Synergistic effects of glutamic acid and cerium oxide nanoparticles on cadmium phytoremediation efficiency in marigold

Mujahid Farid; Muskan Shahzad; Muhammad Zubair; Sheharyaar Farid; Wajiha Sarfraz; Roha Murtaza; Muhammad Munir; Abdulrahman Alasmari; Sarah Owdah Alomrani; Shafaqat Ali

doi:10.1186/s12870-026-08692-7

. 2026 Apr 9;26:844. doi: 10.1186/s12870-026-08692-7

Synergistic effects of glutamic acid and cerium oxide nanoparticles on cadmium phytoremediation efficiency in marigold

Mujahid Farid ^1,^✉, Muskan Shahzad ¹, Muhammad Zubair ², Sheharyaar Farid ³, Wajiha Sarfraz ⁴, Roha Murtaza ¹, Muhammad Munir ⁵, Abdulrahman Alasmari ^6,⁷, Sarah Owdah Alomrani ⁸, Shafaqat Ali ^9,^10,^✉

PMCID: PMC13173739 PMID: 41957562

Abstract

The presence of heavy metals in soil, water, and air is an emerging critical environmental challenge due to their non-biodegradability and toxicity. Therefore, this study was designed to evaluate the phytoremediation of cadmium (Cd) using the combined effect of glutamic acid (GA, 10 mM) and cerium oxide nanoparticles (CeO₂NPs, 150 mgL^− 1) in marigold. Over 28 days in a completely randomized design experiment, the effects of CeO₂ NPs and GA on plant growth, photosynthetic pigments, antioxidant enzymes, reactive oxygen species (ROS), electrolyte leakage (EL), SPAD values, soluble proteins, and Cd accumulation in marigold plants were assessed. Exposure to Cd, especially at 5.0 mM, significantly reduces plant growth, chlorophyll content, and protein levels while increasing ROS, EL, and oxidative damage. However, the combined application of GA and CeO₂NPs alleviated Cd toxicity by improving growth parameters, enhancing chlorophyll and protein content, and boosting antioxidant defenses. Notably, the applied treatment also increased Cd accumulation in roots (108%), stems (106%), and leaves (119%) relative to controls. SPAD values and soluble protein levels increased under the combined treatment, thereby improving photosynthetic efficiency and metabolic resilience. The results also showed a substantial reduction in oxidative stress markers such as hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), confirming a strong protective effect. These findings reveal that GA and CeO₂NPs can enhance both Cd tolerance and uptake in marigold, suggesting potential for improving phytoremediation efficiency under controlled conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08692-7.

Keywords: Foliar application, Antioxidant enzymes, Accumulation, Photosynthetic pigments, Reactive oxygen species

Introduction

Heavy metals (HMs) are emerging pollutants in the human food chain, especially when agricultural lands are irrigated with contaminated water [1]. HMs enter the environment through both natural and human activities. In nature, Cadmium (Cd) is present in sediments, rocks, and minerals, which are released into the water bodies [2]. The HM’s natural sources include volcanic eruptions, soil erosion, and rock weathering [3]. Human activities such as fossil fuel burning, metal refining, waste burning and disposal, and the synthesis and application of phosphate fertilizers release significant amounts of Cd into the environment [4]. Cadmium is a highly toxic and mobile element that accumulates in soil and is readily absorbed by plants [1]. Due to absorption, transmission, and accumulation in crops, Cd is one of the hazardous HMs and also poses a serious threat to human health. Approximately 1.0 × 10⁶ tons of Cd is discharged annually worldwide [5]. Different remediation techniques, including ion exchange, soil washing, adsorption, and bioremediation, are in place to mitigate HMs from soil [6]. In recent years, phytoremediation has gained attention as a sustainable approach for removing heavy metals from contaminated soils [7].

Phytoremediation is a natural process in which plants purify air, water, and soil, which includes methods like phytoaccumulation, phytoextraction, phytovolatilization, phytotransformation, and phyto-stabilization. In phytoextraction, plants absorb heavy metals (HMs) like Cd, Zn, Ni, and Cr from the soil, while phytoaccumulation helps store these elements in the shoots and leaves [8]. Phytoremediation of cadmium-polluted areas using hyperaccumulating plants is an innovative and effective approach for sustaining soil [9]. Recently, ornamental plants have been used for phytoremediation of HMs from soil and water [10]. These plants play a pivotal role in ecological and environmental restoration, in restoring damaged aquatic and terrestrial ecosystems, and in contributing to multifunctional landscapes. As a result, they are widely cultivated for phytoremediation in both rural and urban areas [11]. Although phytoremediation is considered an environmentally friendly approach for heavy metal removal, its efficiency is often limited by reduced plant growth and metal-induced oxidative stress [12]. Therefore, various strategies have been explored to enhance plant tolerance and metal uptake efficiency. Among these strategies, the use of organic acids and nanomaterials has gained increasing attention due to their ability to improve metal mobility, enhance antioxidant defense systems, and support plant physiological performance under stress conditions [13, 14].

The marigold plant is a member of the Asteraceae family, which is a flowering crop cultivated worldwide for essential oils, bedding and potted plants, and photosynthetic pigments used in nutraceutical and pharmaceutical products [15]. Marigold flowers and leaves are widely used in several European and Asian countries, including India, Denmark, Thailand, and Germany, for livestock and human diets, medicinal purposes, cosmetics, and ornamental use. Additionally, numerous studies have demonstrated their ability to hyperaccumulate HMs and other pollutants [16]. Marigold species have also attracted attention as potential phytoremediation plants due to their relatively high biomass production, tolerance to environmental stress, and ability to accumulate certain heavy metals. In addition, the use of ornamental plants for phytoremediation has gained increasing interest because these plants can remediate contaminated environments without entering the human food chain. Therefore, marigold represents a suitable model plant for investigating strategies aimed at enhancing phytoremediation efficiency under heavy metal stress [17]. The hydroponic system provides optimal growing conditions and nutrient supply for plants. Several strategies for hydroponic culture are identified, including circulating and non-circulating solutions, aeroponics, and solid medium culture [18]. Nanomaterials, with their exceptionally small size and large surface area, exhibit diverse functionalities and are effective at removing specific contaminants, such as Cd. Adsorption using nanomaterials is a viable technique for removing hazardous metals from contaminated water [19]. Cerium oxide nanoparticles have attracted considerable attention due to their unique redox properties and catalytic activity. These nanoparticles can undergo reversible redox cycling between Ce³⁺ and Ce⁴⁺ states, enabling them to scavenge reactive oxygen species (ROS) and reduce oxidative stress in biological systems [20]. In plants exposed to heavy metals, excessive ROS production can damage cellular components such as membranes, proteins, and nucleic acids. CeO₂NPs may help mitigate this damage by acting as antioxidant nanozymes that neutralize ROS and improve plant stress tolerance. In addition, CeO₂ nanoparticles may interact with metal ions and influence their mobility, uptake, and detoxification processes in plant tissues. These properties make CeO₂NPs promising candidates for enhancing plant tolerance to heavy metal stress and improving phytoremediation efficiency [21–23].

