Hydrogen peroxide foliar spray alleviates chromium toxicity through modulation of antioxidant defence mechanism, photosynthetic machinery and ions regulation in Brassica oleracea L.

doi:10.21203/rs.3.rs-4876880/v1

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Hydrogen peroxide foliar spray alleviates chromium toxicity through modulation of antioxidant defence mechanism, photosynthetic machinery and ions regulation in Brassica oleracea L.

https://doi.org/10.21203/rs.3.rs-4876880/v1

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The economy of Pakistan largely depends on agriculture. Agriculture lands are facing the challenges of heavy metals contamination. Soil is an important source of nutrients which is continuously polluted with heavy metal due to anthropogenic activities. In this study the effect of hydrogen peroxide in mitigating chromium contamination in cabbage (Brassica oleracea var. capitate) was evaluated. Brassica oleracea var. capitata is a crop that is vulnerable to Cr toxicity. In order to reduce Cr contamination in B. oleracea, this study explored use of hydrogen peroxide (H₂O₂) as a signalling molecule. Plants were subjected to 100 and 200 µM of Cr stress, and they were either treated with H₂O₂ (10 mM) or in combination with Cr. Our studies suggested that H₂O₂ greatly enhanced morphological characters, such as plant growth and development in Cr-stressed environment. Antioxidant activity elevated in response to Cr stress and the use of H₂O₂ enhanced it further. Biochemical factors, such as enzyme activity, elevated under Cr stress but maintained in response to H₂O₂ foliar application. Chromium stress reduced physiological parameters like photosynthesis and water use efficiency, but H₂O₂ treatment upgraded them. With the application of H₂O_2, inorganic ionic strength and gas exchange parameters showed a significant improvement. According to our research, H₂O₂ is efficient for reducing Cr stress in B. oleracea as it boosted physiological, antioxidant, and morphological characteristics. This suggests a possible method for promoting crop tolerance to heavy metal stress.

Globally, environmental pollution is increasing at an alarming rate[1]. Previously, metals having an atomic weight between 63.5 and higher and a density of more than 4.5 g/cm³ have been referred to as "heavy metals”[2]. Among the heavy metals are basic metals, transition metals, and several s metalloids[1]. Due to more urbanization and industrialization, contaminants including heavy metals and hazardous substances are widely dispersed throughout natural resources like air, soil, and water[3]. While several trace elements are required for basic cellular functions, excessive concentrations can be hazardous and exceeds permissible limits[4]. Environment contains heavy metals that are produced by a variety of human activities, such as farming, manufacturing, and household maintenance. These metals have a detrimental effect on the sustainability of agriculture. In regions with limited water supplies, the use of wastewater for irrigation has increased[5]. Investigation has shown that providing irrigation to plants that absorb effluents speeds up their growth and yields more. Thus, farmers in regions where fertilizer is expensive could benefit economically from this approach[6]. Furthermore, areas near factories used soil with polluted water might contain significant level of contaminants from tanning, smelting, and electroplating[7].Chromium is considered as one of the hazardous heavy metals that could be found in nature and frequently used in industrial processes[8]. It is a steel-grey, bright and shiny hard mineral with a maximum melting point[9]. Chromium is not found as a free element in nature, but rather as an ore [10]. While Cr is necessary in little quantities for both plants and animals, it is a severe environmental pollutant at higher concentrations [11].Chromium can be found in water in oxidation states ranging from + 6 to − 2. The trivalent Cr (III) and hexavalent Cr (VI) forms are the most stable and can interconvert with one another [12]. Depending on the current pH and redox potential, either oxidation state may exist. In an alkaline, strongly oxidizing environment, hexavalent chromium predominates, while in an acidic, moderately oxidizing to reducing environment, Cr (III) predominates[13] Cr (VI), the most hazardous form of Cr in the environment, is more readily absorbed by plants, which impacts crop productivity and quality. It’ carcinogenic, mutagenic, and genotoxic properties also have negative effects on humans and animals [14]. The release of chromium into soil, air, and water is caused by both ordinary sources and different anthropogenic activities, which has finally resulted in chromium contamination worldwide[11]. All environmental compartments, including rock, soil, water, and air, contain chromium, however in varying amounts. Despite the fact that sedimentary and acid igneous rocks often only contain trace amounts of Cr[14].Chromium is primarily used by the metallurgical, chemical, and refractory brick sectors, the widespread use of chromium in a number of industries, including metallurgy, electroplating, paint and pigment manufacturing, tanning, chemical production, pulp and paper manufacturing, has also contaminated land and water. Of all of them, tanneries pose the greatest risk for chromium contamination. The discharge of untreated waste into the environment causes significant contamination of Cr since tanneries' wastewater control and treatment facilities are insufficient to handle the waste[15]. China is the world's largest consumer of Cr, mostly for the production of stainless steel. As a result, mining and industry pollutants increase the danger of Cr contamination in these countries' soils [14]. Among HMS, Cr is potentially toxic and serves no critical role in plant metabolism. Thus far, no transporters or channels specific to Cr have been found in plants. Cr is transported by certain important element transporters. After Cr exposure to plants, numerous physiological, morphological, and metabolic features are adversely impacted that ultimately lead to plant death[16]. Cr toxicity inhibits the vital metabolic processes of plants and affects their growth. Typically, Cr toxicity lowers plant growth by generating ultra-structural alterations persuading chlorosis in the leaves, harming root cells, lowering pigment content, affecting water relations and by modifying different enzyme activities[17]. Cereals, legumes, vegetables, forages, and trees are among the plants that are impacted by toxicity. It has been discovered that Cr (VI) affects both terrestrial and aquatic plants, however their responses vary dramatically [18].Because of increased production of reactive oxygen species (ROS), plants with high levels of Cr also experience physiologic and morphological alterations. Plants also have another technique for producing antioxidant enzymes to counteract the high levels of Cr-mediated ROS[9]. In most agronomic plants, Cr is hazardous at concentrations between 5 and 100 mg/g depending on the soil. Generally, plants have a content of less than 1 µg/g of Cr[19]. Due to its structural similarity to many essential elements; Cr can affect the mineral and nutritional status of plants. The plant body lacks a specialized mechanism for absorbing Cr because it is a non-essential element. Because of their structural resemblance to iron (Fe), phosphorus (P), and sulphur (S), Cr can hinder these nutrients' absorption, transport, and accumulation[20].Scientists studying plants have become curious about hydrogen peroxide (H₂O₂) as a signalling molecule in recent years. Louis Jacques Thenard discovered this chemical compound a century ago, and it possesses qualities that support its use as a regulator of plant growth[21]. Although considered to be a hazardous reactive oxygen species (ROS) that might harm a variety of cellular structures, hydrogen peroxide (H₂O₂) has become more prominent as a possible signalling molecule involved in a number of physiological processes[22]. Reactive oxygen species (ROS) generation and removal fluctuate in stressful environmental circumstances as heat, salinity, and water scarcity[23]. Plants generate too many reactive oxygen species (ROS) in adverse conditions, which lead to lipid peroxidation, protein denaturation, DNA mutation, disturbance of cellular homeostasis, and other forms of oxidative damage to cell[24]. H₂O₂ has a longer half-life, is more stable, and can travel freely through cell membranes, making the ROS most suited for the signalling role. It is understood that applying H₂O₂ in little amounts can help plants become less susceptible to stress[25]. A relatively long lifespan due to its low reactivity when compared to other reactive oxygen species (ROS), and its small stature makes it less difficult for it to pass through cellular membranes, assisting in signalling processes. Higher concentrations trigger the beginning of cell death. Furthermore, applying H₂O₂ exogenously may be an effective approach for enhancing crop plants' stress tolerance and productivity in response to a variety of abiotic stressors [21].Look at how the chromium stress influence the physiology, growth and productivity of Brassica oleracea or cabbage. To assess if hydrogen peroxide (H₂O₂) is beneficial for cabbage that has encountered chromium stress. To evaluate the optimum concentration of hydrogen peroxide for mitigating chromium stress in Brassica oleracea and to check out potential interaction between hydrogen peroxide (H₂O₂) and chromium stress.

