Physiological Behavior and Antioxidant Responses of Abelmoschus esculentus (L.) Exposed to Different Concentrations of Aluminum and Barium

Abstract

Soil contamination by trace metal elements, such as aluminum and barium, presents specific environmental risks, particularly to plant health and agricultural productivity. Excessive accumulation of these toxic elements in plant tissues can alter redox equilibrium and affect homeostasis. This study sought to examine the physiological reactions of Abelmoschus esculentus (L.) under aluminum- and barium-induced stress. The plants were exposed to multiple concentrations of Al or Ba (0, 100, 200, 400 and 600 µM) for 45 days; then, the accumulation potential of Al and Ba, oxidative damage, and antioxidative metabolism were assessed. Key findings showed a proportional distribution of the Al and Ba in roots and aerial parts of the plants, with lower accumulation in the fruits. The occurrence of oxidative damage and the involvement of antioxidant enzymes were demonstrated by increased amounts of malondialdehyde and H₂O₂, enhanced activity of superoxide dismutase, and decreased catalase activity. The study also highlighted that GSH played a primary role in Al detoxification in the roots and fruits, while phytochelatins were more active in Ba-treated plants, particularly in roots and shoots, facilitating Ba sequestration.

1. Introduction

Trace metallic elements (TMEs) are widespread in the environment mainly due to both natural occurrences (rock weathering, volcanic activity, and erosion) and anthropic effect, (including military training, electroplating, phosphate fertilizers, and pesticide use) and are commonly found in agricultural soils [1,2,3]. The excessive accumulation of TMEs in soils disrupts ecosystems and can eventually harm public health through the food chain by the contamination of agricultural produce, thus indirectly becoming a major factor that contributes to health-related hazards [4,5].

Among the TMEs, aluminum (Al) and barium (Ba) are ubiquitously distributed in soil. Al is classified as the third most available element in the Earth’s crust [6] and has a significant impact on marine and terrestrial ecosystems [7]. The toxicity of this element mostly depends on its solubility and exposure route [8]. Ba is the 14th most abundant element found in the Earth’s crust [9]. Negative effects on divers’ organs have been connected to an overabundance of Ba [10,11]. Excess Ba may also pose risks to health and the environment, particularly in regions associated with mining and waste disposal [12]. These two elements may profusely affect crop yield and productivity since plants are the most vulnerable organisms to soil pollution. Indeed, the severity of Al or Ba toxicity depends on stress concentration, stress duration, and plant type. It was reported that Al stress prompts a range of biochemical, physiological, morphological, and molecular modifications to growing plants, resulting in a diminishment of processes related to growth, development, and crop productivity [7].

Further investigations have verified that Al stress induces oxidative stress, which causes an increase in reactive oxygen species (ROS) and eventually leads to lipid peroxidation (LPO) [13,14]. Al disturbance within plant tissues could occur in different patterns. For example, in cucumber plants, Al was mostly accumulated in the roots [15]; however, hyperaccumulating plants exhibit a preference for accumulating a significant quantity of aluminum in their shoots [16]. However, Ba has not received a great amount of attention compared to Al; only a few studies have focused on the impact of this element on plants. Peana et al. [17] reported that plants can be severely impacted by high concentrations of harmful cations, such as Ba. This exposure can trigger undesirable alterations in their metabolic processes and stimulate the generation of ROS. Ba can be accumulated in various plant organs, which has been noted in some species such as Cucumis sativus [18], Abelmoschus esculentus (L.) [19], Limbarda crithmoides, and Heliantus annuus [20], where it was noted that the Ba remained in the roots and the shoots.

For plant species, maintaining a harmonious metabolic equilibrium is essential for enhancing productivity, a balance often disrupted by both biotic and abiotic stresses. Consequently, redox balance stands out as a fundamental aspect of life, as it involves the reduction of oxidized compounds to enable subsequent oxidation and the provision of energy [21]. The presence of a well-structured antioxidant system in plants has the potential to alleviate and counteract the impacts caused by oxidative stress [22]. This system comprises enzymatic antioxidants such as ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), along with non-enzymatic antioxidants including reduced glutathione (GSH) and ascorbate (AsA). These antioxidants, in conjunction (AsA-GSH), form a pathway that effectively reduces the levels of ROS, thus preserving cellular homeostasis from TME toxicity [23,24]. Besides the antioxidant system, plants possess the ability to regulate lower levels of metal/metalloid(s) within the cell using the metallothionein (MT) proteins family and phytochelatins (PCs) [21].

Abelmoschus esculentus L. (Okra) is classified under the Malvaceae family. Okra is grown all around the world from the Mediterranean Sea to equatorial areas. The utilization of okra encompasses various purposes, including its consumption as a vegetable, the utilization of its mucilage in industrial applications such as paper production and confectionery, and its medical applications as a blood volume expander or plasma substitute [25]. In Tunisia, it is a valuable crop, possessing cultural and economic value; it is extensively cultivated in the country and is a staple ingredient in traditional Tunisian cuisine. In fact, okra is prone to various abiotic stress factors like waterlogging, drought stress, cold temperatures, and frost conditions. Okra cultivars have developed specific adaptations in response to the prevailing climatic conditions in their growth environment [26].

Taking into consideration all of the above, this study was designed in order to study the behavior of Abelmoschus esculentus (L.) in response to Al and Ba exposure, as well as understanding the adaptation strategies to cope with this harmful situation and assess its tolerance limits. The main objectives were to examinate the accumulating potential of Al/Ba and the disturbance of these elements in the different parts of Abelmoschus esculentus (L.) and to investigate the extent of Al/Ba-triggered oxidative stress by determination of LPO and the endogenous contents of H₂O₂. These objectives were accomplished by the assessment of variations in antioxidant enzyme activities, including SOD, CAT, and GR, along with the levels of GSH and PCs in the various tissues of the investigated plant exposed to increasing concentrations of Al or Ba.