Organic acids and nanomaterials have independently shown potential in improving plant tolerance to heavy metal stress. Organic acids such as glutamic acid can enhance metal chelation, mobility, and detoxification within plant tissues, thereby reducing metal toxicity. Similarly, cerium oxide nanoparticles (CeO₂NPs), due to their redox-active properties, can scavenge reactive oxygen species and improve plant antioxidant defense systems under stress conditions [24–27]. However, studies investigating the combined application of organic acids and nanoparticles in phytoremediation are still limited. It is hypothesized that their combined use may provide a synergistic effect, where organic acids enhance metal availability and transport, while nanoparticles mitigate oxidative stress, thereby improving both metal uptake and plant tolerance. Therefore, this study was designed to evaluate the combined effect of glutamic acid and CeO₂NPs on cadmium phytoremediation efficiency in marigold. The primary objectives of this study are (a) to examine the effects of Cd on the morphology and physiology of marigold (b) to evaluate the impact of GA and CeO₂NPs on marigold growth attributes, and (c) to assess the combined effect of GA and CeO₂NPs in facilitating Cd removal by marigold.

Materials and methods

Preparation of cerium oxide nanoparticles (CeO₂NPs)

Cerium oxide nanoparticles (CeO₂NPs) used in this study were synthesized following the precipitation method [28], where detailed physicochemical characterization of the nanoparticles was previously reported. Briefly, a cerium precursor solution was subjected to controlled precipitation, followed by washing and drying to obtain CeO₂ nanoparticles. According to the original study, nanoparticles prepared using this method typically exhibit nanoscale particle sizes and predominantly spherical morphology as confirmed by X-ray diffraction (XRD) and scanning electron microscopy (SEM). CeO₂NPs were applied as a foliar spray at a concentration of 150 mg L⁻¹, which was selected based on previous studies reporting beneficial effects of CeO₂ nanoparticles on plant growth and stress tolerance under abiotic stress conditions [29].

Experimental treatments and design

Seeds of marigold were procured from the Punjab Seed Corporation, Government of Punjab, Lahore. In the soil-filled styrofoam trays, seeds were sown and raised to full maturity and health. Following fifteen days of seed germination, the juvenile plants were carefully rooted out of the soil, washed with fresh water, and then placed in the hydroponic system that has been set up and built in the research laboratory of the Environmental Sciences Department at the University of Gujrat, Pakistan. Glass containers with a 5.0 L capacity provided the plants with nutrients supplied through Hoagland nutrient solution. The hydroponic system was supplied with Hoagland nutrient solution prepared according to the standard formulation, containing macronutrients (KNO₃, Ca(NO₃)₂, KH₂PO₄, and MgSO₄) and micronutrients (Fe-EDTA, H₃BO₃, MnCl₂, ZnSO₄, CuSO₄, and (NH₄)₆Mo₇O₂₄). Aeration pumps were used to aerate the plants continuously. The hydroponic nutrient solution was not completely replaced during the 28-day experimental period. However, the solution volume was monitored regularly and replenished with fresh Hoagland nutrient solution to compensate for water loss due to evaporation and plant uptake. The pH of the nutrient solution was also monitored and maintained between 5.8 and 6.3 using dilute H₂SO₄ or NaOH. After one week of acclimatization in the hydroponic system, the plants were treated with different concentrations of GA, Cd, and CeO₂ nanoparticles. The plants were grown under controlled conditions with a temperature of 25 ± 2 °C, relative humidity of 60–70%, and a photoperiod of 16 h light / 8 h dark. These conditions were kept stable throughout the experiment. The concentration of glutamic acid (10 mM) was selected based on previous studies reporting its beneficial effects on plant growth and stress tolerance under heavy metal stress conditions [30]. The CeO₂NPs suspension (150 mg L⁻¹) was applied as a foliar spray using a hand sprayer until the leaf surfaces were uniformly wetted (approximately 10–15 mL per plant). Applications were performed once per week throughout the 28-day experimental period. The spraying was carried out in the morning to ensure uniform coverage and minimize evaporation losses. Each treatment was arranged in a completely randomized design with three replicates. Each hydroponic container served as one replicate and contained four marigold plants. In 5-liter glass containers, marigold plants were cultivated with and without combinations of CeO2 NPs, GA, and Cd. Given treatments were applied weekly in triplicate to plants in various concentrations: where

T1: CK, T2: GA10 mM, T3:CeO2NPs 150 mg, T4:GA10 mM + CeO2NPs 150 mg, T5:Cd 1 mM, T6: GA 10mM + Cd 1 mM, T7:CeO2NPs 150 mg + Cd 1 mM, T8:GA 10 mM + CeO2NPs 150 mg + Cd 1 mM, T9:Cd5 mM, T10:GA10 mM + Cd 5 mM, T11:CeO2NPs 150 mg + Cd 5 mM, and T12:GA 10 mM + CeO2NPs 150 mg + Cd 5 mM.

