Optimizing Brassica napus Resilience to Lead Toxicity: The Combined Effects of Fullerenol Nanoparticles and AMF on Antioxidant Systems

doi:10.21203/rs.3.rs-4976100/v1

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Optimizing Brassica napus Resilience to Lead Toxicity: The Combined Effects of Fullerenol Nanoparticles and AMF on Antioxidant Systems

https://doi.org/10.21203/rs.3.rs-4976100/v1

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Crop plants are severely affected by heavy metals (HMs) leading to food scarcity and economical loss. Lead (Pb) is outsourced by use of lead based fertilizers, lead batteries, mining, smelting and metal processing. It greatly reduced growth, development and yield of crops cultivated on contaminated sites. In this study, ameliorative role of carbon based fullerenol nanoparticles (FNPs) along with AMF inoculation was examined on Brassica napus L. grown in Pb contaminated soil. A pot experiment in 3 way completely randomize fashion with three replicates was conducted under natural conditions. For Pb stress, 200 µM PbCl₂ solution was used at rate of 1 L per pot. Fullerenol nanoparticles (FNPs) were purchased from Sigma Aldrich and applied via foliar spray at 3 mM concentration. For AMF inoculation rhizospheric soil was colleccted from Sorghum bicolor fields and used in this experiment. Results of the study showed that Pb toxicity greatly reduced growth of B. napus plants. It lowered photosynthesis and gas exchange related attributes. Pb contamination caused oxidative stress, evident from elevated level of malondialdehye (MDA), lipid peroxidation and electrolytic leakage. It also triggered antioxidant defense system of B. napus. These plants also had high Pb metal ions in their root and shoot compared with control. Foliar application of FNPs along with AMF inoculation effectively mitigated oxidative stress caused by Pb and reduced its accumulation in root and shoot of B. napus plants. These treatments modulated phytosynthetic machinery, antioxidant defense mechanism and nutrients uptake in B. napus plants. It is concluded that use of carbon-based nano particles in combination with AMF can effectively mitigate HMs stress in crop plants grown in contaminated soil.

Physical sciences/Materials science/Nanoscale materials/Carbon nanotubes and fullerenes

Physical sciences/Materials science/Nanoscale materials/Nanoparticles

Biological sciences/Plant sciences

Biological sciences/Plant sciences/Plant physiology

Earth and environmental sciences/Environmental sciences

These contaminants are taken in by the plant's roots and have negative impacts on plant growth and yield, and they are incorporated into human food’s chain, harming human health¹. Lead (Pb) is one of the most dangerous heavy metal due to its obvious deleterious impacts on environment and on living organisms². Pb concentration rises from both geochemical processes, including the weathering of Pb-containing minerals, and human activities involving industrial processes, the manufacturing of lead-acid batteries, and the use of leaded gasoline³.

Pb has a harmful impact on plants including reduction in seed germination, alteration in cellular structures, chlorosis, and significant reduction in plant growth⁴. Pb hinders the absorption of essential nutrients like potassium, magnesium, calcium, manganese, zinc, and iron, which results in disruption in the physiological and biochemical features⁵. Pb toxicity caused oxidative stress by generation of ROS, and interfere with various essential physiological processes such as chlorophyll synthesis, photosynthesis process, hormonal balance, optimal membrane potential, and ultimately, cell damage and death in plants⁶.

Plants employ both enzymatic and non-enzymatic antioxidant as survival mechanisms under Pb toxicity. Enzymatic antioxidants like SOD, CAT, APX, and GR, and non-enzymatic antioxidants includes AsA, GSH, and phenolics well known to their potential role under Pb toxici⁷. In the same way, plants also produced higher concentration of osmolytes to maintain cellular osmotic conditions under ion stress⁸.

Pb toxicity can be managed through the process of phytoremediation, which involves the use of plants to counteract the effects of heavy metal contamination. Arbuscular mycorrhizal fungi (AMF), predominantly from the Glomeraceae family, is significantly incorporated in mitigating plant Pb toxicity. Glomalin-associated soil proteins are produced by AMF, which reduces Pb bioavailability and increases Pb retention in plant roots, hence limiting Pb transfer to aerial portions⁹. Recent research has demonstrated how AMF nutrient absorption and fosters root development, hence boosting plant growth and antioxidant defenses under lead stress¹⁰

Nanotechnology offers innovative solutions for enhancing plant stress tolerance. Fullerenol nanoparticles (hydroxylated fullerenes) have shown promise due to their antioxidant properties and interaction with plant physiological processes¹¹. Fullerenol nanoparticles can mitigate drought and alkaline induced oxidative stress by scavenging ROS and modulating antioxidant enzyme activity¹². Thus, combined application of fullerenol nanoparticles and AMF presents a novel approach to improve plant growth and biochemical attributes of B. napus under Pb stress.

This study aims to explore the synergistic effects of fullerenol nanoparticles and AMF on B. napus under Pb toxicity. We hypothesize that this combined treatment will enhance plant growth, photosynthetic efficiency, transpiration rate, and antioxidant defenses, providing a comprehensive strategy for mitigating Pb stress and improving crop productivity in contaminated environments. The findings could lead to innovative strategies for managing heavy metal pollution and enhancing agricultural sustainability.