2. Material and Methods

2.1. Plant Culture

This research was carried out in the greenhouse at the Faculty of Sciences of Bizerte, with exposure to natural light, average temperatures (day/night) ranging from 25/12 °C, and relative humidity alternating from 60% to 88%. The cultivation was conducted in an inert substrate composed of a combination of gravel and perlite in a 1:2 (v/v) ratio and the plants were regularly watered with Hewitt nutritive solution [27] (pH = 7) at a volume of 100 mL/pot. This standardized nutrient medium provides essential macro- and micronutrients (N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, B, and Mo) to support optimal plant growth under controlled experimental conditions. Thirty days after sowing, the plants were separated into two sets for each treatment (Al and Ba). Each set was then further divided into five subgroups, each consisting of 10 plants, with respective treatments of 0 (control), 100, 200, 400, and 600 μM for each element. The different doses of the treatments were incorporated into the watering solution. The plants were harvested at the mature stage (after 45 days of treatment). Then, plants from each treatment were partitioned into root, aerial parts, and fruit, followed by a rinse with distilled cold water.

Roots were submerged in a cold CaCl₂ solution as described by Stolt et al. [28] to remove the adsorbed TME. The plant material was ground and directly frozen using liquid nitrogen and kept at −80 °C until analysis.

2.2. Growth

2.2.1. Relative Growth Rate

The relative growth rate (RGR) was calculated with Hunt’s formula [29] based on biomass production. The formula considers the fresh weight measured at the beginning (FW₁) and the end (FW₂) of the treatment, the duration of the treatment (Δt = t₂ − t₁), and the natural logarithm (Ln) [30].

RGR = (ln (FW₂) − ln (FW₁))/(t₂ − t₁)

2.2.2. Growth Percentage

The growth percentage (GP) was determined by expressing the growth as a percentage compared to the control, and it is calculated according the subsequent formula [31]:

GP = (FW treated/FW control) × 100

FW: Fresh weight.

2.3. TME Contents

Al and Ba quantification was terminated based on the method by Sleimi et al. [32], Bankaji et al. [33], and Kouki et al. [19]. Concisely, the mineralization process was conducted on 300 mg of the fresh plant material in Teflon bombs, and we exposed them to a 2 h digestion period at 110 °C with 3 mL of an acid blend (HNO₃/H₂SO₄/HClO₄ in a ratio of 10:1:0.5; v:v:v). The resulting solutions were further diluted with 50 mL of nitric acid (0.5%) and subsequently filtered to determine the contents of Al and Ba in the samples using an Atomic Absorption Spectrometer (Perkin Elmer PinAAcle 900T, Waltham, MA, USA).

2.4. Malondialdehyde (MDA)

MDA contents in plant parts were measured following Hodges et al. [34]. Plant material was ultrasonically processed in 80% cold ethanol then subjected to centrifugation at 4500 rpm for 20 min at 4 °C. The supernatant was combined with either 20% trichloroacetic acid (TCA) or a 20% solution of TCA and thiobarbituric acid (TBA). After 1 h of incubation in a water bath at 90 °C and centrifugation at 2000 rpm for 10 min at 4 °C, the absorbance of each sample was measured at 440, 532, and 600 nm.

2.5. Hydrogen Peroxide (H₂O₂)

The H₂O₂ content in okra plant organs was measured following Brennan and Frenkel [35]. Fresh plant material was homogenized in cold acetone using an ultrasonic homogenizer, followed by filtration through filter paper. The filtrate was treated with titanium reagent and ammonia solution to form a precipitate. After centrifugation (14,000 rpm for 15 min), the obtained white precipitate (titanium–hydroperoxide complex) was dissolved in 2 M H₂SO₄ and the absorbance was measured at 415 nm to determine H₂O₂ content.

2.6. Antioxidant Enzyme Activity

2.6.1. Catalase CAT (EC 1.11.1.6)

CAT activity was assessed according to Arbona et al. [36] and Labidi et al. [37]. Concisely, fresh tissue (100 mg) was extracted with potassium phosphate buffer, triton X-100, and polyvinylpyrrolidone and then centrifuged at 14,000 rpm for 10 min. The CAT activity was determined using a sodium phosphate buffer and H₂O₂, with detection based on colored titanium hydroperoxide at 415 nm.

2.6.2. Superoxide Dismutase SOD (EC 1.15.1.1)

Fresh matter (100 mg) was extracted in phosphate buffer (50 mM, pH 7.8) containing diethyl-diamino-pentaacetic acid and centrifuged at 14,000 rpm for 10 min. SOD activity was measured at 560 nm every 20 s for 2 min, following NBT (nitroblue tetrazolium) reduction induced by O₂∙⁻. This method was adapted from Arbona et al. [36] and Labidi et al. [37].

2.6.3. Glutathione Reductase GR (EC 1.6.4.2)

Freshly ground plant material (100 mg) was processed in a 50 mM sodium phosphate buffer (pH 7.5). After centrifugation, the supernatant was used to measure GR activity by determining the absorbance at 412 nm for 2 min. NADPH and oxidized glutathione (GSSG) were used as substrates, along with DTNB as the thiol-derivatization reagent.

2.7. Determination of Reduced Glutathione (GSH) and Phytochelatin (PC) Concentrations

The measurements of GSH and PC contents in okra plant tissues followed López-Climent et al. [38]. Firstly, total non-protein thiol (TNP-SH) was extracted and diluted using 5-sulphosalicylic acid, with absorbance measured at 412 nm. Secondly, the DTNB–oxidized glutathione (GSSG) reductase recycling method outlined by Anderson [39] was used to determine total glutathione (tGSH) and GSSG separately at 412 nm. GSH concentration was intended by deducting GSSG from tGSH, while PC production was estimated by subtracting GSH from TPN-SH content. Calibration curves were employed for estimating GSSG and tGSH contents [40].