Measurement of agronomic characteristics

After 28 days of treatment, marigold plants were harvested, and their morphological and biochemical characteristics were evaluated. The fresh weight (FW) of the stems, roots, and leaves of the harvested plants was measured using an analytical balance (BA-P2104, Infitek Co., Ltd., China). The dry weight (DW) of the stems, roots, and leaves was determined by drying the fresh plant samples at 90 °C until constant weight for biomass and metal analysis. Other parameters, such as the number of leaves (NOL), plant height, and root length under different treatments, were measured using a measuring scale, and data were recorded accordingly.

Determination of soluble protein and antioxidant enzymes

The amount of soluble protein was determined by using the method of Bradford [31], in which following a four-week treatment with a standard (bovine serum albumin) and dye (Coomassie Brilliant Blue G-250). The fresh plants were mashed with a pestle and mortar under cold conditions. Following this, the plants were mixed with a buffered solution of sodium phosphate and 1.0 mM EDTA in 10 mL that included poly-vinylpyrrolidone 40 (2%, w/v). The previously produced extract was utilized to measure the number of proteins and antioxidant enzymes (SOD and POD) by centrifuging the supernatant for 15 min at 11,000 rpm and 4 °C. The absorbance at 595 nm was measured to determine the actual protein concentration. For such purpose, 100 µL of extract was well mixed, and 1.0 mL of Bradford solution was added to create a homogenized solution. For the measurement of the antioxidant enzyme catalase (CAT), the technique provided by Aebi [32] was used. For such a purpose, a solution containing 100 µL of enzyme extract, 100 µL of hydrogen peroxide, and 2.8 mL of phosphate buffer (50 mM with 2.0 mM of citric acid, pH 7.0) was prepared. The quantity of CAT was determined by measuring the variation in light absorbance at 240 nm, which was caused by the loss of hydrogen peroxide (ε = 39.4 mM^− 1 cm^− 1). The ascorbate peroxidase (APX) amount and activity were measured using the method of Nakano and Asada [33]. For such purpose again a 3.0 mL mixture was made up of 2.7 mL of phosphate buffer solution (50 mM with 2.0 mM citric acid, pH 7.0), 100 µL of ascorbate, 100 µL of enzyme extract, and 100 µL of hydrogen peroxide (30 mM). Because of its oxidation, APX’s wavelength variations at 290 nm were measured to determine its quantity (ε = 2.8 mM^− 1 cm^− 1).

Determination of malondialdehyde (MDA) and hydrogen peroxide contents (H₂O₂)

The H₂O₂ concentration was measured by preparing the mixture. To create the mixture, 50 mg of fresh plants were homogenized with a 3.0 mL phosphate buffer solution, and the mixture was centrifuged for 25 min at 4 °C and 6000×g. Further, the extracts of 3.0mL were combined with a solution of 1.0 mL sulfuric acid (20%, v/v) and 0.1% titanium sulfate and again centrifuged at 6000×g and 4 °C for 15 min. Using a wavelength of 410 nm, the changes in the supernatants’ yellow colour were observed. The hydrogen peroxide content was then calculated using an extinction coefficient of 0.28 µmol⁻¹cm^− 1. As reported by Heath and Packer [34], the concentration of malondialdehyde, a product of lipid peroxidation, was evaluated in plant tissues using the reaction of the thiobarbituric acid (TBA) technique, with minor modifications made later by Zhang and Kirkham [35] and Dhindsa et al., [36].

Measurement of chlorophyll and carotenoid content

Following the four weeks of the experiment, the carotenoid and chlorophyll contents of the harvested plant were assessed. By the approach of Metzner et al., [37], Chlorophyll content was assessed, where plant fresh cultivated leaves were nourished at a temperature 4 °C in the solution of acetone (85% v/v) to extract the colour of leaves under the dark, by shaking the solution continuously. After that, the solution was centrifuged for 10–15 min at 4000 rpm at 4 °C of temperature to extract the supernatants. The solution was then further examined using a spectrophotometer device (Dynamic Company, London, Halo DB-20/DB20S, UK) to measure the amount of carotenoid and chlorophyll at 452, 644, and 663 nm. Further measurements were made using the Lichtenthaler [38] equation.

Measurement of SPAD

With assistance from the SPAD-502 SPAD meter, the chlorophyll contents were measured on the second topmost completely grown leaf by means of the analysis of the soil plant growth (SPAD) meter value.

Measurement of heavy metal concentration and accumulation

Following four weeks of treatment, the marigold’s Cd concentration was evaluated. Plant samples were dried at 90 °C until constant weight prior to metal analysis, and after six hours at 600 °C in a muffle furnace, they were reduced to ash. After the addition of 3mL of a strong solution of HCL and HNO₃ to 50 mL of distilled water, the ash samples were dissolved in the solution. The Perkin Elmer Analyst 100 (USA) technique was used to measure the concentration of Cd. The formula below was used to compute the metal concentration

Where,

The concentration of the heavy metal Cd in all parts of the plant was measured by the formula provided by Zayed et al. [39]:

Assessment of electrolyte leakage

The method employed for evaluating electrolyte leakage was provided by Dionisio-Sese and Tobita [40]. When four weeks (28 days) of treatment were over, the topmost leaves of the marigold were divided into long pieces of 5 mm and then placed into 8 mL filled deionized water test tubes. After that, the initial electrical conductivity (EC₁) was measured in a water bath over the course of two hours at 32 °C. First, the test tubes with the same samples as EC₁ were autoclaved for 20 min at a temperature of 121 °C to release the number of electrolytes in a solution. Upon reaching room temperature (25 °C), the samples were allowed to cool before the second electrical conductivity (EC₂) was assessed by utilizing the conductivity/pH meter version (Model-720, Company INCO-LAB, Kuwait). The following formula was used to determine electrolyte leakage:

Statistical analysis

All data obtained was analyzed using Statistix 10.0 software. A one-way analysis of variance (ANOVA) was conducted to determine the significance of differences among treatments. Tukey’s Honest Significant Difference (HSD) test was used for multiple comparisons at a significance level of p < 0.05. Data were expressed as mean ± standard deviation, and treatments with significantly different means are denoted by different lowercase letters in tables and figures.

