by 15–20%, improving growth and pigment retention, while EDTA plus IAA enhanced biomass and pigments by 35–45%, with maize showing the most effective recovery. In remediation, two distinct strategies were identified: maize primarily sequestered Cu in roots (up to 2-fold higher than shoots), reflecting traits suited for phytostabilization, while sunflower efficiently translocated Cu to shoots (translocation factor ~ 3.4), consistent with phytoextraction. The combined application of EDTA and IAA enhanced these inherent strategies. Maize absorbed Cu rapidly at early stages, largely retaining it in roots, whereas sunflower exhibited sustained shoot translocation, highlighting long-term phytoextraction potential.

Conclusions

These findings move beyond simply reporting amendment efficacy, providing a mechanistic, species-specific framework for selecting plants based on remediation goals, rapid stabilization (maize) versus sustained extraction (sunflower), advancing phytoremediation from trial-and-error approaches toward rational, targeted design.

Introduction

The problem of heavy metal contamination of agricultural soils has become a pressing environmental issue worldwide due to intensive industrialization, mining, and the haphazard application of agrochemicals [1, 2]. Chemically, copper is a soft, malleable metal that exists in the + 2-oxidation state (i.e., Cu^{+ 2}, cupric ion) in environmental systems. It has a high affinity for soil organic matter, and while it is relatively immobile and less prone to leaching, this characteristic leads to its persistent accumulation in the topsoil over time [3]. This continuous buildup in agricultural soil increases phytotoxicity and poses a significant threat to food safety [4]. As a micronutrient to plants, it poses toxicity beyond threshold levels, leading to chlorosis, retarded growth, oxidative stress, and interference with essential physiological functions, including photosynthesis, respiration, and enzymatic activity [5, 6]. When soils are exposed to copper over a long period, they may not only lower crop yield but also bring serious health problems to humans and animals due to bioaccumulation in the food chain. Hence, it is essential to fight copper contamination to recover soil health, guarantee the food safety of the soil, and agricultural productivity [2, 7, 8].

The use of plants to extract, immobilize, or detoxify contaminants present in soils has emerged as an environmentally friendly and economical alternative to traditional remediation efforts [9]. Some of the high-biomass crops, such as sunflower and maize, show significant potential in heavy metal remediation, as they grow fast, have extensive root systems, and can survive under metal stress [10, 11]. However, their utility for copper remediation is not equivalent and remains poorly understood in a comparative context. Such crops can store the metals in their roots and shoots, hence decreasing the amount of metal in soils over time [8]. Critically, the strategy for this storage, whether a plant preferentially sequesters metals in roots (Phyto-stabilization) or translocates them to shoots (Phytoextraction), is species-dependent and dictates its practical application [12].

Nevertheless, their inherent ability to take up copper may be constrained by low metal bioavailability and toxicity, which can hinder large-scale remediation [7, 13, 14]. A key knowledge gap is the lack of a parallel analysis linking the physiological basis of Cu tolerance to actual metal partitioning behavior, which would allow strategic crop selection based on remediation goals [15]. Chelating agents such as EDTA form stable, water-soluble complexes with Cu²⁺ ions, increasing copper solubility and phytoavailability [16, 17]. At the same time, plant hormones like indole-3-acetic acid (IAA) enhance the elongation of roots, uptake of nutrients, and general growth of the plant, and alleviate the stress impacts of the heavy metals [18, 19]. The integration of the EDTA and IAA is also a good but relatively under-investigated strategy, as this can enhance the effectiveness of copper accumulation and growth of the plants in the contaminated environment [20]. However, some studies reveal that while co-application has been explored, studies often lack the factorial design necessary to distinguish combined effects from additive ones [21].

Subsequently, they often treat “phytoremediation efficiency” as a singular endpoint without disentangling the underlying species-specific physiological strategies that determine practical outcomes. This complementary approach may accelerate remediation and increase biomass output. Identifying plant species capable of accumulating higher amounts of copper over a short period is particularly important for practical applications such as phytoremediation and phytomining, where economically valuable metals can be recovered from contaminated sites [22]. A systematic comparison of sunflower and maize under the EDTA and IAA conditions can be considered as beneficial information on the species-specific metal uptake processes, tolerance reactions, and plant growth dynamics that may present new contributions to the environmental biotechnology field [23–25].

To move beyond generic comparison, this study tested species-specific Cu remediation strategies in maize and sunflower under identical stress. We hypothesized that maize favors root Cu sequestration (phytostabilization) and sunflower favors shoot translocation (phytoextraction), modulated by EDTA and IAA. Accordingly, both species were evaluated for Cu accumulation, growth, and physiology under Cu stress with EDTA and IAA. This provides a functional basis for selecting crop-amendment combinations for targeted remediation (phytostabilization, phytoextraction, or phytomining).