2.8. Statistical Analyses

All measurements were performed in five replicates. The figures present the average values along with their standard deviations (±). To evaluate the influence of TME on parameter variability, we used a single-factor analysis of variance (ANOVA 1) followed by the comparison of means using Tukey’s HSD test. Significant variations were determined at p < 0.05. To explore the relationships between the studied parameters, an association analysis was conducted using correlation circles derived from a principal component analysis (PCA) in STATISTICA 8.0 software [41].

3. Results

3.1. Growth

Table 1. Impact of the escalating doses of Al and Ba on relative growth rate and growth percentage (relative to control) in roots and shoots of Abelmoschus esculentus (L.).

	RGR (mg·Day−1)
Treatment	Al		Ba
	Roots	Shoots	Roots	Shoots
0 µM	0.0557 ± 0.004 a	0.0485 ± 0.004 b	0.0557 ± 0.0003 a	0.0485 ± 0.003 a
100 µM	0.0550 ± 0.002 a	0.0481 ± 0.003 b	0.0555 ± 0.0003 a	0.0419 ± 0.003 b
200 µM	0.0554 ± 0.003 a	0.0523 ± 0.003 a	0.0564 ± 0.0004 a	0.0423 ± 0.004 b
400 µM	0.0562 ± 0.003 a	0.0478 ± 0.003 b	0.0560 ± 0.0003 a	0.0426 ± 0.003 b
600 µM	0.0567 ± 0.003 a	0.0476 ± 0.003 b	0.0562 ± 0.0004 a	0.043 ± 0.004 b
	GP (%)
Treatment	Al		Ba
	Roots	Shoots	Roots	Shoots
100 µM	101.59 ± 4.53 a	100.75 ± 2.83 b	101.11 ± 1.79 a	88.12 ± 1.84 a
200 µM	103.36 ± 8.17 a	110.45 ± 3.38 a	102.22 ± 5.42 a	87.42 ± 2.05 a
400 µM	103.44 ± 6.30 a	103.40 ± 3.18 b	102.09 ± 5.00 a	87.14 ± 1.04 a
600 µM	101.75 ± 5.34 a	103.49 ± 2.68 b	101.20 ± 2.85 a	87.15 ± 1.82 a

RGR: root growth rate (mg/Day⁻¹). GP: Growth percentage (%). Doses of Al and Ba: 0, 100, 200, 400, and 600 µM. Data were analyzed using one-way ANOVA followed by Tukey’s HSD test for post hoc comparisons. Different letters denote statistical significance at p < 0.05. The results are represented as mean values ± standard error (SE); number of replicates (n) = 10.

presents the quantitative data on the impact of escalating doses of Al and Ba on the relative growth rate (RGR), expressed in mg.day⁻¹, and the growth percentage (GP), calculated relative to the control, in roots and aerial parts of Abelmoschus esculentus (L.). The numbers indicate how Al and Ba treatments affect biomass production over time and compare it to untreated plants. The results suggested that the two metallic cations had opposite impacts on the plant’s growth. Regarding the Al treatment, a significant improvement in crop yield was noticed in the samples irrigated with 200 µM of Al. RGR and GP increased by 7.84 and 10.45%, respectively, in the plant’s shoots compared to the control (Table 1). Conversely, the application of Ba treatment induced a decrease in fresh biomass yield of the shoots across all treatment doses. The main reduction was observed with a 13.61% decrease in RGR when treated with 100 µM of Ba (Table 1).

3.2. TME Contents

Figure 1 illustrates the accumulation of Al and Ba in the roots, shoots, and fruits of Abelmoschus esculentus (L.) plants subjected to increasing concentrations of these elements (0, 100, 200, 400, and 600 µM). The values are presented in mg·kg⁻¹ FW (fresh weight). Al and Ba resulted in a substantial accumulation of these elements within the tissues across all parts of A. esculentus (L.). However, notably lower accumulation was observed in the fruits under both treatments. The increase in the used dose was concomitant with a significant increase in the endogenous contents of Al and Ba (p < 0.05). For plants irrigated with Al, maximum accumulation reached 67.51, 61.43, and 47.6 mg/kg⁻¹ FW with 600 µM in roots, shoots, and fruits, respectively. For the shoots, Al accumulation at 400 µM was lower than at 100 µM and 200 µM, suggesting a saturation threshold or mechanisms limiting Al uptake or transport (Figure 1A). Similarly, plants treated with Ba exhibited a similar disturbance, maximum accumulated amounts were observed with 600 µM: 65.88, 64.63, and 53.13 mg/Kg⁻¹ FW in roots, shoots, and fruits, respectively (Figure 1B).

3.3. Malondialdehyde (MDA)

MDA determination is a widely accepted methodology giving an estimation of oxidative alterations. In the present study, MDA content serves as a broad indicator of oxidative stress. In Figure 2, the MDA levels in Abelmoschus esculentus (L.) organs (roots, shoots, and fruits) are presented across different concentrations of Al and Ba (0–600 µM). The MDA values are expressed in nmol·g⁻¹ FW.

Indeed, Al and Ba induced an increase in the MDA rates in different A. esculentus (L.) organs. However, Al affected only the roots and fruits, while Ba impacted all organs.

The roots treated with 100 μM exhibited the highest MDA value, reaching 223.18% of the control value (p < 0.05) (Figure 2A). The data also revealed that lipid peroxidation occurred in okra fruits, with MDA concentrations significantly elevated at higher doses of Al, particularly at 600 µM, reaching an increase of 79.9%.

The stimulatory effect of Al was also observed in Ba treatment. In fact, Ba stress caused significant increases in MDA levels in the shoots and the roots of okra plants (p < 0.05); all the applied doses caused an increase in lipid peroxidation in the roots with the maximum increase reaching 283.73% compared to the control with 100 µM. The same effect was observed in the shoots, where endogenous amounts of MDA reached 156.46% compared to control groups at 600 µM Ba. Regarding the fruits, the rise in Ba doses during the experiment led a significant level of MDA content, where the maximum increase reached 156.63% at 600 µM (Figure 2B).

