**1. Introduction**

Heavy metals are part of the natural soil system; however, their high concentration accumulated by plants and animals due to agricultural malpractices and other anthropogenic activities poses a serious concern [1]. In plants and algae, copper (Cu) is a crucial micronutrient. Plants utilize copper, particularly in ATP synthesis, photosynthesis, CO2 assimilation, and as a vital component of several proteins. However, the overuse of Cu in industry and mining has increased its concentrations to toxic levels in ecosystems [2]. Cu above the threshold level can cause kidney and liver dysfunction, anemia, intestine and stomach irritation, hypertension, and nervous system problems. Cu-induced phytotoxicity leads to physiological stress and causes stunted growth and leaf chlorosis [3]. Due to the presence of malondialdehyde (MDA), which promotes bilayer lipid and protein

**Citation:** Shah, N.; Irshad, M.; Hussain, A.; Qadir, M.; Murad, W.; Khan, A.; Awais, M.; Alrefaei, A.F.; Ali, S. EDTA and IAA Ameliorates Phytoextraction Potential and Growth of Sunflower by Mitigating Cu-Induced Morphological and Biochemical Injuries. *Life* **2023**, *13*, 759. https://doi.org/10.3390/ life13030759

Academic Editors: Kousuke Hanada, Wajid Zaman and Hakim Manghwar

Received: 4 January 2023 Revised: 8 February 2023 Accepted: 9 March 2023 Published: 10 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

peroxidation, Cu seriously harms plants through oxidation. Reactive oxygen species, such as hydroxyl radicals (OH), superoxide radicals (O2) and singlet oxygen (1O2), exhibit increases due to toxic Cu levels; however, OH, SOD, and POD are crucial antioxidants for the scavenging of ROS [4,5].

The copper content in the soil should be reduced in order to avoid Cu-induced phytotoxicity. In general, heavy metal removal from polluted soil involves physical, chemical, and biological methods [6]. Most chemical and physical methods, such as stabilization, solidification, vitrification, electrokinetics, soil washing, and vapour extraction, are, however, costly and ineffective [7–10]. Due to its extensive use and economic viability, phytoextraction, a green technique that allows the removal of soil contaminants, has emerged as a viable alternative. Fast-growing plants are used in an efficient and eco-friendly manner to remove heavy metals and other dangerous substances from contaminated soils and accumulate in harvestable portions. Exposure duration also impacts the reclamation of metal under certain conditions, i.e., metal accumulates actively as plants grow; however, after a certain growth period, the reclamation remains the same and the plant is unable to accumulate more metal [11]. More than 400 plant species from 45 distinct plant families, ranging from tropical to temperate regions, have been documented and claimed to be able to withstand and absorb heavy metals from soil. Heavy metals are absorbed from soil to shoots through roots, which depends on the species of plant, availability of heavy metals, growth stage and use of fertilizers [12].

Chelates, such as EDTA, lower the pH of soil solutions by forming complexes with heavy metals, thus increasing metal bioavailability and facilitating metal translocation from soil to root and then shoot. Limited amounts of chelators are required to boost metal absorption by plants [13].

Plant hormones have a crucial role in several important physiological processes [14]. Indole-3-acetic acid (IAA), a naturally occurring auxin, has the power to control a variety of aspects of plant growth, including vascular tissue differentiation, growth, and elongation, the production of lateral roots, apical dominance, and fruit formation and ripening. There have been numerous publications on the phytoextraction of various heavy metals from a variety of plant species using EDTA and IAA; however, there is limited literature on the synergistic effects of EDTA and IAA assisted phytoremediation [13,15].

As a novel study, this work was aimed at finding out the phytoextraction capacity of sunflowers in Cu-contaminated soils in the presence of EDTA and IAA. Moreover, the biochemical signatures of the sunflowers exposed to Cu and all other treatments were also investigated.

#### **2. Materials and Methods**

#### *2.1. Preparation of Soil*

The soil used throughout the experiment to grow the sunflowers was a sandy loam soil with composition of sand and clay was used with particle size of approximately 0.5 mm and 0.002 mm, respectively. The sand and clay were mixed with manure with the approximate ratio of 2:1:1 to make the plant growth medium, which was then used to prepare a sandy loam for improved sunflower development. Pots containing 5 kg of soil were maintained in the green house at the department of Botany, Abdul Wali Khan University in Mardan.