2.1 Experimental design and layout

The research was performed to determine the role of hydrogen peroxide in alleviating the chromium stress on cabbage (Brassica oleracea var. capitata). A randomized design research consisting of three replicates and 18 pots in total was performed. Garden soil was collected from the Botanical Garden, University of Education, Lahore. After mashing the soil with a 2 mm sieve, the soil was blended properly. Healthy seeds were collected for sowing. After decontaminating the seeds for 15 minutes with 1% hypochlorite solution, washed them with distilled water. The plastic pots which were selected had a diameter of 22 cm and height of 26 cm. The pots were filled with 5 Kg soil and the seeds were sown at the depth of 1 cm. Three concentrations for chromium (0 µM, 100 µM, 200 µM) and two concentrations for hydrogen peroxide (0 µM, 100 µM) were selected. Growth attributes were examined on 4th week of treatments application.

2.2 Determination of physiological attributes

2.2.1 Chlorophyll and Carotenoid Content

The methods of Arnon were used to calculate the content of carotenoid and chlorophyll. Samples of fully-grown fresh leaves were obtained from each replica. Weighted leaves to 0.5 gram and crushed them. After mashing, the extract was homogenized in 10 millilitres of 80% acetone. Next, filtered the extract with Whatman filter paper. Then, remainder was cooled for whole day at 4℃. The absorbance taken at wavelength 480,645, and 663 nm.

2.2.2 SAPD

A SPAD meter was used to figure out the leaf's SPAD value. Three readings (above, middle, and bottom) were taken from one leaf in each pot that was picked. The mean of these values was used to get each leaf's SPAD reading.

2.2.3 Chlorophyll Fluorescence

An OS30p⁺ Chlorophyll Fluorimeter (Opti-science, Inc. Hudson, NH 03051, and USA) employed to quickly obtain light response curves and measure the attributes of chlorophyll fluorescence. One leaf was taken from each replicate and placed in a chlorophyll fluorometer for investigation after the replicates had all been placed in darkness for fifteen minutes. Measurements were taken instantly after a 15-minute dark period, without exposing leaves to daylight. The PSII photochemistry's highest quantum efficiency (Fv/Fm) was determined.

2.2.4 Relative Water Content

Leaves of the same magnitude were taken from each replica, measured, and then rapidly floated over distilled water. Leaves soaked in 25–26 ions free water for 3 hours. Leaves were turgid after three hours; their mass was recorded. Weight of dry leaves were recorded after placed them in oven for 24 hours at 80°C. RWC of the samples was measured using formula as mentioned by Jones and Turner [26].