Methodology

Experimental design and metal treatments

To conduct the study on the growth and phytoremediation capacity of sunflower and Maize under heavy metal stress, a sandy loam soil mixture (60% sand, 30% silt, 10% clay) was prepared [26]. Five kilograms of this mixture were placed into 10-L clay pots and set in a screened house [27]. The soil copper concentration in the area was previously recorded as 12.2 ± 0.021 mg kg⁻¹ [28], with soil organic matter of approximately 8.7 g kg⁻¹ and a pH of about 7.74 ± 0.2 [29]. Healthy and viable sunflower (HYSUN-33) and maize (HYBRID P4040) seeds were obtained from the Agricultural Research Center (ARC) in Mardan, Pakistan. No specific permissions were required for the use of these commercial cultivars, and the study complied with institutional and national guidelines. The seeds were surface sterilized in 70% ethanol and triple rinsed in sterilized distilled water to make them pure [30]. The experiment evaluated the effects of copper at 0, 25, 50, and 75 mg/kg, applied individually and in combination with EDTA and IAA, using three plants per pot, with three biological replicates per treatment. EDTA was applied as a soil drench at 5 mmol kg⁻¹ soil at the same time as the copper treatment. IAA (5 µmol L⁻¹) was prepared by dissolving in a minimal volume of 1 M NaOH, diluted with distilled water, and the pH was adjusted to 6.5-7.0. IAA was applied as a foliar spray at one-week intervals after sowing. To maintain ideal circumstances, pots were irrigated twice a day with tap water [31]. At 60 days following the exposure to the metals, plants were gathered, and biochemical and agronomic assays assessed the physiological responses, metal accumulation, and phytoremediation capacity [32].

Morphological characteristics

Root and shoot lengths were measured before drying. To determine the dry weight of plants subjected to various metal stresses, the root and shoot materials were air-dried for 48 h and then oven-dried at 80 °C to constant weight. This cautious precaution was needed to guarantee precise measurements of vital morphological variables [33].

Total chlorophyll contents (TCC)

Total chlorophyll content was measured as an indicator of photosynthetic capacity under metal stress. The quantification of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll was done as described [34]. Homogenized fresh leaf tissue (0.1 g) in 10 mL of 80 (v/v) acetone, centrifuged at 10,000 rpm at temperatures of 4 °C, and the absorbance values were recorded at 645 nm, 663 nm, and 470 nm. The calculation of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll, and total carotenoids was done using the formulae of Arnon (1949).

Assessment of antioxidant enzyme activities in plant samples

For the extraction of catalases and ascorbate peroxidase, approximately 0.5 g fresh plant materials were homogenized in 5mL of ice-cold 50mM potassium phosphate buffer (pH 7.2 ± 0.2) containing 1% (w/v) polyvinylpyrrolidone (PVP), 1 mM EDTA, and 1mM ascorbate (for APX assay); no ascorbate was used in the case of the CAT assay. The mixture was centrifuged at 10,000 rpm for 15 min. The supernatant (crude enzyme extract) was used immediately for enzyme assays.

Catalase activity was recorded by measuring the initial rate of hydrogen peroxide (H₂O₂) decomposition at 240 nm (extinction coefficient 39.4 mM^{− 1} cm^{− 1}). The 3 mL reaction mixture consists of 2.55 mL of 50 mM potassium phosphate buffer (pH 7.2 ± 0.2), 0.3 mL of 15 mM H₂O₂, and 0.15 mL of enzyme extract. The decline in the absorbance was recorded for 3 min. The mixture without the enzyme extract was used as a blank. One unit (U) of CAT activity was defined as the amount of enzyme required to decompose 1 µmol of H₂O₂ per minute [35].

Ascorbate peroxidase activity was determined by monitoring the oxidation of ascorbate at 290 nm (extinction coefficient 2.8 mM^{− 1} cm^{− 1}. The reaction mixture 3 mL consists of 2.4 mL of 50 mM potassium phosphate buffer (pH 7.2 ± 0.2), 0.3 mL of 0.5 mM ascorbate, 0.15 mL of 15 mM H₂O₂, and 0.15 mL of enzyme extract. The decline in the absorbance was recorded at 290 nm for 5 min. The mixture without the enzyme extract was used as a blank. One unit (U) of APX activity was defined as the amount of enzyme required to oxidize 1 µmol of ascorbate per minute and was calculated as U = change in 0.1 optical density per minute per gram of protein [35].