3.4. Hydrogen Peroxide (H₂O₂)

In Figure 3, the H₂O₂ concentrations are presented for Abelmoschus esculentus (L.) organs treated with increasing concentrations of Al and Ba (0, 100, 200, 400 and 600 µM). The values are expressed in µmol/g FW. Exposing A. esculentus (L.) plants to Al or Ba stress caused a proportional and significant rise in endogenous levels of H₂O₂ in the studied the plant parts (p < 0.05). For both treatments, the H₂O₂ concentrations were more important in shoots and fruits compared to the roots of okra plants (Figure 3). In fact, in the roots, H₂O₂ showed a higher concentration at 400 µM of Al compared to the control, while Ba stress did not result in any significant variation.

Concerning the parts of okra plants that are aboveground, the maximum increase was 148.7% and 151.14% in shoots and fruits, respectively, at 600 μM of Al in comparison to the control. With Ba stress, the enhancement of H₂O₂ values in the shoots was more obvious than the Al treatment, where the maximum stimulation reached 435.97% compared to the control values with 400 µM. It was also noticed that H₂O₂ content showed an increase in the fruits in the presence of Ba at 600 µM, about 65.29%.

3.5. Enzymatic Activities

Table 2. Impact of the escalating doses of Al and Ba on the activities of SOD, CAT, and GR in roots, shoots, and fruits of Abelmoschus esculentus (L.).

SOD (U/mg protein−1)	(µM)	Al			Ba
		Roots	Shoots	Fruits	Roots	Shoots	Fruits
	0	3758.2 ± 231.1 c	1773.5 ± 59.6 c	1036.8 ± 34.7 b	3758.2 ± 231.1 a	1773.5 ± 59.6 c	1036.8 ± 34.7 c
	100	4936.95 ± 251.5 b	2169.4 ± 55.6 c	1370.01 ± 20 b	3595.4 ± 667.2 a	2325.09 ± 172 c	1266.3 ± 59.9 c
	200	6229.4 ± 498.2 a	2979.1 ± 191.9 c	1973.5 ± 96.5 a	3235.3 ± 228.9 a	2279.2 ± 168.5 c	1161.9 ± 62.7 c
	400	5632.98 ± 453.2 b	3834.2 ± 63.6 b	2025.5 ± 192.6 a	3597.7 ± 540.6 a	3402.6 ± 337.5 b	1325.7 ± 203 b
	600	5512.90 ± 112 b	5961.2 ± 636 a	2330.5 ± 251.8 a	3637.01 ± 379.8 a	4506.7 ± 447.3 a	1693.05 ± 103.4 a
CAT (U/mg protein−1)	(µM)	Al			Ba
		Roots	Shoots	Fruits	Roots	Shoots	Fruits
	0	2.74 ± 0.2 a	9.75 ± 1.04 a	0.82 ± 0.03 a	2.74 ± 0.20 a	9.75 ± 1.04 a	0.82 ± 0.03 a
	100	1.95 ± 0.45 b	9.16 ± 0.68 a	1.00 ± 0.11 a	0.80 ± 0.05 b	0.79 ± 0.03 b	0.79 ± 0.08 a
	200	0.97 ± 0.09 c	0.83 ± 0.09 b	0.83 ± 0.04 a	0.77 ± 0.11 b	0.79 ± 0.05 b	0.71 ± 0.09 a
	400	1.09 ± 0.11 c	0.83 ± 0.15 b	0.85 ± 0.02 a	0.94 ± 0.13 b	0.65 ± 0.07 b	0.84 ± 0.10 a
	600	0.72 ± 0.16 c	1.50 ± 0.02 b	0.82 ± 0.06 a	1.06 ± 0.05 b	0.95 ± 0.18 b	0.62 ± 0.09 a
GR (U/mg protein−1)	(µM)	Al			Ba
		Roots	Shoots	Fruits	Roots	Shoots	Fruits
	0	0.089 ± 0.008 a	0.07 ± 0.002 b	0.018 ± 0.004 c	0.089 ± 0.008 a	0.07 ± 0.002 a	0.018 ± 0.004 b
	100	0.045 ± 0.01 b	0.068 ± 0.003 b	0.031 ± 0.003 b	0.07 ± 0.009 b	0.046 ± 0.003 b	0.046 ± 0.001 a
	200	0.015 ± 0.006 c	0.072 ± 0.003 b	0.045 ± 0.003 a	0.05 ± 0.003 c	0.036 ± 0.002 c	0.05 ± 0.005 a
	400	0.012 ± 0.002 c	0.088 ± 0.003 a	0.047 ± 0.002 a	0.044 ± 0.004 c	0.033 ± 0.001 c	0.047 ± 0.005 a
	600	0.013 ± 0.006 c	0.091 ± 0.004 a	0.05 ± 0.005 a	0.036 ± 0.005 c	0.037 ± 0.002 c	0.052 ± 0.001 a

SOD: Superoxide dismutase; CAT: catalase; GR: glutathione reductase (U/mg protein). Data were analyzed using one-way ANOVA followed by Tukey’s HSD test for post hoc comparisons. Different letters denote statistical significance at p < 0.05. Doses of Al and Ba: 0, 100, 200, 400, and 600 µM. The results are represented as mean values ± standard error (SE); number of replicates (n) = 10.

illustrates the variations in studied antioxidant enzymes activities’ across different parts of A. esculentus (L.) under Al or Ba stress.

SOD: The SOD activity presented notable variations upon Al- and Ba-induced stresses. The data showed that SOD was increased significantly according the increase in Al doses in all the tested parts of A. esculentus (L.) plants, especially in the shoots (p < 0.05). The maximum increase reached 65.75% with 200 µM in the roots, and 236.13 and 124.79% with 600 µM in the shoots and the roots, respectively. This stimulatory effect was also detected in the shoots and fruits of the plants treated with Ba, where the maximum increase was about 154.12 and 63.3%, respectively. However, in the roots, SOD activity did not present any significant variation after the exposure to Ba stress (p < 0.05).