In this context, the combined application of AMF and fullerenol nanoparticles presents a novel approach to promoting the growth of B. napus, a crop of significant economic importance. The synergistic effects of AMF and fullerenol nanoparticles on B. napus growth and its antioxidant defense system have yet to be fully explored, making this a promising area of research. This study aims to elucidate the mechanisms through which this combined treatment enhances plant growth and strengthens the antioxidant system, contributing to improved overall plant health and productivity. The combination of fullerenol and AMF may eventually lead to a complete plan for lowering Pb poisoning and improving the overall health and production of crops like Brassica napus in contaminated regions.

Experimental design and layout

This experiment was conducted in botanical garden, University of Punjab, Lahore, Pakistan. It was a completely randomized design experiment with a total of 24 pots. Brassica napus seeds were procured from Punjab Seeds, Lahore. Ten to twelve seeds were sown in each pot. Four same sized seedlings on equal distance were maintained in every pot. For Pb stress, 200 µM PbCl₂ solution was used at rate of 1 L per pot. Fullerenol nanoparticles (FNPs) were purchased from Sigma Aldrich. FNPs were applied via foliar spray at 3 mM concentration. For AMF inoculation rhizospheric soil was colleccted from Sorghum bicolor fields and used in this experiment. Field survey was conducted, where S. color roots were identified for having vesicles formation. Following that AMF strains were identified and isolated via colony method. These treatments were applied on four leaves stage seedlings. After four weeks of treatments one plant from every pot was uprooted and analyzed for different growth, physiological, gas exchange and biochemical attributes.

Estimation of photosynthetic pigments and chlorophyll fluorescence parameters

For this purpose, 0.5 g leaf was grinded using pestle and mortar. This blend was homogenized in 10 mL of 80% acetone solution. After filtration, absorbance was read at 480, 645 and 663 nm using spectrophotometer. Chlorophyll pigments and carotenoids contents were estimated using this method¹³.

Chlorophyll fluorometer OS30p⁺ (Opti-sciences) was used to check chlorophyll fluorescence parameters. Firstly, young but fully developed leaves were dark adapted for 15 min with help of clips. Following that dark-adapted leaves were given saturation pulse at 3500 µmol and modulation light intensity set to 40%. Maximum quantum efficiency (F_v/F_m)and potential efficiency (F_v/F_o) of PSII were ascertained¹⁴.

Analysis of gas exchange attributes

Infrared gas analyzer (LCpro SD, ADC BioScientific, UK) with broad chamber was used to analyze Pn, Tr, gs and Ci. Readings were taken using PAR from the leaf chamber light source at 900 µmol m^− 2 s^− 1 and ambient CO₂ averaging 400 ppm all-round the observation time¹⁵.

Estimation of stress related parameters, lipid peroxidation and reactive oxygen species

The method described by Cakmak & Horst¹⁶. was used to assess lipid peroxidation that means malondialdehyde (MDA) contents. 0.1 g leaf leaf was frozen, grinded in mortar and homogenized in phosphate buffer (50 mM, 25 mL) having pH 7.8 made in 1% polyethene pyrrole. Sample was centrifuged (10,000 × g) for fifteen minutes. After being heated to 100 ^oC for 15 to 30 minutes, the reaction was stopped by rapid cooling in ice bath. OD was measured at wavelengths of 532 and 600 nm.

$$\:\varDelta\:\:(A\:532\:nm\:-\:A\:600\:nm)/1.56\times\:105\:=\:MDA\:level\:\left(nmol\right)$$

For H₂O₂ the reaction solution was prepared by adding 1 mL of 100 mM K-phosphate buffer, 4 mL reagent (1 mM KI) and 1 mL of 0.1% trichloroacetic acid (TCA). Leaf extract supernatant placed in darkness for 60 min. Afterwards, absorbance of samples along with blank containing 0.1% TCA and reaction mixture was observed at 390 nm. Quantity of H₂O₂ was analyzed by comparing it with a standard curve of H₂O₂¹⁷.

O₂^•− was estimated by homogenizing a 4 g leaf sample in 6 mL of 3% trichloroacetic acid. The resulting solution was subjected to centrifugation at 10,000×g for 22 min. The reaction solution was prepared by adding 1 mM hydroxylamine hydrochloride in 2 mL supernatant and 2 mL of 50 mM potassium phosphate buffer at pH 7.1. The optical density of the reaction solution was observed at 530 nm to estimate O₂^•− level by comparing with the standard curve¹⁸.

For hydroxyl radicals (OH-), fresh leaf (0.5 g) incubated in 1 mL solution of 10 mM Na-phosphate buffer and 15 mM 2-deoxy-D-ribose for 2 hours at 37°C. Reaction mixture was made of 3 mL 0.5% thiobirbuteric acid and 1 mL glacialacetic acid. Incubated sample was added to reaction mixture. The mixture was heated (100°C) in a water bath for 30 minutes. After cooled to 41◦C OD at 550 nm was assessed.