Assessment of stress-related phytohormone salicylic acid (SA) and indole-3-acetic acid (IAA)

Salicylic acid in plant samples, stored at -80 °C, was quantified by thawing 0.5 g of tissue, mashing it in 2 mL of phosphate buffer, and centrifuging at 12,000 rpm for 20 min. The supernatant was mixed with 100 µM tri-potassium EDTA and 2 mL of ethyl acetate, vortexed, and centrifuged at 2000 rpm for 10 min at 48 °C [36]. The top phase was removed, and the aqueous layer was re-extracted and diluted with 0.5 mL of 100 µM EDTA by mixing 2 mL of plant extract with 2.25 g of acid ninhydrin, 30 mL of glacial acetic acid, and 20 mL of 6 M phosphoric acid, heating at 100 °C for one hour, cooling in an ice bath, and measuring absorbance at 520 nm using a spectrophotometer.

The Indole-3-acetic Acid content was determined as described [37]. The homogenate (0.5 g) was mixed with 5 mL of cold 80% methanol and left to extract overnight at 4 °C. The 1 mL of the supernatant was centrifuged, and 2 mL of Salkowski reagent (50 mL 35% HClO₄ + 1 mL 0.5 mL FeCl₃) was added. The mixture was then incubated in darkness (30 min), and the absorbance was measured at 530 nm using a reagent blank. The standard curve of pure IAA was used to study the concentration of IAA.

Assessment of primary and secondary metabolites

Total sugar contents (TSC)

Total soluble sugars were quantified using the phenol-sulfuric acid colorimetric method [38]. Fresh leaf tissue (0.1 g) was homogenized in 1 mL of a methanol: chloroform: water (MCW) extraction buffer (12:5:3, v/v/v) and incubated for 30 min at room temperature. The homogenate was centrifuged at 10,000 rpm for 5 min. A 200 µL aliquot of the clear supernatant was transferred to a clean glass tube. To this, 200 µL of 5% (w/v) aqueous phenol was added, followed by the rapid addition of 1 mL of concentrated sulfuric acid (98%). The mixture was vortexed briefly and allowed to stand at room temperature for 60 min to allow full color development. A standard curve was prepared using D-glucose (0–140 µg). Absorbance was measured at 490 nm using a spectrophotometer (PerkinElmer Lambda 25 double-beam spectrophotometer) with a 3 mL quartz cuvette.

Total lipid contents (TLC)

Total lipids were extracted from fresh tissue (0.2 g) using a chloroform: methanol (1:1, v/v) mixture according to a modified Folch procedure [39]. The homogenate was centrifuged at 10,000 rpm for 5 min, and the supernatant was mixed with 0.3 mL of distilled water to induce phase separation. After a second centrifugation, the lower chloroform layer was transferred to a clean tube and evaporated to dryness at 90 °C.

Lipid content was measured by a sulfuric acid-vanillin colorimetric assay. Dried extracts were reacted with 0.5 mL of 98% H₂SO₄ at 90 °C for 20 min, then cooled and mixed with 1.5 mL of phospho-vanillin reagent (0.6% vanillin in 85% H₃PO₄). After 10 min at room temperature, absorbance was read at 530 nm. Concentration was calculated from a triolein standard curve (r² > 0.995) and expressed as mg triolein equivalents g⁻¹ fresh weight [39].

Total soluble protein contents (TSPC)

Total soluble protein was extracted from fresh leaf tissue (0.1 g) by homogenization in 1 mL of ice-cold 50 mM potassium phosphate buffer (pH 7.5). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. The clear supernatant was collected and used immediately for protein quantification.

TSPC was determined using the Bradford colorimetric assay [40]. A standard curve was prepared using bovine serum albumin (BSA; Sigma-Aldrich, purity ≥ 98%) as the reference protein. Duplicate aliquots of BSA stock solution (0.5 mg mL⁻¹) were diluted in 0.15 M NaCl to prepare standards. For the assay, 100 µL of appropriately diluted sample extract or standard was mixed with 1 mL of Coomassie Brilliant Blue G-250 dye reagent (Bradford reagent). The mixture was vortexed and incubated at room temperature for 2 min. Absorbance was measured at 595 nm using a spectrophotometer. Protein concentration in each sample was determined by interpolation from the linear BSA standard curve (r² > 0.995). Results are expressed as milligrams of protein per gram of fresh leaf weight (mg g⁻¹ FW).