2.2.5 Determination of photosynthesis related attributes photosynthesis rate (Pn), transpiration rate (Tr), stomatal conductance (gs) and intercellular CO ₂ (Ci)

LCpro SD (ADC Bio scientific Ltd Hoddesdons, UK) was used to analysed gas exchange attributes. Measurements were taken during daytime using PAR from the light source of the leaf chamber. Third mature leaf was set in the chamber and acclimation period was set at 3 minutes to take readings.

2.3 Determination of stress markers

2.3.1 Hydrogen peroxide determination

Recently indicated technique used to estimate it Velikova et al.[27] All replicate fresh leaves assembled, measured (0.5 g), and thoroughly combined with TCA (0.1 %, ml) in a mortar and pestle that had been chilled previously. Test tubes were filled with ½ milliliter of potassium phosphate buffer (pH 7), one milliliter of potassium iodide, and half a milliliter of supernatant after shifted extracts to conical tubes and centrifuged at 12,000 g for 15 minutes and 4^◦C). Test tubes filled with 1/2 ml of potassium phosphate buffer (pH 7), one milliliter of potassium iodide, and half a milliliter of produced supernatant. A UV/VIS spectrophotometer used to record absorbance at 390 nm after the mixture had been swirled.

2.3.2 Malondialdehyde

With just a few changes, it was as certained by using the procedure as stated Cakmak & Horst [28]. Every replicate had fresh leaves taken out (0.5 g weighed with a balance), which were then homogenized in 3 ml of 1.0% trichloroacetic acid at 4℃. With just a few changes, it was as certained by using the procedure as stated Cakmak & Horst Every replicate had fresh leaves taken out (0.5 g weighed with a balance), which were then homogenized in 3 ml of 1.0% trichloroacetic acid at 4℃. After that homogenate was transferred to conical tubes, it was centrifuged at 20,000 g for 15 minutes. Following centrifugation, 0.5 ml of the filtrate was taken in test tubes containing 3ml of 0.5%thiobarbituric acid solution produced in 20% trichloroacetic acid solution. All the samples were then heated at 95℃ for 50 minutes using shaking water bath. After immediately freezing the samples in an ice water bath to stop the reaction, all of the samples were centrifuged again for 10 minutes at 10,000g, and a UV/VIS spectrophotometer was used to detect the absorbance at 532 and 600 nm. MDA level (nmol) was calculated for every sample using following formula.

MDA level (nmol) = [(A_{532 nm} – A_{600 nm})/1.56 x 10⁵] x V/W x 100000

2.3.3 Relative membrane permeability

Fresh leaf sample (0.5 g) was taken from each replica, chopped and dipped in deionized water (20 mL) in falcon tubes. Electrical conductivity (EC0) was measured using EC meter. These sample were kept in refrigerator for about 24 hours. Following that EC1 was measured. Then same samples were autoclaved at 120 ^oC for 20 minutes and EC2 was measured. Relative membrane permeability was estimated using the formula [29, 30]

$$\:RMP\:\left(\%\right)\:=\:\left[\right(EC1\:-\:EC0)/(EC2-EC0\left)\right]\:\times\:\:100$$

2.4 Total Phenolic

Samples of dried leaves, measured 1/2 grams, put in centrifuge tubes after being weighed. Samples were homogenized in ten ml of 80% aqueous acetone for one min. Centrifuged mixture at 4000 rpm for 15 min at 4°C. Removed supernatant and dry solid residue was extracted for further use. After that, dried solid residue was combined with ten millilitres of methanol. The Folin-Ciocalteu technique used to calculate the total phenolic content. After the prepared samples (2 ml from every replica) placed in tubes, 0.8 milliliter of sodium carbonate (7.5%) and 1 ml of Folin-Ciocalteu reagent mixed thoroughly, mixture allowed to stand for 30 minutes. Absorbances at 765 nm recorded using UV/VIS’S spectrophotometer. Total phenolic content estimated as gallic acid equivalents per gram dry material.

2.5 Determination Leaf Proline

Proline was calculated using the Bates et al. [31] technique. In the procedure, 0.5 g of leaves was homogenized in 3% of 10 mL sulfuric acid. By using Whatman filter paper the homogenate was filtered. After mixing 1.25 g of ninhydrin into Glacial acetic acid (30 mL) and O-phosphoric acid (6 M, 20 mL), acid ninhydrin reagent was made. In a glass tube, 2 mL of homogenate filtrate was dissolved into galacial acetic acid (2 mL) and ninhydrin acid, then sample solution was incubated at 100 ^oC for 60°C in oven. After this reaction mixture was kept in ice bath. The sample mixture was centrifuged where 2 mL of supernatant was collected. Further, 4 mL of toluene is added in reaction mixture for 1–2 minutes while passing a constant stream of air. From the upper aqueous phase, the chromophore was withdrawn. Absorbance was obtained at 520 nm with the help of UV spectrophotometer by using blank (toluene).

