Physiological, Morphological, and Biochemical Responses of Soybean [ Glycine max (L.) Merr.] to Loquat ( Eriobotrya japonica Lindl.) Leaf Extract Application on Pb-Contaminated Soil

: Lead (Pb) is a non-essential element; however, plants uptake it from soils rich in Pb. Soybean [ Glycine max (L.) Merr.] is an important legume crop, and Pb toxicity exerts negative impacts on its growth and yield. This study investigated the role of foliar-applied loquat ( Eriobotrya japonica Lindl.) leaf extract in improving the morphological, physiological, and biochemical traits of soybean plants under Pb toxicity. Soybean plants were exposed to four Pb concentrations (0, 200, 400, and 800 µ g/L) and supplemented with 0% or 5% loquat leaf aqueous extract (EJLE). Data relating to pigments, proline, total soluble sugars, malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), non-enzymatic antioxidant, i.e., [ascorbic acid (AsA), glutathione (GSH), total phenolic contents (TPC), and total ﬂavonoids content (TFC)] and enzymatic antioxidant, i.e., [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)] were recorded. Total chlorophyll contents and carotenoids were signiﬁcantly decreased by Pb stress, while lycopene and anthocyanin contents were increased. Similarly, proline, total soluble sugars, MDA, H 2 O 2 , AsA, GSH, TPC, TFC, SOD, CAT, POD, APX, and GR were increased under Pb stress. Foliar spray of EJLE lowered MDA and H 2 O 2 accumulation and increased the contents of chlorophylls, carotenoids, lycopene, anthocyanins, proline, total soluble sugars, and the antioxidant system. The increase in the activities of antioxidant enzymes lowered the adverse effects of Pb stress in soybean. Similarly, the application of EJLE lowered Pb accumulation in different plant parts compared to those receiving no EJLE. It is concluded that EJLE can improve the Pb tolerance of soybean plants by enhancing morphological, physiological, and biochemical traits. However, the actual mechanisms behind these improvements warrant further investigation.


Introduction
Environmental damage caused by heavy metal pollution is being observed globally [1]. Human activities are often regarded as the major cause of heavy metal pollution [2]. Wastewater discharge, overuse of fertilizers and pesticides in agriculture, and other anthropogenic activities are increasing the concentration of heavy metals in the rhizosphere [2]. The most dangerous inorganic pollutants are nonbiodegradable heavy metals, including arsenic (As), nickel (Ni), chromium (Cr), and lead (Pb) [3][4][5]. Heavy metals in the soil suppress plant growth by inhibiting the uptake and movement of essential nutrients [6,7]. Intracellular accumulation of non-essential heavy metals such as Pb, cadmium (Cd), mercury (Hg), and silver (Ag) leads to the production of reactive oxygen species (ROS) [7][8][9][10].
Lead is a hazardous and non-biodegradable heavy metal often found in the Earth's crust. It is the second-most dangerous metal after As [11], posing serious threats to all life forms. The use of inorganic fertilizers and pesticides, mining, the burning of fossil fuels, removing the damaged or discolored ones, were selected for the experiment. Selected seeds were thoroughly washed with distilled water and disinfected with a 5% sodium hypochlorite (NaOCl) solution (v/v) for 10 min. Afterwards, the seeds were again washed with distilled water and used in the experiments.
Different Pb concentrations (i.e., 0, 200, 400, and 800 µg L −1 ) were prepared by using Pb(NO 3 ) 2 . The plants were exposed to these concentrations through mixing with the modified Hoagland nutrient solution [30]. Plants in the control treatment (0 µg L −1 ) received only the nutrient solution. Ten seeds were sown in 15 cm plastic pots filled with sand and perlite (1:1). The pots were placed in a greenhouse maintained at 20-25 • C and 16:8 h light and dark duration. The plants were irrigated every second day with modified Hoagland nutrient solution containing different Pb concentrations according to the treatments. Loquat aqueous leaf extract (EJLE) was applied to plants 10 days after emergence. Half of the plants were sprayed with 5% EJLE in each Pb concentration, whereas the remaining half were sprayed with distilled water. Each Pb concentration had 4 replications, and there were 10 pots in each replication for both EJLE concentrations. The plants were harvested at 20 days after emergence by carefully removing them from the pots, and data relating to different growth, biochemical attributes, and Pb uptake were recorded.