Determination of relative water content and relative membrane permeability

Equal sized leaves were taken from each replicate. Measured their fresh weight (FW). These leaves were then dipped in water for three hours in dark and measured turgid weight (TW). Following that, the leaves were oven dried and measured their dry weight (DW).

$$\:RWC\:\left(\%\right)=\left[\right(FW-DW)/(TW-DW\left)\right]\times\:100$$

Same size leaves one from each replicate were chopped and added in 20 mL deionized water in a test tube. Vortexed for 5 sec and electrical conductivity (EC₁) was measured. These samples were then placed in refrigerator and EC₂ was measured on next day. After that, these samples were autoclaved for 20 min at 120 ^oC and EC₃ was measured. RMP was calculated using following formula¹⁹.

$$\:RMP\:\left(\%\right)=\left[\right({EC}_{2}-{EC}_{1})/({EC}_{3}-{EC}_{1}\left)\right]\times\:100$$

Estimation of glutathione and ascorbate

Leaf samples (0.5 g) were homogenized with 10% (w/v) TCA (5 mL) and centrifuged at 15,000 g for 15 minutes to determine reduced glutathione (GSH), as described by Law et al.²⁰. 150 µL of supernatant was mixed with 100 µL of 6 mM DTNB, 50 µL of glutathione reductase (10 units mL^− 1), and 700 µL of 0.3 mM NADPH. A standard curve was used to calculate total glutathione concentration. All reagents were produced in 125 mM NaH₂PO₄ buffer with 6.3 mM EDTA at pH 7.5.

Leaf (1 g) was crushed in 5% TCA and centrifuged (15,000 g for 15 min.). Reaction mixture was consisted of plant homogenate (0.2 mL), phosphate buffer pH 7.4 (0.6 mL, 0.2 M), TCA (1 mL, 10%), H₃PO₄ 0.8 mL, 42%), α,αˊ-dipirydyl (0.8 mL, 4%), and FeCl₃ (0.4, of 3%). For blank 0.2 mL of TCA (5%) was used in place of plant material. OD was measured at 525 nm. AsA were estimated from a curve calibration drawn with standard solutions²¹.

Estimation of soluble proteins and enzymatic antioxidants

Leaf samples (0.5 g) were homogenized in 50 mM potassium phosphate buffer (pH 7.8), then centrifuged at 10,000 g. The supernatant was collected and used for subsequent analysis. The total soluble protein (TSP) content was calculated using Bradford's method²², with bovine serum albumin serving as a standard.

The total superoxide dismutase activity was determined using the Zhang et al.²³ technique. The reaction mixture contained 3 mL of 50 mM potassium-phosphate-buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA, and 100 µL enzyme extract. OD was read at 560 nm. SOD one unit was expressed as quantity to reduce 50% NBT.

Catalase (CAT) activity was evaluated using H₂O₂ (39 mM) extinction co-efficient. OD was read for 120 sec with interval of 30 sec. 3 mL reaction mixture contained 50 mM potassium-phosphate-buffer (pH 7.0), 2 mM EDTA-Na₂, 10 mM H₂O₂, and 0.1 mL enzyme extract, as described by Chance & Maehly²⁴. Peroxidase (POD) activity was defined as a change in guaiacol absorbance at 470 nm. The reaction solution contained potassium-phosphate-buffer (50 mM, pH 7.0), 0.1 mL of enzyme extract, 0.4% H₂O₂, and 1% guaiacol.

Nakano & Asada's assay²⁵ was mounted for ascorbate peroxide (APX) activity. This included phosphate-buffer (100 mM, pH 7), 0.3 mM ascorbic acid, 0.06 mM H₂O₂, 0.1 mM EDTA-Na₂, and 0.1 mL enzyme extract. Spectrophotometer was set at 290 nm, and the absorbance was measured 30 seconds after the addition of H₂O₂.

According to Jiang & Zhang²⁶, the activity of glutathione reductase (GR) was measured by oxidizing NADPH for 1 minute at 340 nm. The reaction mixture consisted of potassium-phosphate-buffer (pH 7.0, 50 mM), 2 mM EDTA Na₂, 0.15 mM NADPH, 0.5 mM GSSG, and 0.1 mL enzyme.

For phenylalanine ammonia-lyase (PAL) activity the methods of Dai et al.²⁷ was employed. Change in OD read at 290 nm was expressed as one unit of PAL. Reaction mixture was having supernatant ().5 mL), sodium borate buffer (2 mL, pH 8.8), and 0.5 mL of 3 mM L-phenylalanine. It was placed in incubator at 30 ◦C for 1 hour. The controls did not include L-phenylalanine. Ruiz et al. (1999) determined polyphenol peroxidase (PPO) activity by measuring the rise in absorbance at 370 nm while using caffeic acid as a substrate. The assay combination included sodium acetate buffer (0.9 mL, pH 5.0), 0.1 mL of catechol, and 0.1 mL of enzyme extract.

Estimation of nutritional cations (Na⁺, K⁺, Ca²⁺ and Mg²⁺)

Plant leaves (0.2 g) were digested with a solution of 5 mL HNO₃ and 1 mL HClO₄. Following that, the resulting solutions were diluted to 25 mL with 2% HNO₃ and filtered. The amounts of Na⁺, K⁺, Ca²⁺, and Mg²⁺ in the filtrate were measured using inductively coupled plasma-mass spectrometry according to a conventional technique.

Pb metal uptake in root and shoot of B. napus

0.5 g root and shoot samples were dry-ashed, extracted with HCl, and centrifuged (3600 rpm) for 15 minutes. Flame atomic absorption spectrometry was used to determine Pb concentrations in the root and shoot. The Pb concentration in root and shoot tissues was determined using the following formula:

$$\:Pb\:concentration\:\left(\mu\:g\:{g}^{-1}\:DW\right)=reading\times\:dilution\:factor$$

Effect of FNPs foliar spray and AMF inoculation on growth attributes of Pb treated B. napus.