2.6 Total soluble protein

Each replicate fresh leaf samples were taken, weighed (0.5 g), and mixed in 10 ml of 50 mM phosphate buffer that had been pre-cooled (pH 7.8). After gathering the extracts in a conical flask, they were centrifuged at 6,000 g for 20 minutes at 4℃. After being removed from the residue, the supernatant was kept in a deep freezer. The method described above was used to measure the protein level in the samples[32]. Bradford solution was prepared by mixing 100 ml of phosphoric acid (85%) with 50 ml of ethanol % after 100 ml of Coomassie Brilliant Blue had been dissolved. One liter was added to the total volume by adding distilled water. 5 ml of Bradford solution were mixed with 0.1 ml of pre-made leaf extracts that had been deep-frozen. Optical density was measured with a UV/VIS spectrophotometer at a wavelength of 595 nm.

2.7 Estimation of antioxidant enzymes activities

2.7.1 Catalase (CAT) and Peroxidase (POD)

To assess the enzymatic activity of catalase and peroxidase, specific reaction solutions were meticulously prepared, and their activities were quantified through spectrophotometric analysis. For the catalase assay, a reaction solution was formulated by diluting a 50 mM phosphate buffer (pH 7.0) to achieve a final concentration of 1 mM to this buffer, 1.9 mL of a 5.9 mM hydrogen peroxide (H₂O₂) solution was added, along with 0.1 mL of the enzyme extract. The total volume of the reaction mixture was adjusted to 3.0 mL. The catalytic activity of the enzyme was monitored by recording the absorbance at 240 nm using a UV/VIS spectrophotometer, with measurements taken every 30 seconds over a total duration of 120 seconds. In a parallel setup for assessing peroxidase activity, a reaction mixture was prepared using 0.7 mL of a phosphate buffer at pH 5.0. To this buffer, 0.6 mL of a 20 mM guaiacol solution and 0.6 mL of a 40 mM H₂O₂ solution were added. The enzyme extract was incorporated at a volume of 0.1 mL, making up a total reaction volume of 2.0 mL. The peroxidase activity was evaluated by recording the absorbance at 470 nm, with data collected every 30 seconds throughout a 150-second period. This detailed approach facilitated precise monitoring and quantification of the enzymatic reactions under investigation.

2.7.2 Ascorbate Peroxidase (APX) and Superoxide dismutase (SOD) activity

APX reaction mixture prepared for every replica, contained (3 milliliter), and included 50 millimolar phosphate buffer 2.70 milliliter, 7.50 millimolar ascorbic acid 0.10 milliliter, 0.10 milliliter of 300 millimolar hydrogen peroxide (H₂O₂) and extract of enzyme 0.10 milliliter. Recorded absorbance at wavelength of 290nm for every 30 sec in time period of 60 sec[33].

Enzyme solution 0.05 ml was added to solution, treated to 4000 lux light for 20 min. Following that, the prepared extract analysed using a UV/VIS spectrophotometer to figure out their absorbance at 560 nm[34].

2.8 Determination of Inorganic Mineral Ions Content

Using a pestle and mortar, samples of dried shoots and roots were obtained and thoroughly crushed. Samples of roots and shoots weighing 0.1 g were digested using H₂SO₄. Test tubes were filled with samples, and 2 ml of H₂SO₄ was added. Samples were cooked on a hot plate for 24 hours, and then H₂O₂ added drop by drop until the liquid took on no colour. Then added distilled water. Final volume elevated to 50 millilitres. Filtered mixture and recorded reading for Na^+, K⁺ and Ca²⁺ using flame-photometer.

3.1 Effect of hydrogen peroxide on growth attributes of Brassica oleracea var. capitata grown under Cr stress

Chromium stress significantly decreased the shoot length (Fig. 1A) of Brassica oleracea var. capitata (cabbage) by 23% and 38% when grown in toxic conditions under 100 µM and 200 µM application of chromium, respectively compared to the control. Foliar application of H₂O₂ showed significant improvement in the shoot length by 26% as compared to the control. In comparison to the stressed plants, the shoot length increased by 11% and 18% with foliar application of H₂O₂. Shoot fresh weight of Brassica oleracea var. capitata was significantly lowered by 43% and 70% under chromium stress of 100 µM and 200 µM respectively compared to the control untreated group (Fig. 1B). The shoot fresh weight was improved by foliar application of H₂O₂ by 9% compared to the untreated control. The fresh weight of shoot was increased by combination of H₂O₂ and chromium (H₂O₂+Cr100 and H₂O₂+Cr200) by 28% and 35% compared to the plants under chromium stress only.

The shoot dry weight of Brassica oleracea var. capitata (Fig. 1C) was significantly reduced by 37% and 53% under chromium stress of 100 µM and 200 µM compared to the control untreated group. The shoot dry weight was upgraded by 19% when spray of H₂O₂ was applied compared to the control. The shoot dry weight was improved under the combination of application of H₂O₂ and chromium by 16% and 37% respectively, compared to the plants under chromium stress of 100 µM and 200 µM. The root length was significantly reduced under 100 µM and 200 µM chromium stress by 39% and 67% compared to the control untreated group. In comparison to the control, spray of H₂O₂ increased the root length by10% as compared to the control. The root length was significantly upgrade by 18% and 33% compared to the plants with 100 µM and 200 µM chromium stress respectively (Fig. 2A).