Preparation of Loquat Leaf Extract
Loquat leaves were washed with distilled water, dried at 60 • C in the oven, and milled into powder. The aqueous extract was prepared by mixing 5 g of leaf powder in 150 mL distilled water. The resultant mixture was heated in an oscillator for 90 min at 80 • C until the solution reduced to 100 mL. The extract was centrifuged at 4000 rpm for 20 min to exclude the contaminants. The resultant supernatant was regarded as 100% concentrated, stored at 4 • C, and used in the experiment. This supernatant was diluted to 5% with distilled water for the foliar spray in the experiment [31].

Growth Traits and Pb Uptake
The harvested plants were divided into roots and shoots. The plants in each treatment were dried in an oven at 70 ± 5 • C and dry weights were recorded. The Pb contents in roots and shoots were quantified by digesting the dried samples in HClO 4 :HNO 3 solution (1:5, v/v). The Pb concentration in the samples was measured on ICP-OES. Translocation factor was computed according to Malik et al. [32].

Pigments
Different pigments were determined by collecting 0.2 g fresh leaf samples from each treatment. Acetone (80%, 4 mL) was used to extract these samples overnight, followed by homogenization. Afterwards, the samples were filtered, and 25 mL of acetone was added to increase the volume of the filtrates. Spectrophotometer (Optima 2100 DV, PerkinElmer, Rodgau, Germany) was used to record the absorbance of the filtrates at different wavelengths (663, 645, 480, and 537 nm). The method of Arnon [33] was used to determine total chlorophyll contents, carotenoids, and anthocyanin. Likewise, the method of Ravelo-Pérez et al. [34] was followed to determine lycopene contents.

Osmolytes
The method of Bates et al. [35] was followed to determine proline contents. A total of 5 mL sulfosalicylic acid (3%) was used to homogenize the leaf samples. Next, 2 mL ninhydrin reagent and 2 mL glacial acetic acid were added to 2 mL leaf extract in a test tube. The mixture was heated at 90 • C for half hour, and the reaction terminated in an ice bath. A total of 5 mL toluene was added to the reaction mixture after it cooled. The mixture was vortexed for 15 s and kept at room temperature for 20 min in the dark so that the toluene layer would separate from the aqueous solution. The UV-1900 BMS (Malvern Panalytical GmbH, Kassel, Germany) spectrophotometer was used to measure the absorbance at 520 nm after collecting each toluene layer in a separate tube. Analytical-grade proline was used to create a standard curve calculated on a mg/g FW basis and to calculate the free proline concentration in each sample.
The method of Irigoyen [36] was followed to extract and determine total soluble sugars. Fresh leaves (0.2 g) were homogenized in 5 mL of ethanol (96% v/v) after rinsing with 5 mL of ethanol (70% v/v). Afterwards, the samples were heated at 80 • C in a boiling water bath for 10 min. The extract was cooled, centrifuged at 4000× g for 10 min, and the supernatant was kept at 4 • C for measurement. Anthrone reagent [150 mg anthrone + 100 mL of sulfuric acid (72%, v/v)] was reacted with 3 mL ethanolic extract in a boiling water bath at 80 • C for 15 min to estimate total soluble sugar content. Once the mixture was cooled, absorbance at 625 nm was measured using a spectrophotometer (UV-1900 BMS, Thermo Fisher Scientific, Duisburg, Germany), and the total soluble sugars (mg/g FW) were determined using a glucose standard curve.

Stress Indicators
Malondialdehyde (MDA) contents were used to assess lipid peroxidation in plant tissues, as described by Zhang and Kirkham [37]. After homogenization in 5 mL TCA (0.1%), 0.25 g leaf sample was centrifuged at 6000× g for 15 min. The resulting aliquot (1 mL) was heated at 95 • C for 30 min, cooled in an ice bath, and centrifuged with 4 mL of thiobarbituric acid (TBA). Absorbance of the supernatant was recorded at 532 and 600 nm. The difference between 532 and 600 nm readings was recorded, and absorption coefficient of 155 mM L −1 was used to express the MDA level in micromoles per gram of dry weight (M/g FW).