Pb stress lowered growth attributes of B. napus significantly. It reduced shoot length, root length, shoot fresh weight and dry weight. Shoot length was reduced by 15% under 200 µM Pb stress. Followed by 25, 40 and 47% reduction in root length, shoot fresh weight and shoot dry weight, respectively, compared to control plants. FNPs and AMF treatments, alone or in combination, successfully retrieved original status of the plants. These treatments improved growth attributes not only in control group but also in stressed plants. In stressed plants, maximum increment in growth attributes was observed with synergistic application of FNPs and AMF. Followed by FNPs alone and then AMF inoculation (Fig. 1A-D).

Effect of FNPs foliar spray and AMF inoculation on photosynthetic pigments and chlorophyll fluorescence attributes of Pb treated B. napus.

Chlorophyll a contents were significantly disintegrated under Pb stress. As depicted in Table 1, it was reduced by 39%, compared to control. In contrast, FNPs and AMF alone or combined treatment elevated chl a contents in both control and stressed plants. Compared to non-treated control, FNPs foliar spray, AMF inoculation and their synergistic application improved chl a contents by 23, 28 and 35%, respectively. While in Pb stressed plants, these treatments incremented chl a by 29, 44 and 43%, respectively. Chlorophyll b contents were slightly uplifted in Pb stress plants. Furthermore, above mentioned treatments also incremented chl b contents under stress conditions. Conversely, FNPs and AMF treatments slightly reduced carotenoids in non-stressed B. napus. Notedly, their application retrieved carotenoids in Pb stressed plants (Table 1).

Table 1

Chlorophyll pigments and fluorescence related attributes of *B. napus* plants, exposed to Pb toxicity, along with FNPs foliar spray and AMF inoculation.
Chlorophyll pigments and flourescence related parametrs
Treatments	Chl a			Chl b			Carotenoids			Fv/Fm			Fv/Fo
Treatments	Mean	±	SE	Mean	±	SE	Mean	±	SE	Mean	±	SE	Mean	±	SE
Control	1.507	±	0.164bc	0.539	±	0.033bc	4.322	±	0.436ab	0.818	±	0.081a	4.064	±	0.147a
FNPs	1.855	±	0.074ab	0.845	±	0.034a	3.504	±	0.092b	0.818	±	0.061ab	3.657	±	0.107a
AMF	1.929	±	0.053ab	0.521	±	0.051bc	3.845	±	0.230ab	0.832	±	0.020a	4.106	±	0.186a
FNPs + AMF	2.035	±	0.081a	0.471	±	0.039c	5.472	±	0.578a	0.741	±	0.033a	4.105	±	0.064a
Pb	0.922	±	0.033d	0.580	±	0.037bc	3.771	±	0.403ab	0.521	±	0.013c	1.390	±	0.127b
P + FNPs	1.193	±	0.063cd	0.574	±	0.008bc	3.513	±	0.253b	0.634	±	0.001c	1.752	±	0.180ab
Pb + AMF	1.332	±	0.024cd	0.667	±	0.049abc	4.225	±	0.054ab	0.675	±	0.013c	2.012	±	0.177ab
Pb + FNPs + AMF	1.319	±	0.066cd	0.695	±	0.054ab	4.349	±	0.220ab	0.826	±	0.033b	3.251	±	0.105a
Values presented in the table are mean of three replicates ± standard error. Different letters obtained after LSD test, showed that mean values are significantly different at p < 0.05.

Chlorophyll fluorescence parameters (Fv/Fm and Fv/Fo) were lowered by Pb stress. This reduction was nearly half-fold. FNPs and AMF treatments had little or no effect on chlorophyll fluorescence in control conditions. Nevertheless, these treatments effectively salvaged reduction of chlorophyll fluorescence attributes in Pb stressed plants. FNPs and AMF alone and combined application incremented Fv/Fm (22, 30 and 59%) and Fv/Fo (26, 45 and 134%), compared to stress only plants (Table 1).

Effect of FNPs foliar spray and AMF inoculation on gas exchange related attributes of Pb treated B. napus.

Gas exchange (Pn, Tr, gs and Ci) attributes were altered significantly in response to Pb toxicity. Net photosynthesis rate was reduced by 38%, when plants were given 200 µM Pb stress. Followed by 25 and 18% reduction in stomatal conductance and transpiration rate, respectively. On other hand, intercellular CO₂ was elevated by 20% in this group of plants, compared to control. FNPs and AMF synergistic treatment had more significant influence on gas exchange attributes, compared to their alone treatment. Their synergistic application over stressed plants enhanced Pn, gs and Tr by 81, 54 and 7%, respectively. While FNPs foliar spray and AMF inoculation alone and their combined treatment reduced Ci by 11, 8 and 3%, respectively, compared to stress only plants (Fig. 2A-D).

Effect of FNPs foliar spray and AMF inoculation on stress markers of Pb treated B. napus.