Results

Agronomic traits of marigold

Significant variations were observed in marigold agronomic traits, including plant height, root length, and leaf number (Table 1). Compared with control treatments, a significant decrease was observed in treatments under Cd stress, particularly at higher Cd concentrations (5 mM). Cadmium application at 5 mM results in a reduction in the average height of the plant 52%, root length by 48%, and number of leaves by 50%, respectively, as compared to control plants. Similarly, application of increased Cd to the fresh weight (FW) of roots, stems, and leaves was reduced by -51%, -65%, and − 62% and dry weight (DW) of roots, stems, and leaves reduced by -63%, -54%, -48% as compared with the control. Moreover, increased plant growth and biomass were observed in such treatments where GA and CeO₂NPs were applied together in combination in the presence and absence of Cd. A notable improvement in the agronomic traits of marigolds, due to the application of GA and CeO2NPs, was observed compared with the control. The application of GA and CeO₂NPs improved the plant height (PH) by 27–43%, roots length (RL) by 28–75%, no. of leaves (NOL) by 43–86%, roots fresh weight (FWR) by 43–101%, stem fresh weight (FWS) by 32–66%, leaf fresh weight (FWL) by 40–68%, root dry weight (DWR) 38–74%, stem dry weight (DWS) by 34–60%, and leaf dry weight (DWL) by 30–64% under increased Cd stress, respectively as compared to control treatments.

Table 1.

Effect of cadmium, glutamic acid and cerium oxide nanoparticles on Agronomic Traits of marigold cultivated hydroponically with various Cd concentrations (0, 1 and 5mM). Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p < 0.05

Cd Concentration in mM
Root Length(cm)	Plant Height (cm)	Number of Leaf plant ^− 1	Roots Fresh Weight (g)	Stem Fresh Weight (g)	Leaf Fresh Weight (g)	Roots Dry Weight (g)	Stem Dry Weight(g)	Leaf Dry Weight (g)
Cd 0(mM)
6.6 ± 0.2^de	15.4 ± 0.2^bcd	9.3 ± 0.6^bc	11.2 ± 1^c	12.4 ± 0.3^d	10.8 ± 0.6^c	6.9 ± 0.2^d	7 ± 0.3^cd	6.6 ± 0.4^c
7.5 ± 0.2^bc	16.6 ± 0.3^bc	10.6 ± 0.6^b	13.2 ± 0.3^b	14.4 ± 0.1^c	13.3 ± 0.6^b	7.7 ± 0.2^c	8 ± 0.2^bc	6.7 ± 0.2^bc
7.8 ± 0.1^ab	17.8 ± 0.3^ab	11 ± 1^b	14.9 ± 0.9^a	15.3 ± 0.3^b	14.3 ± 0.2^ab	8.5 ± 0.3^b	8.4 ± 0.3^b	7.5 ± 0.4^b
8.5 ± 0.2^a	19.5 ± 0.5^a	13.3 ± 0.6^a	16 ± 0.4^a	16.5 ± 0.4^a	15.2 ± 0.2^a	9.5 ± 0.2^a	9.8 ± 0.2^a	8.6 ± 0.4^a
Cd 1(mM)
4.4 ± 0.2ⁱ	10.6 ± 0.6^fg	5.3 ± 0.6^ef	8.3 ± 0.1^f	6.9 ± 0.2^g	7.3 ± 0.5^e	4.6 ± 0.4^f	4.6 ± 0.4^fg	5.5 ± 0.4^d
5.2 ± 0.2^gh	12.2 ± 0.4^ef	6.6 ± 0.6^cd	9 ± 0.2^ef	7.6 ± 0.1^fg	7.8 ± 0.3^e	5.4 ± 0.2^e	5 ± 0.2^efg	5.8 ± 0.3^cd
6 ± 0.2^ef	13.2 ± 0.3^de	8 ± 0^cd	9.7 ± 0.2^de	8.3 ± 0.2^f	8.8 ± 0.3^d	5.9 ± 0.1^e	5.7 ± 0.1^e	6.3 ± 0.2^cd
6.8 ± 0.2^cd	14.6 ± 0.3^cde	8.7 ± 0.6^c	10.6 ± 0.3^cd	9.5 ± 0.2^e	9.8 ± 0.4^c	6.9 ± 0.2^d	6.6 ± 0.2^d	6.7 ± 0.2^bc
Cd 5(mM)
3.4 ± 0.4ⁱ	7.4 ± 0.3^h	4.6 ± 0.6^f	5.5 ± 0.3^g	4.3 ± 0.1ⁱ	4.1 ± 0.1^g	2.6 ± 0.4^h	3.4 ± 0.3ⁱ	3.4 ± 0.3^f
4.6 ± 0.3^hi	8.4 ± 0.2^h	5.3 ± 0.6^ef	6.7 ± 0.3^g	4.3 ± 0.1^hi	5.1 ± 0.1^fg	3.5 ± 0.1^g	3.6 ± 0.5^hi	3.6 ± 0.3^ef
5.3 ± 0.3^fg	9.2 ± 0.1^gh	6.3 ± 0.6^ef	8.2 ± 0.3^f	5.2 ± 0.2^h	5.6 ± 0.3^f	4.2 ± 0.2^fg	4.4 ± 0.3^gh	4.5 ± 0.1^e
6 ± 0.1^ef	10.6 ± 0.5^fg	8.7 ± 0.7^c	11.1 ± 0.1^c	5.1 ± 0.7^g	6.9 ± 0.2^e	4.4 ± 0.1^f	5.4 ± 0.3^ef	5.5 ± 0.4^d

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Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p <0.05

Photosynthetic pigments of marigold

The results show a decrease in photosynthetic pigments with increasing Cd concentration (Fig. 1). The greatest reduction in marigolds was observed at a 5 mM Cd concentration, where chlorophyll a and b decreased by 51% and 61%, respectively; total chlorophyll decreased by 56%; and carotenoid content decreased by 73% compared with the control specimens, as shown in Fig. 1. Application of GA and CeO₂NPs improved the chlorophyll and carotenoid content of marigold which were under Cd stress as compared with the control. Furthermore, the greatest increases in chlorophyll and carotenoid content were observed in treatments in which GA and CeO₂NPs were applied simultaneously, in the absence of Cd, compared with the control. However, chlorophyll and carotenoid contents were also improved in treatments in which GA and CeO2NPs were applied under Cd stress. Low Cd concentrations, such as 1.0 mM, show greater increases in chlorophyll and carotenoids than treatments in which 5.0 mM Cd is applied along with GA and CeO₂NPs. Hence, the application of GA and CeO₂NPs in increased Cd stress increases the contents of chlorophyll a and b by 37–73%, 44–147%, total chlorophyll by 40–101%, and carotenoids by 28–100%, respectively, as compared to the control.