CAT: The introduction of Al and Ba into the irrigation solution resulted in significant alterations in CAT activity. Indeed, the CAT activity was significantly and sharply inhibited in the roots and more importantly in the shoots of okra plants with increasing the doses used in the treatments (p < 0.05). As a matter of fact, in the roots, the maximum decrease reached 73.56% with 600 µM, and this value was 91.54% with 400 µM in the shoots. Similarly, in the roots and the shoots of plants subjected to Ba, CAT activity declined to a maximum of 71.78 and 93.34% in roots and aerial parts with 200 and 400 µM, respectively.

GR: The subjection of okra plants to Al or Ba stress led to significant changes in the activity of GR (p < 0.05). The data showed that plants treated with Al presented a rise in GR activity in the shoots and the fruits, especially with high doses; the maximum increase reached 36.22 and 180.8%, respectively. Meanwhile, the roots presented a sharp and significant inhibition of the activity of this enzyme to 84.84%. Ba treatment had the same inhibitory impact on plants, where the GR activity was significantly reduced (p < 0.05) in roots and shoots to 51.39 and 59.34% with 400 and 600 µM, respectively. However, in the fruits, Ba significantly stimulated the GR activity, with the maximum stimulation reaching 190.8% with 600 µM (p < 0.05).

3.6. GSH and GSSG

In Figure 4 and Figure 5, the levels of GSH and GSSG are measured in the roots, shoots, and fruits of Abelmoschus esculentus (L.) treated with increasing concentrations of Al or Ba (0, 100, 200, 400 and 600 µM). The data are typically expressed in µmol/g FW for each compound.

Our findings indicated that introducing Al or Ba into the irrigation solution led to changes in the GSH levels within various organs of the examined plant. Indeed, for plants treated with Al, GSH contents were significantly stimulated to 36.36% in roots (p < 0.05) (Figure 4(A-1)), whereas, the shoots presented a significant decline in GSH amounts with all the doses, reaching 56.88% with 400 µM (p < 0.05) (Figure 4(A-2)). This decline was also observed with all the doses of Ba in the shoots, whereas in the roots, it was evident only with doses of 400 and 600 µM (Figure 4(B-1,B-2)). The maximum decrease reached 42.04 and 54.75%, respectively, in the roots with 400 µM and in the shoots with 200 µM. The subjection to Al or Ba stress induced an important enhancement of GSH contents in the fruits. GSH rose significantly (p < 0.05) with the increase in the supplied doses of TMEs in the experiment (Figure 4(A-3,B-3)).

As regards the results of oxidized glutathione (GSSG), it was noticed that Al and Ba induced a significant increase only in the shoots, reaching 76.7 and 83.15%, respectively, with 600 µM (p < 0.05) (Figure 5(A-2,B-2)).

The results of the GSH/GSSG ratio are presented in

Table 3. Impact of the escalating concentrations of Al (A) and Ba (B) on GSH/GSSG ratio in different parts of Abelmoschus esculentus (L.).

GSH/GSSG	(µM)	Al			Ba
		Roots	Shoots	Fruits	Roots	Shoots	Fruits
	0	0.64 ± 0.02 c	7.08 ± 0.35 a	0.01 ± 0.001 c	0.64 ± 0.02	7.08 ± 0.35 a	0.01 ± 0.001 c
	100	0.77 ± 0.05 b	3.65 ± 0.15 b	0.01 ± 0.004 c	0.59 ± 0.06	3.05 ± 0.35 b	0.10 ± 0.03 b
	200	0.82 ± 0.02 a	3.92 ± 0.34 b	0.03 ± 0.01 c	0.51 ± 0.05	2.88 ± 0.33 b	0.08 ± 0.03 b
	400	0.77 ± 0.04 b	2.44 ± 0.37 b	0.08 ± 0.03 b	0.39 ± 0.04	2.00 ± 0.11 b	0.07 ± 0.02
	600	0.87 ± 0.02 a	2.63 ± 0.43 b	0.13 ± 0.01 a	0.41 ± 0.04	2.40 ± 0.39 b	0.27 ± 0.02 a

GSH/GSSG: Reduced glutathione (GSH) and oxidized glutathione (GSSG) ratio. Data were analyzed using one-way ANOVA followed by Tukey’s HSD test for post hoc comparisons. Different letters denote statistical significance at p < 0.05. Doses of Al and Ba: 0, 100, 200, 400, and 600 µM. The results are represented as mean values ± standard error (SE); number of replicates (n) = 10.

; the variations observed were similar to GSH variations. In plant roots treated with 600 µM of Al, this ratio was stimulated to a maximum of 36.3%, whereas there was a decrease of 65.48% in the shoots. The same inhibitory effect was detected in the roots treated with 400 µM and in the shoots subjected to 600 µM, reaching a maximum of 38.8 and 71.8%, respectively. It was observed that the GSH/GSSG ratio was noticeably increased with the escalating doses applied in both treatments.

3.7. Phytochelatins (PCs)

In Figure 6, the levels of PCs are measured in the roots, shoots, and fruits of okra plants treated with increasing concentrations of Al or Ba (0, 100, 200, 400 and 600 µM). The values are expressed in nmol/g FW. The subjection of okra plants to increasing doses of Al or Ba generated some changes in PC rates. Plants treated with Al showed that PC contents decreased significantly in the roots to 32.52% (p < 0.05) (Figure 6A).

Conversely, Ba caused a significant enhancement of PC contents in the roots and shoots, reaching 28.15 and 56.42%, respectively, with 600 µM (p < 0.05). However, the fruits presented a decline, reaching 49.38% with 600 µM (p < 0.05) (Figure 6B).