As depicted in Fig. 3, lipid peroxidation greatly enhanced in B. napus with application of Pb stress. This is indicated by 62% increment in MDA contents in Pb treated plants, compared to control. Pb toxicity also caused ROS generation and posed oxidative damages. As depicted from elevated levels of H₂O₂, OH⁻ and O₂^•− reactive species. These reactive species were increased by 60, 103 and 23%, respectively, compared to control. FNPs and AMF treatments alone or in combination successfully detoxified ROS and lowered MDA contents. Hence eliminated lipid peroxidation of cells even under Pb toxic stress. Synergistic application of FNPs and AMF reduced MDA, H₂O₂, OH⁻ and O₂^•− levels by 33, 30, 47 and 20% in Pb treated plants, compared with stress only plants (Fig. 3A-D).

Effect of FNPs foliar spray and AMF inoculation on relative water contents and relative membrane permeability of Pb treated B. napus.

Relative water contents in control plants were 52.97%, while those treated with FNPs and AMF alone and their combined treatment had 62.31, 63.13 and 63.58% respectively. This increment is also reflected in enhanced growth with application of aforementioned treatments. Nevertheless, Pb stressed plants had 36.09% RWC. RWC in Pb stressed plants were significantly improved with FNPs and AMF alone and their synergistic application. These plants had 41.08, 39.28 and 44.51% RWC, respectively. Similarly, relative membrane permeability was also greatly influenced by Pb toxicity and successive treatments of FNPs and AMF. Pb treated plants had 68.90% which is nearly twofold of control plants. FNPs and AMF alone and their combined treatment reduced RMP by 21, 30 and 15%, respectively, in stressed plants compared to stress only plants (Fig. 4A & B).

Effect of FNPs foliar spray and AMF inoculation on glutathione and ascorbate contents of Pb treated B. napus.

Glutathione and Ascorbate contents were higher (32 and 79%) in Pb stress plants compared to control. FNPs and AMF treatments, alone or in combination further enhanced these antioxidants. Synergistic application of FNPs and AMF was more effective in elevation of these antioxidants compared to alone treatments. As depicted in Fig. 4C & D, in Pb treated plants application of FNPs and AMF alone and in combination incremented glutathione by 5, 20 and 20%, respectively. Similarly, ascorbate contents were incremented by 14, 26 and 33%, respectively, compared to stress only plants.

Effect of FNPs foliar spray and AMF inoculation on soluble proteins and enzymatic antioxidants of Pb treated B. napus.

Total soluble proteins were reduced greatly (46%) under the toxic effect of Pb stress compared to control. FNPs foliar spray and AMF inoculation applied alone and in combination significantly enhanced TSP in both control (27, 9 and 22%, respectively) and stress (42, 73 and 62%, respectively) groups, compared with their respective controls. Maximum TSP contents (16.43 mg g^− 1 FW) were observed in control plants treated with FNPs as shown in Fig. 5A.

Glutathione reductase enzyme activity was also reduced (47%) in Pb treated plants compared to control. Nonetheless, FNPs and AMF treatment significantly improved GR activity in both control and stress conditions. It was increased by 38, 40 and 47% with application of FNPs and AMF alone and their combined treatment, respectively, compared to non-treated stress plants (Fig. 5B).

In response to Pb stress, B. napus plants significantly enhanced phenylalanine ammonia-lyase (PAL) and polyphenol peroxidase activities. Activities of these enzymes in Pb treated plants were 0.42 and 0.30 U mg^− 1 protein, respectively. This was 117 and 47% higher than control plants. However, FNPs and AMF treatments either alone or in combination, further incremented activities of these enzymes in both control and stress plants. Pb stressed plants along with following treatments had improved PAL activity by 138, 147 and 169% and that of PPO by 66, 70 and 111%, respectively (Fig. 5C &D).

Activities of CAT, POD, SOD and APX were increased greatly (by 37, 19, 96 and 200%, respectively) under toxic Pb stress (Fig. 6A-D). FNPs and AMF treatments further added in this increment. As depicted in Fig. 6, synergistic application of FNPs and AMF inoculation was more effective in triggering this incrementation. Maximum activities of these enzymes were confined in Pb stressed plant with synergistic effect of FNPs and AMF. This was 120, 97, 130 and 289%, respectively, higher than control plants.

Effect of FNPs foliar spray and AMF inoculation on nutritional cations (Na ⁺, K⁺, Ca²⁺ and Mg²⁺) of Pb treated B. napus.

Cations (Na⁺, K⁺, Ca²⁺ and Mg²⁺) concentrations were significantly influenced Pb stress. Pb stress caused reduction in Na⁺, K⁺, Ca²⁺ and Mg²⁺ ions by 39, 47, 36 and 32%, respectively, compared to control. Conversely, FNPs foliar spray and AMF inoculation alone and their combined effect retrieved these cations in shoot of B. napus. Their synergistic application was more effective among all treatments. As in Pb stressed plants, it increased Na⁺, K⁺, Ca²⁺ and Mg²⁺ concentrations by 73, 68, 53 and 53%, respectively, compared to non-treated ones (Fig. 7A-D).

Effect of FNPs foliar spray and AMF inoculation on Pb metal concentration in root and shoot of Pb treated B. napus.

In B. napus treated with 200 µM Pb, 8.30 and 0.66 mg of Pb metal ions were detected in g^− 1 DW of root and shoot, respectively. FNPs and AMF alone and their combined treatment increased Pb metal ions in shoot of B. napus. It was incremented by 36, 51 and 48%, respectively, following these treatments compared to stress only plants. This indicated that FNPs and AMF treatments increased Pb ions translocation towards shoot (Table 2).