Total flavonoid contents (TFC)

The total flavonoid content was estimated using the colorimetric Aluminium chloride (AlCl₃) method. For the assay, 0.1 mL of the sample extract was mixed with 0.3 mL of 5% (w/v) NaNO₂ and 0.4 mL of distilled water. After 5 min of incubation at room temperature (22 °C), 0.03 mL of 10% (w/v) AlCl₃ was added. Following a further 6-minute incubation, 0.2 mL of 1 M NaOH and 0.24 mL of distilled water were added. The reaction mixture was mixed thoroughly, and the absorbance of the developed pink-orange color was measured at 510 nm. The total flavonoid content was calculated by the linear quercetin standard curve (r² > 0.995) and expressed as milligrams of quercetin equivalents per gram of fresh weight (mg QEs g⁻¹ FW). A standard curve was prepared using quercetin (≥ 95% HPLC grade) as the reference compound. An appropriate dilution series of quercetin in 30% aqueous ethanol (v/v) was used for calibration [41].

Total phenolic contents (TPC)

The total phenolic content was determined using the Folin-Ciocalteu colorimetric assay. For the assay, 0.5 mL of a 1:10 (v/v) dilution of the Folin-Ciocalteu reagent was mixed with 0.5 mL of the sample extract. The mixture was vortexed and allowed to stand for 5 min. Then, 0.5 mL of a 6% (w/v) sodium carbonate (Na₂CO₃) solution was added. The reaction mixture was incubated at room temperature (28 °C) in the dark for 90 min to allow full color development. The absorbance of the resulting blue complex was measured at 765 nm. The total phenolic content was calculated by the linear gallic acid standard curve (r² > 0.995) and expressed as milligrams of gallic acid equivalents per gram of fresh weight (mg GAEs g⁻¹ FW). A standard curve was prepared using gallic acid as the reference [41].

Proline contents

Proline was quantified as a marker of osmotic adjustment under stress (a secondary metabolite). Free proline was extracted from fresh leaf tissue (0.1 g) in 1.0 mL of ice-cold 3% (w/v) sulfosalicylic acid according to Bates [42]. The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. Proline concentration was determined by the acid-ninhydrin assay. The reaction mixture contained 0.1 mL of supernatant, 0.2 mL of glacial acetic acid, and 0.2 mL of freshly prepared acidic ninhydrin reagent (1.25% ninhydrin in glacial acetic acid:6 M orthophosphoric acid, 3:2 v/v). The mixture was incubated at 96 °C for 60 min (tube lids punctured), then cooled on ice. The chromophore was extracted with 1.0 mL of toluene, vortexed for 20 s, and the upper toluene layer was used for absorbance measurement at 520 nm (toluene blank). Proline concentration was calculated from an L-proline standard curve (r² > 0.995) and expressed as mg proline g⁻¹ fresh weight.

Metal quantification in soil and plant biomass

Metal content was determined in plant biomass by drying 500 mg of the sample and subjecting it to acid digestion by nitric acid (HNO₃) and perchloric acid (HClO₄), a method with a metal recovery rate of 96%±2.3% in the current experimental setup. The mixture was cooled down to room temperature, then filtered over a Whatman 42 filter paper and brought to a final volume of 25 mL of distilled water. Control plants were treated the same as test plants to compare them, and a blank solution was also purged using the same process, but no sample was in it [43]. The concentrations of copper in the samples were measured using a Perkin Elmer flame atomic absorption spectroscope (Analyst 700, USA) with an acetylene/air flame, following the manufacturer’s guidelines for operational parameters. The standard curve was plotted from the data using the known concentration of Cu prepared in parallel with the samples mentioned earlier.

Data analysis

The experiments were performed in triplicate using the specified copper concentrations. All data were subjected to factorial analysis using SPSS version 21 (IBM, Armonk, NY, USA). The effects of the main factors, i.e., plant species (sunflower and maize), copper (Cu), and amendment treatments and their interactions were evaluated using a two-way analysis of variance (ANOVA). Where significant main or interaction effects were found (p < 0.05), Tukey’s (HSD) test was applied for pairwise comparisons between treatment groups. Figures were prepared using GraphPad Prism version 8.0.

Results

Copper vs. sunflower and maize

Effects of copper stress on growth of sunflower and maize

Copper stress reduced growth parameters in both maize and sunflower, with different magnitudes and patterns between the two species. In sunflower, root length decreased by 48–53% compared to the control at 25, 50, and 75 mg kg⁻¹ Cu, while shoot length decreased by 15–25%. Fresh biomass declined by 57–71% and dry biomass by 34–50% relative to the control (Fig. 1a-d). In maize, shoot length decreased by 23–35% and root length by 60–76% compared to the control. Root biomass declined by 54–69% and shoot biomass by 59–71% (Fig. 1e-h).

Fig. 1.