#### *2.2. Experimental Design*

Viable and healthy sunflower seeds were purchased and the surface of the seeds was disinfected by 70% ethanol, and the ethanol was then washed off with sterile distilled water. The experiment was in three factorial combinations, i.e., Cu concentrations (25, 50, and 75 mg/kg of soil in the form of CuCl2 as a bioavailable form of Cu), EDTA (5 mM (1.45 g/kg)), and foliar application of IAA (2.5 μM sprayed at intervals of 5 days until harvest), EDTA and IAA (in the same concentrations as used in the separate treatments) were used in combination with different levels of the selected metal. Each treatment consists of three replicates, and every replicate had three plants. The pots received a thorough

watering of tap water every morning and evening. Several biochemical tests were run to determine the effect of copper concentration, IAA, and EDTA treatment on the agronomic and physiological characteristics of the host plant, as well as metal accumulation.

#### *2.3. Morphological Parameters*

The sunflowers roots and shoots were measured in centimeters (cm). The fresh and dry weights of the root and shoot were calculated and represented in grams using an analytical weight balance. To obtain their dry weight, the samples were oven dried at 80 ◦C.

#### *2.4. Estimation of Chlorophyll Contents*

A UV-visible spectrophotometer was used to quantify chlorophyll (Biochrom Libra S22) [16]. For the purpose of extracting chlorophyll, 0.5 g of fresh leaves were crushed with 80% acetone, and Whatman filter paper No. 42 was used for the filtration of mixture. Additional acetone was used to dilute the solution by about 1 mL (approximately 2 mL of acetone). Two wavelengths, 663 and 645 nm, were used to measure the optical density (OD) in comparison to a blank surface.

#### *2.5. Estimation of Phytohormones*

For estimation of IAA, the Salkowski reagent technique [17], and salicylic acid was measured using 1% iron chloride [18]. The gibberellic acid content was determined using a wheat endosperm assay [19].

#### *2.6. Metabolite Determination*

Total flavonoids were determined by the AlCl3 method [20]. Leaf samples of 0.5 g were homogenized using 80% ethanol (5mL) and kept for incubation for 24 h to achieve full flavonoid extraction in the shaker. After that, the mixtures were centrifuged for 15 min at 10,000 rpm at 25 ◦C.

For the determination of total phenolics, 16 mL of ethanol was added to 1 g of crushed plant leaves. Centrifugation of the homogenates were performed at 10,000 rpm after being incubated at an increased temperature (between 20 ◦C and 80 ◦C) for 3 h. The supernatants were concentrated to 1 mL at 40 ◦C by using a rotary evaporator after being filtered through filter paper (Whatman No. 42). Resolving the concentrations in 10 mL distilled water allowed for the measurement of phenolics [20].

In order to extract proline content, the solutions were centrifuged at 10,000 rpm after being incubated for 24 h at 4 ◦C for 5 min following the protocol of Bates et al. [21].

#### *2.7. Determination of Antioxidant Response*

Catalase activity (CAT) and ascorbate peroxidase (APX) were used for the determination of antioxidant responses. The cleavage of H2O2 was determined for CAT activity using the procedure of Radhakrishnan and Lee [22]. Approximately 0.1 mL of supernatant, 0.4 mL of 3% H2O2, and 0.1 mM EDTA were added to 2.6 mL of 0.05 M phosphate buffer (pH 7). The drop in H2O2 was accompanied by a reduction in absorbance at 240 nm, which was quantified as M H2O2 min−<sup>1</sup> cleavage.

To estimate APX in the leaves, the Asada [23] procedure was used. The reaction mixture consists of 0.1 mL ascorbic acid (0.5 mM), 0.6 mL PBS (50 mM, pH 7.0), 0.1 mL H2O2 (0.1 mM), and 0.2 mL leaf extract. A decrease in optical density was measured at 290 nm. Protein content was calculated for each extract using the technique of Bradford [24].

#### *2.8. Estimation of the Copper in Plant Biomass*

Oven dried 0.5 g samples were mixed with perchloric acid (HCLO4) and nitric acid (HNO3) in the ratio of 1:4 in order to prepare samples for metal analysis using an atomic absorption spectrophotometer. After cooling, the mixture was filtered, and the final volume of the mixture was raised to 25 mL by adding distilled water. Control plant samples were treated using the same method as the positive control solution. With the exception of the

inclusion of sample, the blank solution was created in the same manner as the sample solution. For quantification of the copper contents in the biomass, Amin et al.'s [5] method was followed using atomic absorption spectrophotometer (Perkin Elmer 700) to determine selected heavy metals.