Ascorbic Acid
Ascorbic acid was extracted and determined from the leaves by following the methodology of Kampfenkel et al. [39]. Liquid nitrogen was used to homogenize leaf samples (1 g) before extraction with 10 mL TCA (5% w/v), followed by centrifugation (15,000× g) at 4 • C for 5 min. The concentration of AsA in the supernatant was immediately assayed after being transferred to a clean reaction vessel using 1 mL reaction mixture.

Glutathione, Total Phenolics, and Flavonoids
The amount of glutathione was measured in accordance with the methodology of Anderson [40]. Fresh leaves (0.5 g) were homogenized in 5% sulphosalicylic acid (2 mL) under cool temperature, followed by centrifugation (12,000× g) for 10 min. The absorbance was recorded at 412 nm to determine glutathione content. Folin-Ciocalteu reagent (0.75 mL) was used to estimate total phenolic contents [41]. Similarly, total flavonoids were recorded by following the method of Zhishen et al. [42].

Activities of Antioxidant Enzymes
A spectrophotometer was employed to measure the activities of various antioxidant enzymes, i.e., SOD, CAT, POD, APX, and GR. The SOD activity was measured according to Zhang [43]. Similarly, the methodology of Aebi [44] was followed to determine the activity of CAT, and the methodology of Zhou and Leul [45] was followed to measure POD activity. The APX activity was recorded by employing the methodology of Nakano and Asada [46]. Likewise, GR activity was measured by following the methodology of Rao et al. [47].

Statistical Analysis
The data on all of the recorded test were subjected to a normality test [48], which indicated a normal distribution. Therefore, two-way analysis of variance (ANOVA) was employed to infer the significant differences among individual and interactive effects of Pb and EJLE concentrations [49]. Least-significant-difference post hoc test at 95% probability level was used to differentiate the means where ANOVA indicated significant differences among individual and interactive effects of Pb and EJLE concentrations. Lastly, principal component analysis was executed on the growth and biochemical attributes for the easier interpretation of the results. All statistical computations were performed using SPSS statistical software version 20.0 [50].