Effect of different copper concentrations, EDTA, and IAA on growth parameters of sunflower (a-d) and maize (e-h). Bars represent the means of triplicate with standard error (±), and different letters indicate significance among treatments at the levels of p ≤ 0.05. Cu.1, Cu.2, and Cu.3: 25, 50 and 75 mg/kg correspondingly of CuCl₂, Cu.4, Cu.5 and Cu.6: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA, Cu.7, Cu.8 and Cu.9: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA and IAA

The combined application of EDTA and IAA partially alleviated Cu-induced growth inhibition, with species-specific recovery. In sunflower, EDTA + IAA increased root length by 13–18% and shoot length by 8–10% compared to Cu-alone treatment, with the greatest recovery at 25 mg kg⁻¹ Cu. Fresh and dry biomass improved by 16–23% relative to Cu-alone treatment, but remained below untreated control levels. Maize showed a greater response: shoot and root fresh biomass increased by 20–40% and 25–44%, respectively, compared to Cu-alone treatment. Shoot length improved by 6–11% and root length by 13–20% relative to Cu alone. The visual effects of the different treatments on sunflower and maize growth and morphology are shown in Fig. 2.

Fig. 2.

Effects of Cu supplementation, EDTA and IAA on the Growth of sunflower and maize

Effects of copper on photosynthetic pigments of sunflower and maize

In sunflowers, copper stress reduced chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content (TCC) in a concentration-dependent manner. Total chlorophyll decreased by 19%, 40%, and 47% compared to the control at 25, 50, and 75 mg kg⁻¹ Cu, respectively. EDTA application partially alleviated these reductions, and the combined EDTA + IAA treatment further increased TCC by 7–18% relative to Cu-alone treatment across the three Cu concentrations. However, pigment levels remained below untreated control values.

In maize, TCC was reduced by 28–34% compared to the control at the selected Cu concentrations. The reduction was steeper at low Cu concentrations. EDTA application improved pigment stability, and EDTA + IAA further increased TCC by 27–33% relative to Cu-alone treatment, nearly reaching untreated control levels (Fig. 3a, b).

Fig. 3.

Effects of Cu supplementation, EDTA, and IAA on photosynthetic pigments of sunflower (a) and maize (b). Bars represent the means of triplicate measurements with standard error (±), and different letters indicate significant differences among treatments at p ≤ 0.05. Cu. 1, Cu.2, and Cu.3: 25, 50 and 75 mg/kg correspondingly of CuCl₂, Cu.4, Cu.5 and Cu.6: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA, Cu.7, Cu.8 and Cu.9: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA and IAA

Phytohormone response to copper stress

A varied phytohormonal response to copper stress was observed in sunflower and maize. Indole-3-acetic acid (IAA) production increased in both species in a dose-dependent manner, reaching 561.91 µg/g in sunflower and 361.78 µg/g in maize. IAA levels were further amplified with EDTA treatment. Foliar IAA levels were lower in sunflower compared to maize.

The two species differed in their salicylic acid (SA) dynamics. In sunflower, SA production increased to 1387.3 µg/g, reaching a maximum of 2155.4 µg/g at 75 mg kg⁻¹ Cu. EDTA and IAA treatments reduced this increase (Fig. 4a). In maize, SA increased more gradually, reaching 950 µg/g at 75 mg kg⁻¹ Cu. EDTA and IAA partially reduced SA levels in maize, with maximum values of 752.7 and 830.2 µg/g at higher Cu levels, which were lower than in Cu-only treated plants (Fig. 4b).

Fig. 4.

Effects of Cu supplementation, EDTA, and IAA on phytohormones and antioxidant enzymes of sunflower (a) and maize (b). IAA; SA; CAT; APX. Cu.1, Cu.2, and Cu.3: 25, 50, and 75 mg/kg correspondingly of CuCl₂, Cu.4, Cu.5, and Cu.6: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA, Cu.7, Cu.8, and Cu.9: 25, 50 and 75 mg/kg correspondingly of CuCl₂ with EDTA and IAA

Antioxidant response of sunflower and maize

Catalase (CAT) activity decreased with increasing Cu concentration. In sunflower, CAT decreased by 33%, 38%, and 74% compared to control at 25, 50, and 75 mg kg⁻¹ Cu, respectively, with the lowest value at 75 mg kg⁻¹. In maize, CAT decreased by 71%, 83%, and 94% compared to the control, with the lowest level at 75 mg kg⁻¹. EDTA application improved CAT activity in sunflower by 10% at 25 mg kg⁻¹ Cu relative to Cu-alone treatment, but levels remained below control. In maize, EDTA or IAA application increased CAT by 11–19% relative to Cu-alone treatment, though levels remained below control.