Fresh root weight was reduced by 31% and 56% with 100 µM and 200 µM chromium stress, compared to the control. Root FW increased by the application of H₂O₂ by 29% compared to the control. Likewise, the plants with treatment of H₂O₂ showed improved root fresh weight by 32% and 47% compared with only chromium treated plant (Fig. 2B). The root dry weight of Brassica oleracea var. capitata was significantly lessened by 32% and 56% under chromium stress of 100 µM and 200 µM compared to the control untreated group. The root dry weight was increased by 12% when spray of H₂O₂ was applied compared to the control. The root dry weight was improved under the combination of application of H₂O₂ and chromium by 18% and 34% compared to the plants under chromium stress of 100 µM and 200 µM respectively (Fig. 2C).

3.2 Effect of hydrogen peroxide on physiological attributes of Brassica oleracea var. capitata grown under Cr stress

3.2.1 Chlorophyll a & b

During the present study, the reduction in the chlorophyll a observed in Brassica oleracea var. capitata by 28% and 50% compared to the control. The foliar treatment of H₂O₂ show improved chlorophyll a content by 26% compared to the control group. The chlorophyll a content was also improved by combine application of H₂O₂ and chromium by 15% and 23% compared to the only chromium stress plants. The current study showed reduction in the chlorophyll b content in Brassica oleracea var. capitata by 41% and 52% as compared to the control. The foliar treatment of H₂O₂ shown increased chlorophyll b content by 29% compared to the control group. The chlorophyll b content was also improved by combine application of H₂O₂ and chromium by 34% and 29% compared to the plants under chromium stress only (Fig. 3A & B).

3.2.2 Carotenoids

The carotenoids content was significantly decreased by 30% and 51% in Brassica oleracea var. capitata under chromium stress of 100 µM and 200 µM compared to the control untreated group. The spray of H₂O₂ showed increased carotenoids content by 27% as compared to the untreated plants. The carotenoids were also improved by the combination of H₂O₂+chromium by 10% and 21% compared to the plants under only chromium stress (Fig. 3C).

3.2.3 Relative water content

The relative water content in Brassica oleracea var. capitata was significantly lowered by 18% and 35% under chromium stress of 100 µM and 200 µM compared to the control untreated. The RWC level was improved by 8% with the foliar spray of H₂O₂ as compared to the untreated plants. With the combination of H₂O₂+chromium (Cr100 + H₂O₂ and Cr200 + H₂O₂), the RWC was enhanced by 13% and 9% compared to the only chromium stress plants (Fig. 3D).

3.2.4 SPAD value measurement

The SPAD value (chlorophyll value) decreased by 16% and 29% in B. oleracea under chromium stress 100 µM and 200 µM as compared to the control. The spray of hydrogen peroxide (H₂O₂) increased the SPAD value by 20% as compared to the control. Combine application of H₂O₂ and chromium improved the SPAD value. (chlorophyll value) by 11% and 13% compared to the chromium stress only (Fig. 4A).

3.2.5 Chlorophyll fluorescence (Fv/Fm)

The chlorophyll fluorescence (Fv/Fm) in B. oleracea reduced by 22% and 38% under chromium stress 100 µM and 200 µ compared to the control. The foliar application of H₂O₂ improved the Fv/Fm value by 15% compared to the control. While the combine treatment of H₂O₂ and chromium improved 15% and 9% compared to the only chromium stress (Fig. 4B).

3.2.6 Photosynthesis related attributes (Pn, Tr, gs and Ci)

Results in the Fig. showed that Pn, Tr and gs were reduced greatly under Cr stress. For instance, reduction in Pn, Tr and gs were recorded by 31, 47 and 52%, respectively, under 200 µM Cr stress. On the other side, foliar spray of H₂O₂ increased Pn, Tr and gs under all conditions i.e. control, low stress level (100 µM) and high stress level (200 µM). Contrarily, Ci was increased in Cr exposed plants by 13 and 24%, respectively, at 100 and 200 µM Cr stress. While H₂O₂ treatment greatly reduced Ci in control and stress conditions compared with their respective controls (Fig. 5A-D).

3.3 Effect of hydrogen peroxide on stress markers (H₂O₂, MDA and RMP) in Brassica oleracea var. capitata grown under Cr stress

Under chromium stress of 100 µM and 200 µM in Brassica oleracea var. capitata H₂O₂ level was significantly enhanced by 14 and 41% compared to the control. On the other side the treatment of H₂O₂ reduced the hydrogen peroxide level by 17% compared to the control. Likewise, the combine application of H₂O₂ and chromium decreased the H₂O₂ level by 23 and 27% compared to the stressed plant (Fig. 6A). The current study showed the significant increase of malondialdehyde level by 41 and 60% under chromium stress of 100 µM and 200 µM compared to the control. The foliar application of H₂O₂ significantly decreases the MDA level by 20% compared to the control. However, the combine application of H₂O₂ and chromium decreased the MDA level by 17 and 31% compared to the stress plants (Fig. 6B). RMP was increased in plants given Cr stress by 64 and 127% at 100 µM and 200 µM Cr stress, respectively. Whereas foliar application of H₂O₂ reversed the impact of Cr stress by reducing the RMP up to 31% in plants grown in 200 µM Cr stress (Fig. 6C)

3.4 Total phenolics

In the present study, the phenolic content in the leaves was decreased in Brassica oleracea var. capitata by 25% and 42% under chromium stress of 100 µM and 200 µM as compared to the untreated group. The foliar application of H₂O₂ increased the phenolic content in the leaves by 4% as compared to the control. Anyhow, the combine application of H₂O₂ and chromium enhanced the phenolic content by 8% and 19% compared to the plants with chromium stress of 100 µM and 200 µM (Fig. 7A).