Table 2

Lead uptake in *B. napus* plants, exposed to Pb toxicity, along with FNPs foliar spray and AMF inoculation.
Pb uptake in B. napus plants
Treatments	Pb in root			Pb in shoot			TF
Treatments	Mean	±	SE	Mean	±	SE	Mean	±	SE
Control	2.132	±	0.050d	1.014	±	0.044c	0.477	±	0.029b
FNPs	2.958	±	0.074d	2.093	±	0.086c	0.707	±	0.019a
AMF	2.842	±	0.259d	1.936	±	0.029c	0.694	±	0.074a
FNPs + AMF	3.035	±	0.223d	1.880	±	0.082c	0.631	±	0.078a
Pb	8305.830	±	147.746c	664.762	±	19.396b	0.080	±	0.001c
P + FNPs	9166.499	±	218.628bc	906.856	±	68.212a	0.099	±	0.010c
Pb + AMF	9451.615	±	116.419ab	1005.967	±	37.606a	0.107	±	0.005c
Pb + FNPs + AMF	10523.073	±	503.151a	984.205	±	42.641a	0.094	±	0.008c
Values presented in the table are mean of three replicates ± standard error. Different letters obtained after LSD test, showed that mean values are significantly different at p < 0.05.

Pearson’s correlation and principle component analysis

Pearson’s correlation test in Fig. 8 represented that Pb concentration in root and shoot of B. napus plants was highly correlated with stress markers (MDA, H₂O₂, OH⁻ and O2^•−), antioxidants activities (both enzymatic and non enzymatic) and membrane permeability. This depicted that increased Pb concentration in B. napus plants resulted in lipid peroxidation of cells. As MDA level was increased under Pb stress. This caused severe oxidative damage and disturbed cell membrane integrity. Ultimately, this led to reduced growth. Wherease, Pb concentration in B. napus plants was in sigficant negative correlation with growth indices, chlorophyll pigments, quantum efficiency of PSII and photosynthesis related attributes. As stated earlier, FNPs and AMF synergistic application effectively improved these attributes even in Pb stress plants (Fig. 1–2 & Table 1). Also, in Fig. 3 it is shown that FNPs and AMF trreatments reduced stress markers. This indicated stress amelioration. While, in Fig. 4–6 these treatments elevated antioxidants activites (enzymatic and non enzymatic) in Pb stress plants. Nonetheless, in Table 2, it is shown that FNPs and AMF applications incremented Pb uptake in roots and its translocation towards shoot. From this, it can be argued that, FNPs and AMF treatments adjusted plants’ metabolism in a way that detoxify Pb stress.

Principle component analysis also validated the Pearson’s correlation results (Fig. 9). PCA plot for individuals expressed as numerics (Where; 1 = control, 2 = FNPs 3 mM, 3 = AMF, 4 = 2 + 3, 5 = Pb 200 µM, 6 = 2 + 5, 7 = 3 + 5 and 8 = 4 + 5) revealed that Pb stress (5) is separated well from all others treatments. This showed that Pb stress application surely had its impact on all studied attributes. Among all the possible principle components, PC1 and PC2 contributed the maximum (84.84%). From this, all studied attributes can be divided in two groups. In group I, attrbutes were aligned with PC1 while in group II the attributes were aligned with PC2. Attributes of PC1 were positively correlated to each other. But these were in negative correlation to attributes aligned with PC2.

Lead (Pb) is a potent environmental and health-hazard non-essential heavy metal that exists commonly in contaminated soils, mostly in industrial, mining, and urban regions²⁸. Pb toxicity owing to the inhibition of physiological and biochemical processes which are vital for growth and development of plants²⁹. Unlike other micronutrients, Pb has no known utilization in the functioning of plants other than being toxic to the plant; in fact, it can greatly retard plant growth and development as well as hinder fundamental physiological processes³⁰.

The present study examined that Pb toxicity reduce the growth of B. napus, which has great economic importance due to its diverse utilization. The results presented in the work show a significant decrease in a number of restored growth characteristics, such as root and shoot length, and biomass production. The level of growth reduction endorsed in this study can be mainly emphasized as having stemmed from Pb-induced root damage. Pb is well recognized to be transported to roots, and altering the cell membrane permeability which consequently halt the necessary functioning of transport proteins which directly linked with absorption of essential nutrients and water³¹. This disruption probably results in nutrient deficiencies and hampers the regular physiological processes that are essential for the growth and development of a plant³².

Pb toxicity also has detrimental effects on the photosynthetic apparatus of plants, and the extent rises by increasing Pb concentration. More specifically, Pb can interfere with the PSII electron transport chain which is basic component of the photosynthesis process and, therefore, reduce levels of photosynthetic proficiency and biomass production⁴. This inhibition is considered to be attributed to the replacement of essential metal cofactors in the photosynthetic enzymes by Pb and resulted into the generation of oxidative damage to the chloroplasts, thus leading to a decline in the overall photosynthetic activity of the plant³².