3.8. Statistical Analyses

PCA was conducted to investigate how Al and Ba affect the studied parameters according to the Al and Ba contents: MDA and H₂O₂ levels, the activities of antioxidant enzymes (SOD, CAT, and GR), and GSH and PC amounts in okra plants. This analysis was conducted to highlight the relationships between these parameters. Figure 6 represents the multiple-factor analysis of the Al and Ba effects on okra plants.

Our data displayed a high level of significance. Specifically, in the case of plants subjected to Al, the total variance was found to be 81.17% in roots, 81.55% in shoots, and 88.27% in fruits. On the other hand, when it came to barium treatment, the total variance accounted for 91.32% in roots, 95.77% in shoots, and 87.93% in fruits. The Al content presented a positive correlation with both GSH and GSH/GSSG ratio in the roots, while it exhibited a negative correlation with CAT activity and PC content, as illustrated in Figure 7(A-1).

In the shoots, Al contents were negatively correlated with GR activity, GSH/GSSG ratio, and PC contents (Figure 7(A-2)). In addition, SOD and GR activities, H₂O₂, MDA, and GSH amounts, and GSH/GSSG were positively correlated with Al contents in the fruits of okra plants (Figure 7(A-3)).

Regarding Ba treatment, it was shown that Ba contents in the roots presented a positive correlation with H₂O₂ and PC contents while exhibiting a negative correlation with GR activity and GSSG, GSH, and the GSH/GSSG ratio (Figure 7(B-1)). In the shoots, Ba contents showed a positive correlation with SOD activity, GSSG rates, and PC contents and a negative correlation with GR activity (Figure 7(B-2)). The data indicated a positive correlation between Ba content and the levels of MDA and GSSG in the fruits (Figure 7(B-3)).

4. Discussion

4.1. Growth

In the current study, the influence of Al and Ba on the development of A. esculentus (L.) was determined and the results showed a contrasting effect on the productive biomass. A. esculentus (L.) exhibited vigorous growth and demonstrated resilience to aluminum exposure. Overall, the effect of Al on crop yield is complex and depends on certain factors, including the plant species, the soil acidity, and the concentration of Al. High concentrations of Al could be toxic to many plants, leading to reduced growth and yield, as well as root and shoot distortions. However, some plants have evolved to withstand high levels of Al, being known as Al accumulators.

Al-accumulator plants can supported high Al concentrations without suffering phytotoxicity. For example, Camellia sinensis (L.) plants tolerated up to 3200 µM of Al through increased scavenging and detoxification of ROS [42], while Camellia japonica showed increased growth and photosynthesis at 500 and 1000 µM of Al [43]. In several plants such as Cucumis sativus [15] and A. esculentus (L.) [19], Al was found to have a stimulatory effect on growth. The advantages of Al in relation to plants have been linked to their ability to improve phosphorus accessibility, enhance utilization efficiency, and alleviate issues with excess H⁺, Mn, and Fe in acidic environments. Additionally, Al was found to activate genes (ALMT1, SbMATE, and HvAACT1) associated with abiotic stress tolerance, the ability to respond to oxidative stress, and low phosphorus response [44]. The application of Al was also shown to increase chaperone protein rates in citrus plant leaves. These proteins are crucial for preventing protein misfolding and ensuring cellular stability when exposed to Al [45]. Incorporating Al-accumulator plants into crop rotations or intercropping systems can enhance soil resilience in acidic environments, while low-dose aluminum applications may boost crop productivity and support sustainable agriculture by improving nutrient uptake and phosphorus efficiency and alleviating Mn, Fe, and H⁺ toxicity.

Ba is not considered a key nutrient for plant development. In fact, high levels of Ba could be phytotoxic. Ba toxicity can induce a range of symptoms in plants, including leaf chlorosis, wilting, stunted growth, and necrosis. The effects of Ba on plant growth are influenced by various factors such as the plant species and the soil conditions. Different plants may exhibit varying levels of sensitivity to barium, and the existence of additional compounds in the soil can modify how barium influences plant growth [17].

In fact, our study on the impact of Ba exposure on okra plant growth showed a reduction in biomass productivity, particularly in the aboveground parts, as the Ba doses increased. This element exhibited a clear inhibitory effect on growth and altered development, indicating its phytotoxicity. Comparable results were observed in okra [19] and Cakile maritima [46], where Ba led to a significant decrease in the production of dry biomass and inhibited shoot growth, respectively. Moreover, elevated concentrations of Ba resulted in phytotoxic effects on Tanzanian guinea grass, disrupting multiple processes associated with nutrition and growth. This led to the demise of young shoots and a decline in the vigor of mature plants [47]. Suwa et al. [48] attributed the decrease in growth under Ba stress to diminished CO₂ assimilation stemming from a decline in photosynthetic activity. Identifying Ba-sensitive crops and avoiding their cultivation in Ba-contaminated soils could prevent yield losses, while promoting more tolerant varieties or implementing soil remediation techniques could improve agricultural outcomes.

4.2. TME Contents

The studied plant showed an important capacity for retaining Al and Ba; the endogenous concentrations increased concomitantly with the increase in the used doses. Following a 45-day period of exposing okra plants to Al/Ba stress, both metals were found to be accumulated in the different parts of okra plants in equal proportions. This capacity suggests potential applications in phytoremediation, where okra plants could be utilized to extract or stabilize Al and Ba in contaminated soils, particularly in areas where these metals pose a threat to agricultural productivity.

Kumara et al. [49] reported that the efficiency of TME accumulation is closely linked to the various processes involved in plant absorption, translocation, detoxification, sequestration, and storing mechanisms. Indeed, as the plant grows, Al in the soil is relocated to various plant components. The distribution of Al in the plant varies among different species, with some having a tendency to accumulate greater concentrations of Al in their roots, while others exhibit higher levels of Al accumulation in their leaves [50].