3.5 Proline content

The proline content was enhanced in the in Brassica oleracea var. capitata by 52% and 81% under chromium stress of 100 µM and 200 µM as compared to the control. The proline content was increased by the foliar spray of H₂O₂ by 131% compared to the control. While the combined treatment of H₂O₂ and both chromium level also boosted the proline content by 116% and 91% as compared to the only chromium stressed plants (Fig. 7B).

3.6 Total soluble protein

The chromium toxicity decreases the total soluble proteins by 39 and 54% under 100 µM and 200 µM stress condition respectively compared to control. The proteins content in Brassica oleracea var. capitata was improved significantly by 11% when foliar application of H₂O₂ was applied compared to the control. The total soluble proteins in cabbage were enhanced by 24% and 21% when foliar application of H₂O₂ was given compared to the plants under chromium stress (Fig. 7C).

3.7 Effect of hydrogen peroxide on antioxidants enzymes activities in Brassica oleracea var. capitata grown under Cr stress

3.7.1 Catalases (CAT) and Peroxidases (POD) activities

The catalase activity in the Brassica oleracea var. capitata was upgraded by 27% and 55% as compared to the plants with chromium stress of 100 µM and 200 µM, respectively. The foliar treatment of H₂O₂ boosted the activity of CAT by 91% compared to the untreated plants. The combination of H₂O₂ and chromium (Cr100 + H₂O₂ and Cr200 + H₂O₂) also perk up the activity of CAT by 55% and 91% as compared to the chromium stressed plants (Cr100 and Cr200) (Fig. 8A). Toxicity of chromium has significantly enhanced the peroxidase activity in Brassica oleracea var. capitata by 33% and 60% under stress of 100 µM and 200 µM as compared to the plants without any treatment. The POD activity became significantly enhanced by 97% when we apply foliar application of H₂O₂ compared to the control. However, the combine treatment of H₂O₂ and chromium increase the POD activity by 91% and 67% compared to the chromium stress plants only (Cr100 and Cr200) respectively (Fig. 8B).

3.7.2 Ascorbate peroxidase (APX) and Superoxide dismutase (SOD) activities

Under chromium stress of 100µM and 200µM, the ascorbate peroxidase activity in Brassica oleracea var. capitata was enhanced by 55% and 99% compared to the control (Fig. 8C). Likewise, the spray of H₂O₂ increased the APX activity by 171% as compared to the control. The activity of APX seems boosted with the combination H₂O₂ and chromium by 136% and 116% as compared to the plants under chromium stress. Superoxide dismutase activity in the Brassica oleracea var. capitata was increased by 53% and 90% compared to the plants with chromium stress of 100 µM and 200 µM, respectively. The foliar treatment of H₂O₂ upgraded the activity of SOD by 121% compared to the untreated plants. The combination of H₂O₂ and chromium (Cr100 + H₂O₂ and Cr200 + H₂O₂) also improved the activity of SOD by 118% and 90% as compared to the chromium stressed plants (Cr100 and Cr200) (Fig. 8D).

3.8 Effect of hydrogen peroxide on inorganic ions (Na⁺, Ca²⁺ and K⁺) in Brassica oleracea var. capitata grown under Cr stress

In Brassica oleracea var. capitata, the K⁺ ions reduced by 19% and 36% under chromium stress of 100 µM and 200 µM as compared to the control untreated. The foliar treatment of H₂O₂ enhanced the K⁺ ions by 10% as compared to the control. Likewise, the combination of H₂O₂ and chromium boosted the K^₊ ions by 17 and 7% compared to the only chromium stressed plants (Fig. 9A). The Ca²⁺ ions decreased in Brassica oleracea var. capitata by 27% and 37% with chromium stress of 100 µM and 200 µM as compared to the control. The foliar spray of H₂O₂ increased the Ca²⁺ ions by 29% as compared to the control. However, the combine treatment of H₂O₂ and chromium upgraded the Ca²⁺ ions by 26% and 7% as compared to the chromium stressed plants (Fig. 9B). Na⁺ ions significantly reduced in Brassica oleracea var. capitata by 33% and 45% under chromium stress of 100 µM and 200 µM respectively, compared to the control untreated group. The foliar spray of H₂O₂ increased the Na₊ ions by 1% as compared to the control. While the combine treatment of H₂O₂ and chromium enhanced the Na₊ ions by 23% and 32% as compared to the plants under chromium stress (Fig. 9C).

3.9 Correlation and Principle component analysis

Pearson’s correlation (Fig. 10A) showed that growth attributes were in positive correlation with photosynthesis related parameters and nutritional contents in B. oleracea var. capitata grown in Cr stress along with foliar application of H₂O₂. Growth attributes studied in B. oleracea var. capitata were negatively correlated with stress markers lipid peroxidation and membrane permeability. This showed that Cr stress mediated changes in cellular metabolism disrupted growth of plants. In PCA analysis, PCA for individual parameters in Fig. 10B showed all the treatments applied in this study (labelled as 1, 2, 3…, 6) are distributed successfully in first two PCs. Figure 10C showed percentage of explained variables in total of five principle components (PCs). Out of these, PC1 and PC2 contributed the most 69.21 and 17.67%, respectively. Figure 10D showed that all explained variables are grouped into two, parameters in the first group are aligned to PC1 and the parameters in second group are aligned to PC2. Parameters in one group are positively correlated with each other.