SPAD value and soluble proteins in marigold

The soluble proteins and SPAD value of the marigold were measured in control and in the presence of Cd. A measurable decrease in the value of SPAD and soluble protein was recorded in marigolds with increased concentrations of Cd (Fig. 2). The maximum decrease in soluble protein content and SPAD value was observed under Cd 5 mM where SPAD value decreased by -51%, and soluble protein in roots and leaves by -48% and − 52%, respectively as shown in Fig. 2. The maximum observed SPAD value was in the treatment in which GA and CeO₂NPs applied in the absence of Cd. Moreover, the application of GA and CeO₂NPs under Cd stress improved the SPAD value of marigold by 12–50%, and the content of SP leaves by 33–56%, and SP roots by 38–65%, respectively as compared with the control.

Fig. 2 — Effect of cadmium, glutamic acid and cerium oxide nanoparticles on SPAD value (A) and Soluble Protein in root and leaf (B, C) in marigold cultivated hydroponically with various Cd concentrations (0, 1 and 5mM). Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p < 0.05

Oxidative stress biomarkers in marigold

For the assessment of oxidative stress in experimental marigolds influenced by Cd stress, reactive oxygen species (ROS), H₂O₂, MDA, and EL were measured in plant leaves and roots. Electrolyte leakage was used to assess cell membrane damage under Cd stress, thereby indicating the stress-tolerant levels of marigold across different treatments. H₂O₂, MDA, and EL increase in plants as the concentration of Cd increases (Fig. 3). The results show that, with increasing Cd concentration, electrolyte leakage (EL) % in leaves and roots increases by 82% and 115%, H₂O₂ in roots and leaves by 274% and 501%, and MDA in roots and leaves by 162% and 161%, as shown in Fig. 3. The highest electrolyte leakage (EL) % was observed in treatments with only Cd concentrations, compared with treatments that also included GA and CeO₂NPs. The results depict that application of GA and CeO₂NPs under Cd stress decreases the electrolyte leakage from roots and leaves of marigold by -7% to -16%, H₂O₂ by 41% to -13% and 66% to -17%, MDA by -37% to -13% and − 34% to -24% respectively, as compared with control.

Fig. 3 — Effect of cadmium, glutamic acid and cerium oxide nanoparticles on Electrolyte leakage in root (A), Electrolyte leakage in leaf (B) and MDA in root (C), MDA in leaf (D), H₂O₂ in root (E) and H₂O₂ in Leaf (F) of marigold cultivated hydroponically with various Cd concentrations (0, 1 and 5mM). Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p < 0.05

Cadmium concentration and accumulation in marigold

Results revealed that an increase in Cd accumulation accompanied an increase in Cd concentration in the experimental medium of marigolds. As previously observed, a similar trend was observed for the accumulation of Cd in marigolds in the presence and absence of GA and CeO₂NPs (Table 2). The concentration of Cd increased in roots by 89%, stem by 101%, and in leaves by 120%. Accumulation of Cd in roots increased by 20%, stem by 48%, and leaves by 36%. Maximum Cd accumulation in stems, roots, and leaves of marigold was observed in the treatments where GA and CeO₂NPs were applied simultaneously, as shown in Table 2. A significant accumulation of Cd in roots, stems, and leaves was observed in the treatments using 5.0 mM Cd, compared with the control. However, the application of GA and CeO₂NPs improved the rate of concentration of Cd in marigold roots by 82 − 37%, in stem by 28–29%, and in leaves by 18–34%, as compared with the respective control. GA may facilitate Cd chelation and improve its translocation and compartmentalization within plant tissues, while CeO₂NPs act as redox-active nanomaterials that enhance antioxidant defense systems and reduce oxidative stress. This synergistic interaction allows plants to tolerate higher Cd levels while maintaining growth and improving overall phytoremediation efficiency. Furthermore, the application of GA and CeO₂NPs also improves the rate of Cd accumulation in marigold roots by 77–108%, stem by 70–106%, and leaves by 54–119%, which were under stress by different concentrations of Cd, as compared with the control treatment, respectively.

Table 2.

Effect of cadmium, glutamic acid and cerium oxide nanoparticles on Cd concentration and accumulation of marigold cultivated hydroponically with various Cd concentrations (0, 1 and 5mM). Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p < 0.05