Ascorbate peroxidase (APX) activity increased with Cu concentration. In sunflower, APX increased by 18% and 56% at 50 and 75 mg kg⁻¹ Cu compared to the control. However, with EDTA or EDTA + IAA application, APX showed the lowest values in a dose-dependent manner. In maize, APX increased more steeply, reaching a 198% increase at 75 mg kg⁻¹ Cu compared to the control. A similar multifold increase was observed in maize with increasing Cu concentration. EDTA and EDTA + IAA treatments also increased APX in a dose-dependent manner (Fig. 4a, b).

Metabolites production of sunflower and maize

Total flavonoids

In sunflower, TFC decreased by 62–76% compared to control, with the minimum at 75 mg kg⁻¹ Cu. In maize, TFC decreased by 56–82% compared to the control, with the lowest value at 75 mg kg⁻¹. EDTA application increased TFC in sunflower by 4–12% relative to Cu-alone treatment. Foliar IAA also increased TFC, but levels remained below those of the untreated control. In maize, EDTA and foliar IAA increased TFC by 49–51% relative to Cu-alone treatment, with IAA generally producing lower levels than control, except at 25 mg kg⁻¹ Cu, where TFC was similar to untreated plants (Fig. 5a, b).

Fig. 5.

Effects of Cu supplementation, EDTA, and IAA on metabolites of sunflower (a) and maize (b). Bars represent the means of triplicates with standard error (±), and various letters indicate significance among treatments at the levels of p ≤ 0.05. Cu. 1, Cu. 2, and Cu. 3: 25, 50, and 75 mg/kg correspondingly of CuCl₂, Cu. 4, Cu. 5, and Cu. 6: 25, 50, and 75 mg/kg correspondingly of CuCl₂ with EDTA, Cu 7, Cu 8, and Cu 9: 25, 50, and 75 mg/kg correspondingly of CuCl₂ with EDTA and IAA)

Total phenolics

In both species, TPC increased with Cu treatment. EDTA application increased TPC compared to the untreated control. Foliar IAA reduced TPC in sunflower, but levels remained higher than Cu-free control. In maize, foliar IAA also increased TPC, and levels were higher than in controls.

Proline

Proline accumulation increased with Cu concentration in both species. In sunflowers, EDTA application substantially reduced proline, though levels remained above those of the untreated control. Foliar IAA further reduced proline, reaching the lowest level at 75 mg kg⁻¹ Cu, comparable to the control. In maize, proline increased multifold with increasing Cu up to 75 mg kg⁻¹. Both EDTA and foliar IAA reduced proline, but proline levels increased with metal concentration and remained above control levels.

Total soluble protein (TSPC) and total lipid (TLC)

In sunflower, TSPC decreased by 66–77% and TLC by 61–86% compared to the control. EDTA application increased TSPC by 12–41% and TLC by 11% and 12% relative to Cu-alone treatment, but levels remained much lower than those of the untreated control. Foliar IAA also improved TSPC and TLC. In maize, TSPC and TLC decreased by 31–53% compared to the control. EDTA and foliar IAA enhanced these contents, but levels remained significantly lower than those of untreated controls.

Total soluble sugars

In sunflower, total sugars decreased by 40–60% with increasing Cu compared to the control. EDTA and foliar IAA produced minor improvements of 2% and 3%, respectively. In maize, sugars decreased by 30–44% compared to control, with improvements of 14–18% (EDTA) and 3–9% (IAA) relative to Cu-alone treatment. Despite these improvements, sugar levels in both species remained significantly lower than control (Fig. 5a, b).

Copper accumulation and phytoremediation potential in maize and sunflower

Copper uptake increased with increasing Cu concentrations in both maize and sunflower (Fig. 6a, e). At 25 mg kg⁻¹ (Cu1), maize roots accumulated 62% more Cu than sunflower. At Cu1 and Cu3 (75 mg kg⁻¹), total root Cu accumulation increased by nearly 100% in maize and 74% in sunflower compared to the control. Sunflower root Cu content reached 2.2-fold higher than that of maize at 60 days. EDTA application (Cu4–Cu6) enhanced Cu uptake in both species, increasing accumulation by 28–33% in maize and 36–42% in sunflower relative to Cu alone treatments. The combined EDTA + IAA treatment (Cu7–Cu9) further increased Cu accumulation, with total Cu content in sunflower rising by 55% compared to Cu3.

Fig. 6.