Results
The individual and interactive effects of lead (Pb) and loquat leaf extract (EJLE) had significant effects on growth traits, pigment contents, Pb accumulation, and Pb translocation factor (Table 1). The shoot and root dry biomass were significantly decreased by increasing Pb concentrations. However, application of 5% EJLE significantly improved shoot and dry biomass under all Pb concentrations. The highest shoot and root dry biomass were noted under 0 µg Pb with 5% EJLE application, whereas plants exposed to 800 µg Pb concentrations without EJLE application produced the lowest shoot and root dry biomass. Application of 5% EJLE improved shoot dry biomass by 10 (Figure 1).
The Pb accumulation in roots and leaves was significantly increased under increasing Pb concentration. Higher Pb accumulation was recorded in roots compared to shoots. The application of 5% EJLE significantly reduced Pb accumulation in roots and leaves. The application of 5% EJLE reduced Pb accumulation in roots by 62.6%, 36.3%, and 34.4% under 200, 400, and 800 µg Pb concentrations, respectively, compared to no ELJE application ( Figure 1). In the same way, application of 5% EJLE reduced Pb accumulation in leaves by 100.1%, 58.5%, and 79.3% under 200, 400, and 800 µg Pb concentrations, respectively, compared to no ELJE supplementation ( Figure 1).
The Pb translocation factor was increased with increasing Pb concentrations. The lowest translocation factor was noted for 0 µg Pb concentration with or without EJLE application, whereas 800 µg Pb concentration without EJLE application noted the highest translocation factor. The application of 5% EJLE reduced Pb translocation factor by 23.     Increasing Pb concentrations significantly reduced total chlorophyll contents, carotenoids, anthocyanins, lycopene, flavonoids, and total phenols. However, application of 5% EJLE significantly improved these contents. The application of 5% EJLE increased carotenoids by 41.4%, 36.7%, 42.2%, and 57.7% under 0, 200, 400, and 800 µg Pb concentrations, respectively, compared to no ELJE application. Chlorophyll contents were improved by 16.7%, 11.5%, 31.3%, and 33.9% under 0, 200, 400, and 800 µg Pb concentrations, respectively, with the application of 5% EJLE compared to no ELJE application (Figure 2). Sustainability 2023, 15, x FOR PEER REVIEW 9 of 18 Total soluble sugars, proline, ascorbic acid, and glutathione were increased with increasing Pb concentration. The increase in these contents was higher with 5% EJLE application compared to no EJLE application. Similarly, MDA and H2O2 contents increased under higher Pb concentrations, and this increase was less in the plants sprayed with 5% EJLE compared with no application (Figure 3).
Total soluble sugars, proline, ascorbic acid, and glutathione were increased with increasing Pb concentration. The increase in these contents was higher with 5% EJLE application compared to no EJLE application. Similarly, MDA and H 2 O 2 contents increased under higher Pb concentrations, and this increase was less in the plants sprayed with 5% EJLE compared with no application (Figure 3).  The activities of different antioxidant enzymes were significantly affected by individual and interactive effects of Pb and EJLE concentrations (Table 3).    Figure 3).
The activities of different antioxidant enzymes were significantly affected by individual and interactive effects of Pb and EJLE concentrations (Table 3). The activities of antioxidant enzymes increased under higher Pb concentrations, and this increase was higher in the plants sprayed with 5% EJLE compared to those receiving no EJLE (Figure 4). The highest activities of SOD, CAT, POD, APX, and GR enzymes were noted in the plants grown under 800 µg Pb concentration with the application of 5% EJLE, whereas the plants grown under 0 µg Pb concentration with no EJLE application recorded the lowest activities of these enzymes.
The CAT activity was improved by 19 (Figure 4). that the relevant individual or interactive effect was significant for the corresponding trait.
The activities of antioxidant enzymes increased under higher Pb concentrations, and this increase was higher in the plants sprayed with 5% EJLE compared to those receivin no EJLE (Figure 4). The highest activities of SOD, CAT, POD, APX, and GR enzymes wer noted in the plants grown under 800 μg Pb concentration with the application of 5% EJLE whereas the plants grown under 0 μg Pb concentration with no EJLE application recorde the lowest activities of these enzymes.  The principal component analysis divided the traits into three distinct groups ( Figure 5). The first group contained root and shoot dry biomass, total chlorophyll contents, and carotenoids grouped together with 0 and 200 µg Pb concentrations with 5% EJLE concentration. The second group had stress indicators, i.e., MDA and H 2 O 2 , Pb accumulation in roots and leaves, and Pb translocation factor, which were grouped together with 400 and 800 µg Pb concentrations with no EJLE application. The rest of the traits were in the third group, coupled with 400 and 800 µg Pb concentrations with the application of 5% EJLE ( Figure 5). The results of the PCA revealed that the application of 5% EJLE lowered the production of stress indicators compared with no application. Similarly, the activities of antioxidant enzymes and non-enzymatic antioxidants were improved with the application of 5% EJLE. The PCA further revealed that the shoot and root dry biomass, chlorophyll contents and carotenoids were higher in the plants grown under no-and low Pb toxicity with the application of 5% EJLE ( Figure 5). 25.5% under 0, 200, 400, and 800 μg Pb concentrations, respectively, with the application of 5% EJLE compared with no application. In the same way, application of 5% EJLE increased APX activity by 28.6%, 24.6%, 20.5%, and 20.4% under 0, 200, 400, and 800 μg Pb concentrations, respectively, compared with no application. Similarly, GR activity was improved by 30.0%, 26.1%, 23.3%, and 24.3% under 0, 200, 400, and 800 μg Pb concentrations, respectively, with 5% EJLE application compared with no application (Figure 4).
The principal component analysis divided the traits into three distinct groups ( Figure 5). The first group contained root and shoot dry biomass, total chlorophyll contents, and carotenoids grouped together with 0 and 200 μg Pb concentrations with 5% EJLE concentration. The second group had stress indicators, i.e., MDA and H2O2, Pb accumulation in roots and leaves, and Pb translocation factor, which were grouped together with 400 and 800 μg Pb concentrations with no EJLE application. The rest of the traits were in the third group, coupled with 400 and 800 μg Pb concentrations with the application of 5% EJLE ( Figure 5). The results of the PCA revealed that the application of 5% EJLE lowered the production of stress indicators compared with no application. Similarly, the activities of antioxidant enzymes and non-enzymatic antioxidants were improved with the application of 5% EJLE. The PCA further revealed that the shoot and root dry biomass,