Treatments	Cd Concentration (µg g^− 1)			Cd Accumulation (µg plant^− 1)
Treatments	Root	Stem	Leaf	Root	Stem	Leaf
Cd 0(mM)
CK	1.8 ± 0.5ⁱ	1.3 ± 0.1^h	1.2 ± 0.1ⁱ	12.3 ± 3.7^f	9.8 ± 0.2^f	7.8 ± 1^g
GA	2.4 ± 0.3ⁱ	1.5 ± 0.1^h	1.2 ± 0.1ⁱ	18.4 ± 2.1^f	11.8 ± 0.7^f	8.3 ± 0.6^g
CeO₂NPs	2.6 ± 0.2ⁱ	1.7 ± 0.1^h	1.5 ± 0.1ⁱ	21.7 ± 0.1^f	14.4 ± 1.1^f	11 ± 0.9^g
GA + CeO₂NPs	3.2 ± 0.4ⁱ	1.7 ± 0.1^h	1.4 ± 0.1ⁱ	31 ± 4.3^f	16.7 ± 1^f	12 ± 0.8^g
Cd 1(mM)
CK	142.8 ± 4.7^h	124.1 ± 4.5^g	102.3 ± 4^g	659.3 ± 48^e	568 ± 60^e	563 ± 15^f
GA	159.4 ± 3.9^g	137.9 ± 3.6^f	116 ± 3.4^g	859 ± 11.8^cd	712 ± 44^de	677 ± 22^ef
CeO₂NPs	176.5 ± 2.9^f	152.8 ± 3.4^e	128.7 ± 2.5^f	1035 ± 32^c	876 ± 41^cd	818 ± 42^de
GA + CeO₂NPs	201 ± 10^e	181.6 ± 9.9^d	149 ± 5.5^e	1395 ± 114^b	1201 ± 79^b	1007 ± 24^c
Cd 5(mM)
CK	269 ± 1.9^d	248 ± 4.1^c	224 ± 5.1^d	789 ± 112^de	843 ± 88^cd	767 ± 80^de
GA	282 ± 3.5^c	264 ± 3.5^b	237 ± 3.7^c	974 ± 45^c	944 ± 119^c	866 ± 61^cd
CeO₂NPs	339 ± 5.5^b	309 ± 6.1^a	291 ± 3.9^b	1415 ± 99^b	1366 ± 102^b	1320 ± 25^b
GA + CeO₂NPs	369 ± 3.1^a	320 ± 4.1^a	301 ± 3.1^a	1639 ± 54^a	1732 ± 107^a	1681 ± 136^a

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Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p<0.05

Antioxidant enzyme activities in marigold

Activities of the major antioxidant enzymes such as SOD, POD, CAT, and APX were assessed in the leaves and roots of criterion marigold to determine the effect of Cd stress on the antioxidant defense mechanism of plants by the application of GA and CeO₂NPs (Fig. 4). The maximum increase was seen under Cd stress for SOD in root by 395%, SOD in leaf by 668%, POD in root by 144%, POD in leaf by 408%, APX in root by 492%, APX in leaf by 332%, CAT in root by 242%, CAT in leaf by 246%, respectively, than control as shown in Fig. 4. Applications of GA and CeO2 NPs improve plants’ defense mechanisms against Cd stress compared with Cd application alone. Application of GA and CeO₂NPs increased the concentration of SOD in root by 30–51%, SOD in leaf by 23–111%, POD in root by 21–64%, POD in leaf by 30–126%, APX in root by 62–150%, APX in leaf by 25–85%, CAT in root by 24–33%, CAT in leaf by 23–76% under Cd stress as compared with the control, respectively.

Fig. 4 — Effect of cadmium, glutamic acid and cerium oxide nanoparticles on Antioxidant enzymes SOD in root (A) SOD in leaf (B), POD in root (C), POD in leaf (D), APX in root (E) APX in leaf (F), CAT in root (G), CAT in leaf (H) of marigold, cultivated hydroponically with various Cd concentrations (0, 1 and 5mM). Values are expressed as means ± standard deviation (S.D) of three replicates. Different lowercase letters within each graph bar denote significant differences at p < 0.05

Discussion

Ornamental plants are known for their natural ability to accumulate and uptake HMs [10]. Previous studies have demonstrated that marigolds accumulate HMs and other pollutants [41–45]. Marigold (T. patula) accumulates heavy metals such as Cd due to its inherent tolerance to these contaminants [44, 46], and the addition of GA can improve marigold plant growth, biomass, and HM uptake [47–49]. The application of CeO₂NPs improves marigold growth and enables their translocation and accumulation in marigolds [50]. The present research was conducted primarily to investigate the synergistic effect of GA and CeO₂NPs on marigold plants under Cd stress.

Increased Cd concentrations significantly reduce marigold growth. Recent studies indicate that the threshold level for marigolds’ tolerance to Cd is between 75 and 150 mgkg^− 1 of plant mass [51], suggesting that higher Cd concentrations can inhibit marigold growth [52]. The present study demonstrates that the application of GA (10 mM) enhances the agronomic characteristics of marigold, whereas the combined application of GA (10 mM) and CeO₂NPs (150 mgL^− 1) further promotes growth and biomass accumulation. Similarly, a study reported that the application of GA at 5.0 mM affects the growth of sunflower plants under Cr stress [53]. Saffari et al. [54] demonstrated that chelating agent application in Cd-spiked soil improves and boosts plant physiological characteristics, but higher Cd concentration inhibits plant physiological features. The results of this study also highlight the importance of the combined application of GA and CeO₂NPs compared with their individual treatments. Although both GA and CeO₂NPs independently increased plant growth and reduced Cd-induced stress, the combined treatment consistently showed greater enhancements in growth parameters, photosynthetic pigments, antioxidant enzyme activities, and Cd accumulation. This suggests a synergistic interaction between GA and CeO₂NPs, where GA may enhance Cd chelation and mobility, while CeO₂NPs mitigate oxidative stress through ROS scavenging mechanisms. Such combined strategies may therefore provide a more effective approach for improving plant tolerance to heavy metal stress compared with single remediation approaches.

The study shows that increasing the Cd dose (1 mM, 5 mM) causes a significant reduction in leaf greenness, which is attributed to the disruption of photosynthetic pigment activity, specifically carotenoids and chlorophyll, in marigold compared to the control and treatments with GA and CeO₂NPs. Carotenoids play a protective role by safeguarding plant membranes, genetic material, and chlorophyll from reactive oxygen species (ROS). Metal stress in plants reduces carotenoid concentrations by inhibiting photosynthesis and inducing oxidative stress [55, 56]. A decrease in the chlorophyll and carotenoid content in marigolds was reported by Fatima et al. [41]. Application of GA affects the content of chlorophyll in marigold, which shows the effect on photosynthesis and biomass as reported by Farid et al., in a research of phyto-management of Cr with sunflower [30]. Furthermore, the decrease in photosynthetic pigments may be attributed to the production of reactive oxygen species (ROS) under Cd exposure. However, in treatments where CeO₂NPs were applied, the activity of photosynthetic pigments improved, likely due to their antioxidant properties [50, 57–59].