Moreover, the phytotoxicity of Pb is specifically due to the fact that it provokes the formation of oxidative stress in plant cells⁴. Pb toxicity can induce the formation of ROS, which are highly reactive molecules that can easily oxidize lipids, proteins, and DNA³³. Such oxidative stress can breakdown fundamental metabolic activities like chlorophyll biosynthesis and the regulation of antioxidant enzymes, which in turn would lead to further damaging the growth and development of plant⁴. The earlier studies demonstrate that nutrient, photosynthesis and energy uptake decreases and caused oxidative stress in B. napus growth under Pb toxicity³⁴.

Due to the problems associated with heavy metal toxicity, scientists have had to seek out new technologies, one of which is the implementation of nanoparticles (NPs) in agriculture sector³⁵. NPs, because of their size and physicochemical characterization, have been found to have immense potential for improving plant resistance to metal toxicity³⁵. Fullerenol nanoparticles (FNPs), which are a functionalization of fullerene (C60) without any charge, have been studied to enhance plant tolerance to drought stress. cucumber tolerance under Cu toxicity³⁶. Similar potential of FNPs have been observed in current findings in where it significantly reduced the toxic effect of Pb toxicity by increasing the growth parameters and improvement in antioxidants and photosynthetic rate.

Among all the synthesized nanoparticles, fullerenol nanoparticles are further characterized by extremely high antioxidant activity, which enables them to eliminate ROS and reduce oxidative damage in plants³⁶. As applied on B. napus, FNPs have effects in boosting up the antioxidant defense mechanism in the plant, which leads to the minimization of the oxidative stress that produced in response to Pb toxicity. On the same note, FNPs are also in a position to influence the plant’s physiological processes by enhancing nutrient acquisition and general plant growth under stress. This is the reason why FNPs have opportunities to enter plant tissues easily due to their small size and high level of polyhydroxy and to perform protective functions at the cellular level³⁶.

This paper envisages the use of FNPs in farming as a modern strategy for managing Pb toxicity in agriculture. This study contributes to the knowledge on the role played by FNPs in improving the growth and stress tolerance of B. napus under Pb toxicity, a valuable contribution that could be useful to future works dealing with the use of FNPs in increasing crop resistance to hazardous metal stress. Although the particular mechanisms through which FNPs reduce metal stress in B. napus are not yet understand. FNPs are highly reactive as well as relatively small in size and thus can enter the inner structures of plants through the cuticle, stomata, or cell walls in addition to the plasma lemma. Thus, these nanoparticles can be actively transported through the tissues, such as the phloem and plasmodesmata within the cell, where they play important roles in sub-cellular metabolisms, including the detoxification of ROS³⁷. The augmentation of plant capacity to withstand Pd toxicity can be described with the following concept: The mode of action of FNPs applied as foliar spray involves the direct modulation of physiological processes in the plant³⁷.

In this investigation, the plant growth-promoting effect of FNPs and AMF under Pd toxicity was shown for the first time, leading to a marked enhancement in the growth parameters of B. napus, such as shoot and root fresh weight, shoot and root length, and number of leaves. These changes can be associated with the decline of the negative Pb effect on the level of oxidative stress, which generally affects plant growth. Similar observations have been noted in Brassica napus, where it was found that TiO and ZnO nanoparticles decrease Pb uptake and increase growth, which may be due to the improvement in the antioxidant defense system¹¹. The conclusion made in this research study is in agreement with the study conducted by Ahmadi et al.³⁸ suggesting that FNPs enhance Capsicum annuum growth and chlorophyll under drought stress. The observed positive effects of FNPs are in accordance with the observed literature regarding the stress-reducing effects of nanoparticles on plants³⁷.

Thus, FNPs and AMF restored the growth of B. napus under Pb toxicity. AMF has a mutualistic association with the plant roots, which helps in the better uptake of nutrients and reduce the detrimental effects of Pb toxicity. This work represented that AMF inoculation along with FNPs developed have more symbiotic effect with root system of B. napus that promotes plant growth and biomass production. In line with results reported by Gazzar et al.³⁹ which described that AMF along with TiO₂ nanoparticles improves the growth parameters of Phaseolus vulgaris under salinity stress. The similar findings noted that the combined application of AMF and nanoparticles enhanced the growth of cowpea under Cd stress⁴⁰.

Function of AMF in root system expansion is very crucial under Pb toxicity. Even in soil polluted with lead, AMF like Rhizophagus intraradices and Funneliformis mosseae have been revealed to develop symbiotic association with a diversity of plant species³². These fungi escalation the surface area of the roots and boost the growth of large hyphal networks in the soil, which improves the plant's capacity to absorb essential nutrients. In addition to enhancing nutrient absorption, hyphal networks also contribute to immobilization of Pb, which sinks plant bioavailability⁴¹. It has been demonstrated that this procedure enhances plant growth and survival under heavy metal contamination, which is essential for preserving plant health under heavy metal stress⁹.

Pb inhibits photosynthesis as well as reduces chlorophyll contents, damaging the parameters related to gas exchange in plants. In this regard, exposure to Pb toxicity reduced the photosynthesis rate of B. napus, which agrees with earlier research on heavy metals like Pb that significantly reduced chlorophyll formation consequently decreasing the vital photosynthesis process⁴¹. Pb stress leads to a reduction of the photosynthetic rate due to direct damage to the photosynthetic apparatus, the PSII reaction center, and the inhibition of chlorophyll biosynthesis⁹.