Effects of Cu supplementation, EDTA, and IAA on Cu bioaccumulation and translocation by sunflower (a-d) and maize (e-h). Bars represent the means of triplicate measurements with standard error (±), and different letters indicate significant differences among treatments at p ≤ 0.05. Cu.1, Cu.2, and Cu.3: 25, 50, and 75 mg/kg correspondingly of CuCl₂, Cu. 4, Cu. 5, and Cu.6: 25, 50, and 75 mg/kg correspondingly of CuCl₂ with EDTA, Cu.7, Cu.8, and Cu.9: 25, 50, and 75 mg/kg correspondingly of CuCl₂ with EDTA and IAA

Phytoremediation efficiency differed over time (Fig. 6c, g). Maize removed 67–70% of soil Cu within 15 days, whereas sunflower removed 42–46% during the same period. After 60 days, sunflowers achieved 85–88% removal, compared to 72% in maize. EDTA application increased Cu removal efficiency by approximately 20% for both species relative to Cu alone. EDTA + IAA treatment resulted in the highest Cu removal, with maximum efficiencies of 95% in sunflower and 82% in maize at Cu9.

Copper translocation factors (TF) differed between the two species (Fig. 6d, h). In maize, TF values increased from 1.3 at Cu1 to 2.0 at Cu3, then stabilized. In sunflower, TF values increased continuously from 1.1 at Cu1 to 3.4 at Cu3, and increased further under EDTA + IAA treatments. Copper distribution also differed between species (Fig. 6b, f). In maize, root Cu concentrations were 1.8–2.0 times higher than shoot concentrations. In sunflowers, shoot Cu concentrations reached 85–90% of root levels under Cu9 treatment. Cu accumulation in maize seeds remained below 5% of total plant Cu content across all treatments.

Discussion

Maize and sunflower were subjected to copper stress. Growth and physiology were adversely altered in both species, but they exhibited contrasting acclimation and coping strategies [44]. Maize showed higher root and shoot mass than sunflower at equivalent Cu concentrations, indicating greater tolerance [45]. This difference in tolerance may be related to the ability of maize to regulate Cu uptake at the root surface and sequester it in vacuoles, which could help prevent cytosolic toxicity and maintain metabolic activity [46]. Previous studies have reported that maize can homeostatically restore ionic balance and immobilize Cu in the roots [47–49]. This tolerance was enhanced by the addition of EDTA and IAA. The improvement is likely due to increased Cu solubility and auxin-mediated root growth stimulation, which together improve nutrient uptake and reduce metal-induced growth inhibition, leading to more effective nutrient acquisition [50, 51].

Despite having greater biomass, maize consistently had lower total chlorophyll content (TCC) than sunflower across all treatments. This may be because of different photosynthetic metabolism and stress response among species [52]. Sunflower is a C3 plant, which depends mainly on chlorophyll-dependent carbon fixation and may favor the maintenance of pigment biosynthesis under stress. By contrast, maize, being a C4 plant, has higher CO₂ utilization efficiency and lower photorespiration rate; it might change the allocation of metabolic energy used for pigment synthesis, antioxidant enzymes, and detoxification mechanisms with Cu treatment [53, 54].

Consistently, Sunflowers maintained higher chlorophyll and photosynthetic capacity, whereas maize instead focused on biochemical defense and oxidative stress management [55]. The phytohormone analysis supported this observation. Concentrations of IAA and salicylic acid were higher in sunflower than in maize, suggesting activation of hormone-mediated stress signaling for cell protection [56]. In addition, changes in metabolites such as flavonoids, phenolics, proline, protein, and low-molecular-weight sugars contributed to stress mitigation. Flavonoids, phenolics, and proline act as direct ROS quenchers, helping the plant to counter excessive ROS production [57]. These metabolites also help maintain membrane integrity through direct and indirect ROS quenching, preventing nutrient loss and aiding stress tolerance. Similarly, proteins and sugars act as osmolytes, preserving cellular integrity and water balance under unfavorable conditions [58]. Nevertheless, sunflower growth was disproportionately more suppressed than maize growth, suggesting that hormonal compensation was insufficient to protect against oxidative damage when enzymatic support was limited. Catalase activity was also greater in maize than in sunflower. ROS toxicity from spontaneous decomposition may be detoxified by catalase, which remained active [59]. Auxin application may enhance these enzymatic defenses by improving redox homeostasis and promoting the induction of antioxidant genes, particularly in maize [60].

The antioxidant system data supported these trends. Maize exhibited high ascorbate peroxidase activity and stable levels of primary metabolites (proteins, lipids, sugars), indicating efficient ROS elimination and metabolic stability. Aly et al. similarly reported that total soluble sugars, glutathione, cysteine, and lipid peroxidation increased under copper stress [61]. Increased tolerance with reduced growth was observed, consistent with the lower biomass of sunflowers after Cu exposure. The high tolerance of maize can be attributed to its better capacity to maintain primary metabolism under Cu accumulation, likely due to enhanced enzymatic detoxification [62, 63]. Notably, the increase in antioxidant indices was greater when both crops were treated with EDTA and IAA, suggesting that chelating agents and plant hormones act synergistically to reduce oxidative stress [20].