A similar work on Bamboo using titanium dioxide nanoparticles (TiO2 NPs) was performed for phyto-protection against abiotic stress by [60] The results provide a similar trend of research findings that while Cu and Pb increased the generation of reactive oxygen species (ROS) compounds in plants, TiO2 NP treatments played a positive role in reducing oxidative stress, as indicated by the decrease in ROS compounds, the extent of lipoperoxidation, and soluble proteins. Additionally, the use of TiO₂NPs increases the total antioxidant capacity, chlorophyll content, and general plant biomass, and it may be concluded that TiO₂NPs can reduce oxidative stress in plants by increasing the antioxidant capacity. In another similar work [61], give a similar outcome with sodium nitroprusside (SNP), to alleviate plant stress against metals.

Plants can accumulate and take up heavy metals (HMs) depending on the chemical nature and behavior of metals and their cofactors. Marigold is widely recognized as a hyperaccumulator due to its strong tolerance to Cd metal stress [56, 62–65]. In the present study, Cd accumulation gradually increases on increasing Cd concentration in the medium of growth. Many studies reported accumulation of Cd in different parts of marigold in the order roots > stems > leaves [62, 66]. In our results, using GA under Cd stress dramatically boosted Cd accumulation and absorption. Many studies reported Cd concentrations in marigold TFs > 1 [45, 55, 62, 63], indicating strong tolerance of marigold to Cd stress. This higher accumulation might be attributable to the GA’s chelating characteristics, which can detach metals such as Cd from organic components in water and form a soluble complex that plants can readily take up [53]. Our results have similarities with recent studies showing that certain treatments like GA and CeO₂NPs, can simultaneously enhance heavy metal uptake and mitigate metal-induced toxicity. This synergistic effect is due to the role of GA as a chelating agent, which binds to Cd ions, forming less toxic complexes that reduce the immediate physiological damage to the plant while still allowing uptake. Similarly, CeO₂NPs possess antioxidant properties that scavenge reactive oxygen species (ROS), protect cellular structures, and support photosynthetic activity under stress conditions. Therefore, while Cd accumulation increases, the toxicity is buffered by the presence of GA and CeO₂NPs, enabling the marigold to maintain or even improve growth performance. These findings show consistency with earlier research demonstrating that stress-mitigating agents can allow plants to tolerate and accumulate higher levels of heavy metals without suffering typical growth reductions [53, 57]. Chelating chemical GLDA in a study has proven its capacity to increase Cd accumulation in plant cells (French marigold) by facilitating its transfer from roots to shoots and promoting the fixation of cell walls [49]. Furthermore, the application of CeO₂NPs in the current study modified the uptake and accumulation of Cd across various treatments, as reported in many previous studies in which CeO2NPs have shown an effect on Cd uptake and accumulation by plants [57, 59]. In the combined application of GA and CeO₂NPs, a significant synergistic effect is observed, improving marigold tolerance to Cd stress. However, the specific chemical or physical interactions between GA and CeO₂NPs in the hydroponic solution were not characterized in the present study. It is possible that GA can influence the dispersion, surface charge, or reactivity of CeO₂NPs, thereby modifying their bioavailability or uptake in plants. Future studies employing spectroscopic or particle-characterization techniques (e.g., zeta potential analysis, DLS, FTIR) would be valuable for elucidating the dynamics of this interaction and its influence on metal mobility, plant uptake, and nanoparticle behavior in aqueous systems.

The SPAD value and protein synthesis shows a decrease in plants of marigold when treated with higher concentration of Cd, the decrease may occur due to the over production of reactive oxygen species (ROS) because of the metal ions and other environmental factors, Results obtained in the present work have strong agreement with the conclusions of Shi et al., [64] and Fatima et al., [41] whose studies depict decrease in SPAD value and content of soluble protein of marigold when treated with higher concentration of Cd. However, in the present study, the application of GA and CeO₂NPs contributed to increased SPAD values and soluble protein content across various marigold treatments. Adding chelating agents such as Citric acid in T. erecta can improve the carotenoids and protein content, as proved by Aghelan et al. [43] and Fatima et al. [41]. Farid et al. [53], who support the present study to concluded that the GA application in plants greatly enhances the SPAD value. Furthermore, Mohammadi et al. [67] studied that the application of CeO2NPs increases the protein synthesis in plants, which also supports our research work.

Plants possess a suite of antioxidant defense mechanisms that comprise both enzymatic and non-enzymatic components for scavenging ROS [68–70]. The present study concluded that the controlled application of Cd, in combination with GA and CeO₂NPs signify the activity of antioxidant enzymes, as our results indicate. Sharma et al. [71] also reported that antioxidant enzymes in plants, such as SOD, APX, and POD, are released due to metal accumulation. Collectively, these mechanisms prevent plant cells from oxidative damage. Farooq et al. [52] reported that the application of 0.1 g/kg of Cd in soil increased the activities of POD, SOD, APX, and CAT by 60%, 150%, 97%, and 79%, respectively, in Calendula calypso.

As reported in earlier studies, plants experience oxidative stress and damage to cellular proteins, nucleic acids, and lipids due to increased ROS production within cellular compartments under transition metals and other environmental stressors [72]. The protective effect observed in this study may be related to the redox-active nature of CeO₂ nanoparticles, which can cycle between Ce³⁺ and Ce⁴⁺ states and act as ROS scavengers, thereby reducing Cd-induced oxidative damage in plant tissues. Many researchers have reported that increased H₂O₂ and MDA generation are under metal stress. Some indicators of damaged cells include the generation and accumulation of MDA [73, 74]. Stress responses are influenced by the kind of heavy metal, concentration, absorption, and accumulation in the plant tissues [67, 75]. In C. officinalis, MDA and H₂O₂ generation increased with increasing Cd concentration; however, metal stress was mitigated by chelating agents such as TA and CA, and H₂O₂ production also decreased [53, 54]. The application of GA can decrease the EL content in sunflowers.