In the present work, the foliar application of FNPs enhanced the photosynthetic rate in B. napus, probably owing to the improvement in chlorophyll contents. This boast corresponds with the results presented by Shafiq et al.³⁷ ho noticed that the use of polyhydroxy fullerene nanoparticles raised chlorophyll contents as well as increase photosynthesis rate in wheat under salt stress. The improvement in photosynthesis brought by FNPs can be associated with the promotion of enzyme activities in carbohydrate, amino acid, and secondary metabolite metabolism³⁷. NPs may also form associations with the PSII reaction center complex so as to facilitate electron donation and increase the overall photosynthetic rate⁴².

Stomatal conductance, intracellular CO₂ concentrations, and transpiration rates were also reduced by Pb toxicity in the current study. Such results are supported by Ahmed et al.⁶ who observed that increased Pb levels lead to stomatal closure and reduced transpiration, CO₂ uptake, and photosynthetic photocurrent. The amelioration of the parameters related to gas exchange as seen by the application of FNPs points to the fact that these nanoparticles are involved in the regulation of stomata and enhancement of photosynthesis in plants under Pb toxicty. Improvements in exact similar gas exchange parameters have also been observed by the application of ZnO nanoparticles under Pb toxicity⁴³. It may raise RuBisCO activase, the enzyme in the Calvin cycle that increases CO₂ assimilation and leading to significant increase in the process of photosynthesis⁴⁴.

One of the most important components of the antioxidant defense system is its capability of preventing or reducing oxidative stress induced by Pb toxicity. Pb toxicity could also promote the generation of higher amount of ROS, which ultimately causes oxidative injury in the cells of the plant. In response, plants initiate their antioxidant defense system, which consists of enzymes like catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). This present work also revealed that Pb intoxication enhanced the activities of these enzymes and total soluble protein levels. It is well understood that plants under Pb toxicity increase the antioxidant enzyme machinery that helps in the detoxification of ROS and the alteration of cellular structures⁴⁵.

Similarly to the previous study, the presence of FNPs in this study also upregulated the activities of these antioxidant enzymes, and it can be concluded that AMF and FNPs can therefore augment the plant’s own defense strategies against Pb-induced oxidative stress. These observations are supported by findings from other investigator Shakoor et al.⁴⁵ who pointed out that nanoparticles improve the antioxidant system of Brassica juncea exposed to heavy metals. The combined application of FNPs and AMF also led to a significant improvement in antioxidant enzyme activities, indicating a synergistic effect in mitigating Pb toxicity. The ability of AMF to enhance the antioxidant defense system has been demonstrated various studies, including those by Ding et al.⁴⁶ .who showed that AMF can improve plant resilience to heavy metal stress by boosting antioxidant enzyme activities.

Based on the findings of this study, it can be concluded that the FNPs and AMF may be useful for reducing Pb toxicity in B. napus. Besides, the use of the combined agents enhanced the plant growth parameters and the photosynthesis efficiency and stimulated the antioxidant defense system of the plant for a comprehensive solution to the heavy metal stress. This paper makes a contribution to the development of knowledge on the employment of effective nanoparticles and AMF in agriculture, concluding new ideas and providing the perspective for the following analyses.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

We declare that the manuscript reporting studies do not involve any human participants, human data or human tissues. So, it is not applicable.

Our experiment follows with the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

Researchers Supporting Project Number (RSP2024R182) King Saud University, Riyadh, Saudi Arabia

Author Contributions

AAS; Supervision and Validation, SU; Experimentation and Methodology, ZN; writing-original draft preparation, MK; Statistical analysis, SS & MAE; Conceptualization, Data curation and Formal analysis and ZI & SS; Resource acquisition and Investigation. All authors have read and approved the final manuscript.

Acknowledgments:

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R182) King Saud University, Riyadh, Saudi Arabia.

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No competing interests reported.

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You are reading this latest preprint version

Optimizing Brassica napus Resilience to Lead Toxicity: The Combined Effects of Fullerenol Nanoparticles and AMF on Antioxidant Systems

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Experimental design and layout

Estimation of photosynthetic pigments and chlorophyll fluorescence parameters

Analysis of gas exchange attributes

Estimation of stress related parameters, lipid peroxidation and reactive oxygen species

Determination of relative water content and relative membrane permeability

Estimation of glutathione and ascorbate

Estimation of soluble proteins and enzymatic antioxidants

Estimation of nutritional cations (Na⁺, K⁺, Ca²⁺ and Mg²⁺)

Results

Pearson’s correlation and principle component analysis

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Optimizing Brassica napus Resilience to Lead Toxicity: The Combined Effects of Fullerenol Nanoparticles and AMF on Antioxidant Systems

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Experimental design and layout

Estimation of photosynthetic pigments and chlorophyll fluorescence parameters

Analysis of gas exchange attributes

Estimation of stress related parameters, lipid peroxidation and reactive oxygen species

Determination of relative water content and relative membrane permeability

Estimation of glutathione and ascorbate

Estimation of soluble proteins and enzymatic antioxidants

Estimation of nutritional cations (Na+, K+, Ca2+ and Mg2+)

Results

Pearson’s correlation and principle component analysis

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Estimation of nutritional cations (Na⁺, K⁺, Ca²⁺ and Mg²⁺)