Metal accumulation patterns provided further insight into species-specific remediation potential [64]. Maize roots accumulated more Cu than shoots, consistent with a phytostabilization strategy that limits translocation to aboveground tissues [65]. Daryabeigi Zand et al. also found higher root Cu accumulation than shoot Cu in Z. mays during remediation of Cu-contaminated soil [66]. In contrast, sunflowers accumulated more Cu in their shoots, indicating a phytoextraction response for metal removal from soil [67]. The rapid and high root accumulation in maize points to efficient metal sequestration through the synthesis of chelating substances (phytochelatins, metallothionein), forming stable Cu-thiol complexes that restrict Cu to vacuoles [68]. In fact, the sunflower taproot improves the upward transport to the shoot, where it accumulates [69, 70]. Both crops showed greater Cu bioavailability and uptake with EDTA and increased root elongation with IAA, which minimized phytotoxicity and improved the Cu dynamics in the crops [71, 72].

Interestingly, the contrasting Cu partitioning between maize and sunflower also suggests a temporal component of metal absorption and internal regulation. Cu accumulation was rapid, particularly in the early stages, suggesting a higher initial uptake followed by possible immobilization in root tissues [73]. As growth progressed, a decline in Cu accumulation was recorded. This decline may reflect a homeostatic response to restrict further influx and protect photosynthetically active tissues [46]. In contrast, sunflowers showed a gradual but sustained increase in Cu uptake over time, accompanied by enhanced root-to-shoot translocation. This progressive accumulation pattern is consistent with a phytoextraction strategy, where the plant may mitigate stress by increasing biomass and efficient vascular transport, supporting long-term metal removal from the soil [74]. This time-dependent difference in Cu dynamics provides a possible explanation for the stabilization behavior in maize versus the extraction behavior in sunflower, supporting the biological plausibility of the reported results. Although Pearson correlation analysis was not performed in this study, future investigations could employ such correlative approaches to further explore relationships between metal accumulation and antioxidant responses.

Overall, two contrasting adaptive strategies emerged: maize sequesters copper in root tissues, thereby reducing toxicity [75]. This metal confinement supports enzymatic detoxification and sustains primary metabolism [76]. Sunflower does not prevent copper toxicity but instead allows metal loading into secondary metabolites and translocates copper to aerial organs [77, 78]. These distinct strategies underpin their suitability for different remediation goals. By integrating physiological, biochemical, and metal distribution patterns, and by assessing the roles of EDTA and IAA in modulating Cu stress responses, the findings suggest that maize is a promising candidate for Cu phytostabilization, whereas sunflower is more suited for Cu phytoextraction. This comparison provides a basis for selecting plant-amendment combinations tailored to contamination levels and soil conditions, and supports an integrated approach that combines stabilization and extraction to enhance restoration of Cu-polluted soils.

Conclusion

This study demonstrates that maize and sunflower employ distinct, species-specific phytoremediation strategies under copper stress. Maize sequesters Cu primarily in roots (phytostabilization), while sunflower translocates Cu to shoots (phytoextraction). The combined application of EDTA and IAA enhanced these inherent strategies, significantly mitigating Cu-induced physiological stress and boosting metal uptake and tolerance. Improved biomass, photosynthetic pigments, and antioxidant activity were observed in both species compared to Cu stress alone. Within the controlled pot conditions, maize showed characteristics suitable for short-term phytostabilization or phytomining (higher biomass, rapid root accumulation), whereas sunflower exhibited increasing translocation and higher Cu uptake at harvest, indicating greater potential for sustained phytoextraction. Rather than a single “superior” species, our findings highlight complementary, species-specific functional roles. The choice of species and amendments can be tailored to remediation objectives (stabilization vs. removal), contamination level, and time frame.

Authors’ contributions

1. Concept, methodology, and original draft by N.S 2. Statistical analysis and graphs edited by W. G 3. Visualization, analysis by M.A 4. Data validation, editing by L.B 5. Review and editing by J. C 6. Supervision, funding, and final editing by H.L.

Funding

This work was financially supported by the Natural Science Foundation of China (42267059), the Yunnan Fundamental Research Projects (202501AS070148), and the Yunnan International Joint Laboratory of Research and Development of Crop Safety Production on Heavy Metal Pollution Areas (202403AP140035).

Data availability

The data supporting the findings of this study are included within the article. Additional datasets are available from the corresponding author upon reasonable request.”

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.