The findings of our results showed that morphological characters i.e. shoot length, root length, shoot and root fresh weight, shoot and root dry weight and no of leaves declined with the chromium stress in Brassica oleracea var. capitata as compared to the control. Chromium ions may disrupt with normal cell division and elongation processes, resulting in shortening of growth parameters. It might decrease plant growth by preventing the absorption and movement of essential minerals including calcium, magnesium, and potassium. These nutrients are critical for plant growth and development[35]. Ahmed et al.[36] supported the findings of our results that chromium stress decreased the morphological parameters. According to our findings the treatment of hydrogen peroxide (H₂O₂) enhanced the above-mentioned parameters. The primary root of this is H₂O₂ function as a Signalling molecule, which lowers oxidative stress and increased the growth parameters[18].

The findings of our results indicated that chlorophyll a and chlorophyll b and carotenoids diminished under chromium stress as compared to the control untreated. Hadif et al.[37] supported our findings that chlorophyll a, chlorophyll b and carotenoids reduced under chromium stress, the reason is that a crucial enzyme in the production of chlorophyll, “aminolaevulinic acid dehydrates, could be negatively influenced by chromium, which then affects its production. (ALA) itself, causing an increase in ALA concentration and a corresponding drop in chlorophyll levels. Our results shown that hydrogen peroxide improves the content of Chl a and b and carotenoids as compared to control Olowolaju et al.[38] supported our finding as hydrogen peroxide was crucial for the formation of chlorophyll and carotenoids.

Our investigation showed that chlorophyll value (SPAD value) lowered with the chromium stress as compared to the control. Fariduddin et al.[21] supported our result that SPAD value decreased under Cu stress, this declined in the chlorophyll value may be the result of oxidative stress, which destroys organic molecules through oxidation by light and inhibits enzymes involved in the manufacture of chlorophyll, blockage of absorption and movement of other metal elements.

Our results showed that the Fv/Fm value decreased due to the chromium stress as compared to our respective control. Singh et al.[18] supported our findings that the Fv/Fm value declined under chromium stress as it reduces the photosynthetic pigments which are essential to photosynthesis. A drop in the photosynthetic pigments may be the primary cause of the Cr stress-mediated drop in Fv/Fm ratio. Over reduction of quinone pools mediated by Cr-stress could be another factor contributing to the drop in the Fv/Fm ratio. While our results indicated that hydrogen peroxide treatment improves the chlorophyll fluorescence. The findings of our results showed that in Brassica oleracea var. capitata the relative water content was decreased as compared to our respective control. Our findings were supported by Kumar et al.[11] demonstrated that under various Cr treatments, we noticed a drop in the RWC in sweet potato leaves, indicating that the plants were stressed. Optimal water levels are not retained by sensitive plant species, which impacts the plant's ability to alter its osmotic pressure.

Cr toxicity reduced Pn while it increased Ci which is attributed to non-stomatal restriction as major cause of reduction in Pn. Tr and g_s were also reduced under Cr toxic conditions. This suggests that mesophyll cell injury under oxidative stress lowered absorption of CO₂ and raised Ci ultimately suppressed photoreactions [39]. Cr toxicity could reduce Pn by many of ways including stomatal closure and reduced number of stomata [40]. Severe conditions under oxidative stress could lead to abnormal stomatal development [41]. This resulted in reduced Tr and g_s as evident from results of our study. Previous study also revealed that S. oleracea given Pb stress had reduced Pn, Tr and g_s. Results showed that application of chelate successively recovered gas exchange attributes in Pb-stressed plants [42]. Similarly, in our study application of H₂O₂ greatly increased Pn, Tr and g_s in control as well as Cr stressed plants.