Discussion
Increasing Pb concentrations significantly suppressed the growth and biochemical attributes of soybean plants as hypothesized. Similarly, the application of 5% EJLE notably improved these traits, which also supported our hypothesis. Higher Pb accumulation was recorded in roots compared to leaves in the current study, whereas application of EJLE significantly lowered Pb accumulation in roots and leaves. The increased Pb accumulation significantly reduced root growth and pigment contents, and this decrease was higher under no application of EJLE. Plants readily absorb Pb from Pb-contaminated soils, with the greatest accumulation observed in roots, whereas other parts (stems, leaves, and seeds) accumulate lower amounts [7,51]. The main obstacles to Pb entry into cells are cell walls and membranes [52]. Phytochelatins may decrease Pb entry into the cells [53]. Several strategies have been tested in earlier studies to lower Pb accumulation in various plant species. The application of endogenous substances has been reported to mitigate the adverse effects of heavy metal on growth of different plant species [4,7,54]. The combined application of asparagine and thiourea improved the Pb tolerance of wheat [7].
Various phenolic acids are known to improve heavy metal tolerance of different plant species [55]. The EJLE is a phenolic compound; thus, it has considerable potential to improve the heavy metal tolerance of plants. The EJLE has metal chelating, DPPH and ABTS radical scavenging activities, and higher antioxidant enzyme activities [56]. The formation of the EJLE-Pb complex might have reduced Pb accumulation and increased soybean growth and biochemical attributes. However, this inference warrants further investigation. Unfortunately, there is no report relating to the role of EJLE in improving the heavy metal tolerance of crop plants. Thus, future studies must explore the chemical composition of EJLE and the actual mechanisms involved in the improved growth of plants with the application of EJLE.
Chlorophyll concentration is a key measure of a plant's photosynthetic efficiency. Pb toxicity causes significant morphological and physiological changes in plants, including reduced photosynthesis, distributed nutrient uptake and water balance, and abrupt changes in the activates of essential enzymes [57]. Pb toxicity is known to decrease chlorophyll a and b, while it increases chlorophyll a:b ratio and chlorophyll degradation rate [7]. Reduced nutrient and water uptake may result in lower photosynthesis, which would result in slower plant development overall. Furthermore, elevated MDA levels and electrolyte leakage may damage chloroplast membranes and result in decreased accumulation of photosynthetic pigments under Pb stress. Increased Pb concentrations in the current study reduced total chlorophylls and carotenoids, whereas they increased lycopene and anthocyanin. Foliar application of 5% EJLE increased the contents of these pigments. The results of the current study are similar to the findings of Shu et al. [58] who reported decreased photosynthetic pigments in Jatropa curcas under Pb stress. The reduced photosynthetic contents were linked with stomatal closure. Stomatal closure is a result of Pb interaction with guard cells or due to the early effects of Pb toxicity on roots and stems. Plants may shift their stomata in response to signals produced by the ABA under Pb stress [59]. The high redox potential of some heavy metals likely hinders the reductive steps in the production pathways of photosynthetic pigments, leading to a decrease in these pigments [60].
The metabolism of plant cells frequently results in the production of reactive oxygen species (ROS). The oxidative damage, ultimately leading to cell death, is caused by an excessive quantity of ROS produced under heavy metals' toxicity. Plants exposed to higher Pb concentrations produce significant amounts of ROS, which oxidize nucleic acids and proteins and degrade their structures [61]. Plants capable of producing higher phenolic compounds under heavy metal stress are regarded good candidates for phytoremediation [62]. These phenolic compounds may serve as antioxidants because their hydroxyl groups can donate hydrogen and react with ROS in the termination reaction, which breaks the cycle of producing new radicals. Phenolic compounds serve