For example, when examining the allocation of Al in cucumber plants, it was primarily concentrated in the roots, with only negligible translocation to the aboveground parts [15]. In contrast, certain plant species, like the tea plant, have been observed to selectively accumulate a significant quantity of Al in their shoots [16]. Plant roots passively uptake Al in an ionic form [7]. It has been hypothesized that Al crosses lysosomal membranes via channels or pores as electroneutral hydroxide complexes [51]. Additionally, a comparable mechanism, which may include aquaporin-like channels, has been observed for Al transportation across tonoplast and cell membranes [52]. Al was also found to be widely accumulated in edible vegetables; the highest Al amounts were accumulated in cabbage, carrots, marrow, squashes, spinach, and watercress, with a mean Al content of 27.5 mg·kg⁻¹ [53].

Concerning Ba, this element is considered as a strong plant-biophilic element. In the plant roots, it is taken up through active transport, either due to its association with essential plant nutrients or because it might act as a plant nutrient by itself [17]. Okra plants exhibited a dose-dependent accumulation of barium, in equal proporions between roots, shoots, and fruits.

Previous studies have also reported that Ba is widely retained and accumulated by several plant species. Ba amounts reached 6.93 and 6.62 mg·g⁻¹ DW in cucumber roots and shoots, respectively [18]. In the investigation by Dridi et al. [20], it was shown that L. crithmoides and H. annus were able to retain more than 3000 µg·g⁻¹ DW and over 1000 µg·g⁻¹ DW, respectively. Besides that, Ba can be accumulated in vegetables and fruits; for example, in eggplant and marrow sampled from Ba-contaminated areas, Ba levels were found in amounts of 3.02 and 2.64 mg·kg⁻¹ DW, respectively [54]. The highest accumulated amounts of Ba were documented in Brazilian nuts, ranging from 3000 to 4000 mg·kg⁻¹ [55].

A theoretical framework explaining the uptake of Ba through plant roots was presented by Amtmann and Rubio [56]. According to their proposed model, Ba absorption is facilitated by potassium channels, with a particular emphasis on the AtAKT1 channel, as suggested by [57]. This is attributed to the structural similarities between Ba and potassium ions, which allow for effective Ba absorption through this channel. Furthermore, it has been reported that plants can absorb Ba, and it can be easily transported upward through the xylem via the movement of water, mainly because of its resemblance to magnesium/calcium cations [9].

4.3. Malondialdehyde (MDA)

MDA is a compound formed when reactive oxygen species interact with lipids. Consequently, elevated levels of MDA in tissues serve as an indicative sign of oxidative stress, which is commonly associated with an increase in free radical generation [58]. Al and Ba exposure caused a certain degree of oxidative stress in this study. It was noticed that these metal elements caused an enhancement of MDA rates in different parts of okra plants.

For example, Al was a main factor causing an increase in MDA levels in numerous species including Hordeum vulgare L. [14] and Triticum aestivum [59]. Hosseini et al. [60] revealed that exposure to aluminum toxicity led to an elevated accumulation of a lipid peroxidation byproduct. This byproduct indicates compromised structural integrity in membranes exposed to metallic stress. Ba exposure was also able to create a disturbance in MDA amounts in Panicum maximum through stimulation of lipid peroxidation [47]. In horticultural practices, elevated MDA levels can serve as a biochemical marker to assess plant health under metal stress, allowing for early detection of stress impacts and the implementation of mitigation strategies. Our findings align with prior research that has mentioned this effect in plants subjected to other forms of TME stress. For example, Pb stress markedly increased MDA levels in A. esculentus [61]. Furthermore, Atriplex halimus L. exhibited elevated MDA content in its root tissue when subjected to irrigation with a solution containing Cd²⁺ or Cu²⁺ [62].

4.4. Hydrogen Peroxide (H₂O₂)

When plants are subjected to high levels of TME, one of the most immediate consequences is the generation of ROS, including superoxide anions and hydroxyl radicals, along with non-radicals such as singlet oxygen and H₂O₂ [63].

Our research uncovered that exposure to Al/Ba led to an accumulation of H₂O₂ within plant tissues. In fact, H₂O₂ levels increased in a manner dependent on the dose, with the highest concentrations observed in the shoots and the fruits. These elevated H₂O₂ levels exhibited a strong correlation with the levels of LPO according to MDA analysis. In accordance, the application of La and Ce stress caused a significant stimulation of H₂O₂ concentrations in Helianthus annuus plants [64]; also, exposing Tanzania guinea grass to Ba augmented the H₂O₂ amounts [47], and the same effect was found in okra plants exposed to Pb [61].

Apart from the effect of metallic exposure in our experiment, MDA and H₂O₂ rates in the fruits of okra plants were clearly much higher as compared to the roots and the shoots. In fact, according to Huan et al. [65], the respiratory rate increased during the maturation of peach fruits simultaneously with the production of ROS, occurring just before the rapid expansion phase.

Therefore, it appears that ROS are probably included in the signalization that initiates the process of fruit expansion. Additionally, the study also asserted that in the later stages of the maturation of fruit, when H₂O₂ contents are at their peak, ROS cause oxidative damage, which is evident through increased electrolyte leakage and elevated MDA levels. Similarly, the research of Resende et al. [66] revealed that the ripening process of “Golden” papaya was also associated with increased levels of lipid peroxidation.

4.5. Antioxidant Enzymes

The observed alterations in antioxidant enzymes in this study could be elucidated by the prior findings, implying that the experimental conditions likely prompted a degree of oxidative stress. Actually, an excess of TMEs can have a negative impact on plants. Consequently, plant cells have established some mechanisms to prevent the toxic buildup of these elements or mitigating TME-induced oxidative damage [67]. SOD and CAT, two essential antioxidant enzymes, serve as a key component of the antioxidant defense mechanism. Consequently, their activities were measured to assess the extent of cellular stress induced by Al and Ba and to appraise the antioxidant capacity of A. esculentus (L.). The initial protective barrier consists of SOD, which triggers the conversion of superoxides O∙⁻ into H₂O₂, a reaction subsequently facilitated by CAT and peroxidases [68]. Consequently, the stress imposed by Al and Ba induced a significant fluctuation in CAT and SOD activities.