The findings of our result described that malondialdehyde and hydrogen peroxide level increase in Brassica oleracea var. capitata under chromium stress as compared to our respective control. Singh et al.[15] supported our results that MDA and H₂O₂ level increased under Cr stress is associated with increased levels of H₂O₂ and MDA were observed to correlate with a decrease in GST activity due to Cr. According to these findings, increased GST activity is most likely essential for giving Cr stress resistance. Application of H₂O₂ along with Cr stress, however, greatly lowered MDA and H₂O₂ while also increasing GST activity. Our results indicated that chromium stress declined the total soluble proteins in Brassica oleracea var. capitata as compared to the control. The results of Fu et al.[43] supported our findings that chromium stress reduced the total soluble proteins in cabbage because chromium triggers oxidative damage to plant which substantially decrease the total soluble protein content in plants. Our results indicated that hydrogen peroxide treatment increase the protein level in plant as compared to the control. This finding was supported by Habib et al.[44] as described that hydrogen peroxide increased the accumulation of TSP, by maintain a higher cellular water content through osmotic adjustment which enhance wheat plants capacity to bear drought stress and protect plant from oxidative damage as it is a Signalling molecule. Our findings were also supported by which demonstrated that protein content decrease in tomato plant under copper stress. It has been discovered that Cu stress breaks down the disulphide association in proteins irreversibly, which makes it easier for their structure and function to be disrupted. However, root dipping treatment with hydrogen peroxide improves its structural integrity. The findings of our study indicated that antioxidants like SOD, POD, CAT and APX boosted in Brassica oleracea var. capitata under chromium stress. Our findings were supported by Fu et al.[43] in response to external chromium stress, plants' activities of POD, SOD, and CAT were enormously raised. This is known as a shift in the antioxidant defence system. By preventing plants under chromium stress from producing too much ROS, exogenous silicon, selenium, and the silicon-selenium compound treatments could increase plant tolerance to chromium and preserve the plant's internal redox equilibrium as we gave hydrogen peroxide application to upgraded the antioxidants to create defines mechanisms. Our findings were also supported by Hassan et al.[45] which demonstrated that amount of SOD, POD, CAT and APX rose linearly with soil concentrations of heavy metals and urbanization. An abundance of metals will induce plants to encounter oxidative stress, which can encourage the production of ROS. The antioxidant enzymes may convert H₂O₂ to H₂O in plant cells antioxidant enzymes therefore increased proportionately to protect cells from oxidative stress, which is also consistent with our findings. The findings of our results showed that proline level upgraded in Brassica oleracea var. capitata under chromium stress as compared to the control. Fu et al.[43] supported our results that typically found in plants, proline is an osmotic pressure-regulating chemical that serves to maintain redox metabolism by eliminating excess reactive oxygen species. Proline synthesis also supports the plant redox cycle and enhances the antioxidant defence mechanism of plants under stress from the environment. These findings suggested that plants do not require an excessive amount of proline to get rid of ROS that are formed by plants under heavy metal stress, and that silicon, selenium, and the silicon-selenium compound treatments can reduce ROS by starting other antioxidant defence processes as our treatment demonstrated that hydrogen peroxide act as Signalling molecule prevented the production of proline. The findings of our results indicated that in Brassica oleracea var. capitata inorganic mineral ions reduced under chromium stress as compared to the control. This finding was supported by the results of Ahmad et al.[36] which showed that calcium has a variety of crucial roles in plants, such as maintaining ionic balance, serving as the building block of middle wall lamella, acting as an essential cofactor and regulator of enzymes, and being a part of cell Signalling pathways. It is already known that K⁺ plays an active part of sustaining ionic homeostasis, acting as an osmolyte, and activating and modulating enzymes. The findings showed that chromium significantly affects K⁺ and Ca²⁺ absorption and translocation. Therefore, metal-induced decrease in calcium levels may cause abnormalities to these cellular functions, which in turn may promote growth retardation and aberrant metabolism.

This research concluded that hydrogen peroxide (H₂O₂) plays a crucial part in alleviating the chromium (Cr) stress in Brassica oleracea var. capitata. The results illustrated the potential of H₂O₂ as a stress mitigant by establishing a considerable improvement in morphological, oxidative and physiological parameters in Cr-stressed plants. According to our study, H₂O₂ increased the plant tolerance for chromium stress by adjusting enzyme activity and stimulating antioxidant defenses. These findings have significant implication for the formulation of future strategies that enhanced plant durability and productivity in the face of heavy metal stress. Future studies should be emphasized on examining the molecular processes that enable the H₂O₂-mediated stress tolerance and its possible uses in crop production.

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.

Statements & Declarations

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 (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

Author Contributions

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

Acknowledgments: Authors are thankful to Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

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Competing interest reported. one of the authors have conflict of interests with Pervaiz Ahmed, the editor of BMC Plant Biology.

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Hydrogen peroxide foliar spray alleviates chromium toxicity through modulation of antioxidant defence mechanism, photosynthetic machinery and ions regulation in Brassica oleracea L.

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Abstract

Figures

1 Introduction

2 Material and methods

2.1 Experimental design and layout

2.2 Determination of physiological attributes

2.2.1 Chlorophyll and Carotenoid Content

2.2.2 SAPD

2.2.3 Chlorophyll Fluorescence

2.2.4 Relative Water Content

2.3 Determination of stress markers

2.3.1 Hydrogen peroxide determination

2.3.2 Malondialdehyde

2.3.3 Relative membrane permeability

2.4 Total Phenolic

2.5 Determination Leaf Proline

2.6 Total soluble protein

2.7 Estimation of antioxidant enzymes activities

2.7.1 Catalase (CAT) and Peroxidase (POD)

2.7.2 Ascorbate Peroxidase (APX) and Superoxide dismutase (SOD) activity

2.8 Determination of Inorganic Mineral Ions Content

3 Results

3.2.1 Chlorophyll a & b

3.2.2 Carotenoids

3.2.3 Relative water content

3.2.4 SPAD value measurement

3.2.5 Chlorophyll fluorescence (Fv/Fm)

3.2.6 Photosynthesis related attributes (Pn, Tr, gs and Ci)

3.4 Total phenolics

3.5 Proline content

3.6 Total soluble protein

3.7.1 Catalases (CAT) and Peroxidases (POD) activities

3.7.2 Ascorbate peroxidase (APX) and Superoxide dismutase (SOD) activities

3.9 Correlation and Principle component analysis

4 Discussion

5 Conclusion

Declarations

References

Additional Declarations

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