Previous studies have demonstrated that GA functions as a chelating agent, enhancing metal uptake in plants, protecting plants against ROS overproduction, and promoting plant growth [53]. The addition of GA to the medium enhances plant physiological indices against Cd, and the combination of GA and CeO2NPs increases the maximum accumulation level. Similar findings were reported by Fatima et al. [41]. These results indicate that exogenous administration of CeO2NPs at moderate doses may reduce oxidative stress while optimizing marigold development, particularly photosynthetic characteristics and leaf greenness. While this study assessed key morphological, physiological, and biochemical indicators such as biomass, chlorophyll content, antioxidant activity, and metal accumulation, some additional parameters, particularly those related to nutrient uptake (e.g., nitrogen, phosphorus, potassium), gas exchange, and hormonal signaling, could provide a more comprehensive understanding of marigolds’ growth response. Future research should incorporate a broader range of nutritional and physiological markers to further elucidate the mechanisms underlying enhanced growth under Cd stress and under GA and CeO₂NPs treatment. Although the present study focused on physiological and biochemical indicators of Cd stress mitigation, future studies integrating transcriptomic or proteomic approaches would help to further elucidate the molecular mechanisms underlying the interaction between glutamic acid, CeO₂ nanoparticles, and heavy metal stress responses in plants. It is important to note that this study was conducted under hydroponic conditions, which allow controlled evaluation of plant responses but may not fully represent the complexity of natural soil environments. Therefore, future studies should evaluate the combined effects of glutamic acid and CeO₂ nanoparticles under soil and field conditions to better assess their practical application in phytoremediation.

Conclusions

This study demonstrated that marigold plants can effectively uptake and accumulate Cd in a hydroponic system, which negatively impacts their growth and physiological traits. Cd exposure led to reductions in root length, plant height, leaf number, and both fresh and dry weights, along with decreased chlorophyll and carotenoid content and SPAD values, and increased EL. The generation of SOD, POD, CAT, APX, and ROS also increased with higher Cd concentrations. The application of GA and CeO₂NPs mitigated these adverse effects by improving plant growth, SPAD values, soluble protein content, and antioxidant defense mechanisms, while reducing electrolyte leakage and ROS and enhancing photosynthetic pigments. These treatments also promoted Cd accumulation, particularly in the roots. In conclusion, while Cd exposure adversely affects marigold plants, GA and CeO₂NPs alleviated Cd stress and enhanced Cd accumulation under controlled hydroponic conditions, suggesting their potential for enhancing phytoremediation strategies. Although the current findings demonstrate a promising synergistic effect of GA and CeO₂NPs in enhancing Cd phytoremediation, the underlying interaction between these two agents in the aqueous environment remains unexplored. However, further studies under soil and field conditions are required to evaluate the practical applicability and scalability of this approach.

Supplementary Information

Supplementary Material 1.^{(2MB, pdf)}

Author contributions

[Mujahid Farid]: Conceptualization, Project administration, Supervision, Writing- original draft. [Mujahid Farid][Muskan Shahzad]: Conceptualization, Writing – review & editing. [Muhammad Zubair]: Writing – original draft, Writing – review & editing. [Muskan Shahzad][Muhammad Zubair]: Methodology, Writing – review & editing. [Wajiha Sarfraz][Sheharyaar Farid]: Conceptualization, Project administration, Writing – original draft, [Mujahid Farid][Roha Murtaza]: Data curation, Formal analysis, Methodology, Writing – original draft. [Muhammad Munir][ Abdulrahman Alasmari] Data curation, Formal statistical analysis and data validation [Shafaqat Al]: Writing – original draft, Writing – review & editing. [Sarah Owdah Alomrani], Writing – review & editing. [Roha Murtaza][Mujahid Farid], Conceptualization, Project, Funding Acquisition, review & editing. All authors gave their feedback and input during the write-up, experimentation, analysis, data validation and proofreading of the present research work.

Funding

The authors extend their gratitude to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Kingdom of Saudi Arabia, for funding the publication of this work under the Ambitious Researcher program (Project No. KFU261412).

Data availability

All data generated or analyzed during this study is included in this article.

Declarations

Ethics approval and consent to participate

All authors gave their consent to participate in this research.

Consent for publication

All authors gave their consent to publish this research work.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Mujahid Farid, Email: mujahid726@yahoo.com.

Shafaqat Ali, Email: shafaqataligill@gcuf.edu.pk.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(2MB, pdf)}

Data Availability Statement

All data generated or analyzed during this study is included in this article.

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[CR74] 74.Zeeshan M, Lu M, Sehar S, Holford P, Wu F. Comparison of biochemical, anatomical, morphological, and physiological responses to salinity stress in wheat and barley genotypes differing in salinity tolerance. Agronomy. 2020;10(1):127. [Google Scholar]

[CR75] 75.Mittler R, Zandalinas SI, Fichman Y, Van Breusegem F. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol. 2022;23(10):663–79. [DOI] [PubMed] [Google Scholar]

PERMALINK

Synergistic effects of glutamic acid and cerium oxide nanoparticles on cadmium phytoremediation efficiency in marigold

Mujahid Farid

Muskan Shahzad

Muhammad Zubair

Sheharyaar Farid

Wajiha Sarfraz

Roha Murtaza

Muhammad Munir

Abdulrahman Alasmari

Sarah Owdah Alomrani

Shafaqat Ali

Abstract

Supplementary Information

Introduction

Materials and methods

Preparation of cerium oxide nanoparticles (CeO₂NPs)

Experimental treatments and design

Measurement of agronomic characteristics

Determination of soluble protein and antioxidant enzymes

Determination of malondialdehyde (MDA) and hydrogen peroxide contents (H2O2)

Measurement of chlorophyll and carotenoid content

Measurement of SPAD

Measurement of heavy metal concentration and accumulation

Assessment of electrolyte leakage

Statistical analysis

Results

Agronomic traits of marigold

Table 1.

Photosynthetic pigments of marigold

Fig. 1.

SPAD value and soluble proteins in marigold

Fig. 2.

Oxidative stress biomarkers in marigold

Fig. 3.

Cadmium concentration and accumulation in marigold

Table 2.

Antioxidant enzyme activities in marigold

Fig. 4.

Discussion

Conclusions

Supplementary Information

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Determination of malondialdehyde (MDA) and hydrogen peroxide contents (H₂O₂)