The subjection to Al or Ba stress caused an enhancement of SOD activity with all the concentrations used in both treatments. In accordance, the enhancement of SOD activity was also noticed in other plant species such as F. kashmirianum, F. tataricum [69] and Pennisetum glaucum [70] under Al stress, and also in A. esculentus subjected to Cd and Hg [71]. However, in the roots of seedlings of rice, the increase in Al concentrations induced a decline in SOD activity [72]. Alongside SOD, CAT is crucial for the detoxification of peroxides by decomposing H₂O₂ (a byproduct of SOD) into water (H₂O) and molecular oxygen (O₂), thereby mitigating the phytotoxic effects of H₂O₂. Our results also showed that CAT activity declined in the shoots and roots of okra plants with the increase in Al and Ba doses, while the concentration of H₂O₂ stayed high; this ineffectiveness of CAT in H₂O₂ scavenging can be attributed to the degradation initiated by activated peroxisomal proteases or explained by the photoinactivation of the enzyme [71]. The same findings were noted in Pennisetum glaucum [70] and in cucumber [15] under Al stress, as well as in the leaves Brassica juncea subjected to Ba stress [46]. Likewise, CAT activity decreased in okra plants subjected to Pb [61], Cd, and Hg [71].

Furthermore, the ascorbate–glutathione cycle has been demonstrated to enhance plant osmoregulation, the efficiency of water use, nutrient status, photosynthesis effectiveness, and plant yield [21]. GR is an essential enzyme within the ascorbate–glutathione cycle, preserving cells from oxidative harm while upholding an elevated GSH/GSSG ratio [73]. Preserving the cellular reserve of GSH depends on the significance of this enzyme, and an elevation in the activity of GR due to abiotic constraints has been found to improve plant resilience in adverse conditions [74].

Under the effect of the treatments applied to okra plants, it was revealed that GR activity experienced some fluctuations and subsequently a variation in GSH rates and the GSH/GSSG ratio. It was revealed that GR activity and thus GSH concentrations were decreased under the effect of Al/Ba stress, and this could be ascribed to the degradation of GR caused by the overabundance of peroxides and free radicals [75]. Moreover, the GR activity and the levels of GSH exhibited a rise in the fruits and roots in the presence of Al, as well as in fruits harvested from plants exposed to Ba. Similarly to our findings, Al caused an increase in GSH levels in rice [76].

4.6. Reduced Glutathione (GSH) and Phytochelatins (PCs)

Indeed, it is a well-known fact that glutathione is considered as a non-protein thiol antioxidant present in plant tissues, and it serves a significant dual function when it comes to abiotic stress, such as metallic stress, by keeping ROS under control and therefore reduces cell damage [77].

Data showed that GSH amounts declined sharply with Al treatment in the shoots; on the other hand, the elevation in GSSG levels could be attributed to either the swift oxidation of GSH or a disruption in its biosynthesis pathway. In particular, PC rates remained unchanged, despite being considered a fundamental component of PCs [78].

For Ba treatment, the levels of GSH were decreased in both the roots and the shoots, whereas PC rates presented an enhancement. The same results were also observed in Atriplex halimus [62] and in citrus [38]. This decline can be explained by the depletion of this tripeptide since it functions as a precursor of PCs. This fact was confirmed in this work by the increase in PC amounts simultaneously with the reduction in GSH concentrations. During metallic stress, PCs are recognized as the predominant category of non-protein thiols (NPTs), produced through the utilization of both GSH and cysteine [79]. PCs chelate TMEs by forming low- and high-molecular-weight complexes. These complexes will be sequestered in the vacuole, thereby reducing their concentration in the cytosol and maintaining appropriate homeostasis in the cell, ensuring metal detoxification and stress tolerance [24]. Compared to Al, Ba seems to be more deleterious to okra plants. The enhancement of PC rates can be considered as a protective behavior of A. esculentus (L.) exposed to Ba stress to restrict the movement of free Ba from the roots to the shoots, and also to minimize its high cytosolic concentrations in the cells of the aboveground photosynthetic organs, since TME accumulation inhibits photosynthesis [80], which is a process that requires a regular electron flux. This suggests the potential for utilizing plants with enhanced PC synthesis as part of soil management strategies to mitigate TME contamination in agricultural systems.

5. Conclusions

From a global perspective, Abelmoschus esculentus (L.) exposed to Al and Ba exhibited better performance under Al-induced stress than Ba-induced stress when exposed to two different TMEs. Al facilitated plant growth, while Ba hindered it. The studied plant stresses were able to accumulate important amounts of TMEs in both treatments. The distribution of Al and Ba in the endogenous tissues of okra plants occurred in the roots and the shoots in equal proportions and less importantly in the fruits. This accumulation generated a disruption of the redox which was manifested by an enhancement of H₂O₂ and MDA rates. To mitigate the phytotoxic impacts of the induced metallic stress, okra plants showed noticeable variations in the activities of SOD, CAT, and GR. It was found that SOD and GR were the most effective enzymes besides GSH in the detoxification process, especially in plants treated with Al, reflecting a better ability to tolerate Al stress compared to Ba stress. It seems like Ba was more deleterious to okra plants compared to Al. These findings suggest that managing Al levels in soil can potentially enhance crop growth, while minimizing Ba exposure through soil amendments or crop rotation could help reduce Ba toxicity. Ba treatment caused the plant to develop a detoxification mechanism involving the sequestration of this element in the vacuole using PCs and the maintenance of proper homeostasis in the cell. This highlights the importance of promoting phytochelatin synthesis in crops to manage metal stress more effectively, suggesting a potential avenue for breeding or genetically modifying plants to improve metal detoxification capacity. To achieve a better understanding of the response of the studied plant to Al and Ba, it is necessary to explore the effect of both elements on the growth, mineral assimilation, and photosynthesis, and also to examine other detoxification strategies.