as significant antioxidants under stressful environments, and these are generated through the shikimic acid or phenyl propanoid pathways [63]. Loquat leaves are comprised of several active compounds with anti-inflammatory, anti-tumor, and antioxidant properties, including roseoside, procyanidin B-2, triterpene acids, chlorogenic acid, and flavonoids [28]. Production of osmolytes (i.e., proline and total soluble sugars) increased under Pb stress with the application of 5% EJLE. Plants producing more proline could resist lipid peroxidation and membrane alteration caused by Pb toxicity [64,65]. Several earlier studies have reported increased accumulation of osmolytes in plants under heavy metal stress, including Pb [66,67]. The increased proline in Pb-exposed seedlings might be linked with protein breakdown. Proline mitigates metal-induced oxidative stress because of its ability to scavenge ROS [68]. Higher production of proline and total soluble sugars with the application of 5% EJLE lowered ROS, which improved Pb tolerance of soybean plants.
The current study indicated that leaf MDA and H 2 O 2 contents increased under Pb toxicity, while foliar application of 5% EJLE significantly reduced MDA and H 2 O 2 contents. Plant extracts possessing bioactive compounds may serve as powerful antioxidants. The improved Pb tolerance of soybean plants through the application of 5% EJLE might be attributed to its high levels of phenolic (benzoic acid and hydroxycinnamic derivatives) and tocopherol components [69].
Non-enzymatic antioxidant contents (i.e., AsA, GSH, TPC, and TFC) increased under Pb stress with the application of 5% EJLE. Due to electron donating abilities, TPCs serve as hydrogen donors, reducing agents, and singlet oxygen quenchers, in addition to imparting heavy metal tolerance [70]. The increased TPC in the current study can provide a mechanistic aid to soybean plants for improved Pb tolerance [71]. Similarly, the activity of antioxidant enzymes significantly increased under Pb stress with the application of 5% EJLE. The increased activity of these enzymes enhanced the tolerance of soybean plants to Pb toxicity. Improved activity of these enzymes shields plants from oxidative damage; hence, defending the photosynthetic apparatus [72]. Plants produce antioxidants such as proline, APX, glutathione, and GPX under Pb-induced oxidative stress to counteract the negative effects of ROS [73]. Antioxidant activity in plants is achieved by the production of ROS in the mitochondria through the membrane-coupled electron transport chain during oxidative phosphorylation [74]. Increased activity of antioxidant enzymes may have contributed to the reduced membrane leakage [75] due to EJLE application, which preserved the membrane's composition and ultrastructure. Reduced oxidative damage with EJLE application can be linked to the efficient functioning of the antioxidant defense system. Increased antioxidant enzyme activity can be linked to higher antioxidant potential of ELJE [56]. However, no earlier reports are available regarding the functioning of EJLE. Therefore, our results indicate that EJLE could impart Pb tolerance to soybean plants through enhanced activities of antioxidant enzymes. However, field studies exploring the underlying mechanisms of EJLE are needed to reach concrete conclusions. Moreover, future studies must analyze EJLE and test more concentrations of it to reach concrete conclusions.

Conclusions
This study indicated that Pb stress had negative effect on growth, total chlorophyll contents, and carotenoids in soybean plants, whereas it promoted lycopene and anthocyanin. Pb toxicity exhibited a significant induction effect on the proline, total soluble sugars, ascorbic acid, glutathione, total phenolic contents, total flavonoid contents, and activity of antioxidant enzymes (SOD, CAT, POD, APX, and GR). The toxicity of Pb to pigments, osmolytes, and the antioxidant system of soybean plants was significantly mitigated by foliar spray of 5% EJLE. The EJLE decreased MDA and H 2 O 2 contents and promoted pigments, osmolyte contents, and the antioxidant system of soybean plants, thus reducing Pb toxicity. Foliar application of 5% EJLE could effectively improve the Pb tolerance of soybean plants. However, field studies exploring the underlying mechanisms of EJLE are needed to reach concrete conclusions.