Morpho-physiological and Biochemical Responses of Fenugreek Towards Cadmium Stress

Introduction

Heavy metal contamination and their subsequent impact on plants, humans and animals is a matter of global concern. Their persistence and non-biodegradable nature make them consistent and a great threat to the environment. Cadmium (Cd) ranks among the most hazardous heavy metals, and its biological functions in both plants and animals remain unidentified.¹ Nonetheless, it poses a major threat to plant development and productivity.² There are two major sources of cadmium: natural, which include soil erosion and volcanic eruption; and anthropogenic activities such as mining, smelting and agriculture activities.^3,4 Rapid industrial revolution in recent years has vastly contributed to the Cd deposition in the soil. The cadmium contamination of the soil is responsible for its accumulation in plants, as Cd is easily absorbed by the plant’s root.² Cadmium is readily absorbed by the plant roots through the transport pathways of microelements such as Fe, Mn or Zn and disrupts their uptake leading to their crisis in the plants.^5-7 Crop plants growing in high level of Cd contaminated soil exhibited various growth, physiological and biochemical disorder such as reduction in seed germination, root elongation, nutrient uptake, photosynthetic pigments, sugars and soluble protein, changes in membrane permeability, modulation in enzyme activities and reactive oxygen species (ROS) activity.^8-10 The generation of reactive oxygen species (ROS) induced by cadmium, including hydrogen peroxide (H₂O₂), superoxide ( ), singlet oxygen (¹O₂), and hydroxyl radical , occurs at both cellular and subcellular levels. This process is responsible for the oxidative degradation of macromolecules such as nucleic acids, proteins, and lipids, as well as the compromise of membrane integrity and the disruption of metabolic processes, ultimately leading to the inhibition of plant growth.^11-13 To scavenge and alleviate the negative effects of heavy metal-induced oxidative stress, plants produce and employ a variety of defensive enzymes such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), as well as accumulate osmotic substances such as proline.^14,15 Superoxide dismutases neutralize to produce H₂O₂ and O₂, whereas CATs and peroxidases detoxify H₂O₂.^16,17 Proline reduces oxidative stress by upregulating the antioxidant defense enzymes, making the defense system active. However, plant genotypes, age and environmental conditions may influence the antioxidative response of plants towards heavy metals.¹⁸ Many have reported the elevation in the antioxidant activities under Cd stress, while some have observed the reduction in the activities.¹⁹ Fenugreek (Trigonella foenum-graecum L.) is a valuable leguminous crop grown in many countries, including India, because of its food value and medicinal importance.^20,21 Fenugreek, also known as methi in India, is produced in quantities of approximately 55 thousand tons per year.^22,23 Heavy metal-induced oxidative stress has a detrimental effect on a variety of agricultural crops, causing not only decrease in plant growth and yields but even plant deaths.²⁴ Present study aims to evaluate the impact of different levels of Cd concentration on morpho-physiological and biochemical attributes of fenugreek at its early stage of growth and alteration of antioxidant defense activity in response.

Materials and Methods

Plant treatment and growth measurement

Fenugreek seeds taken from Raiganj Beej Bhander, Mohanbati Raiganj, Uttar Dinajpur, West Bengal were surface sterilized with 0.1% HgCl₂ for 3 min before being washed three or four times with distilled water. After the sterilization, fifteen seeds were placed in the sterilized petriplates (90 mm) in between filter papers moistened with different concentrations of Cd (5 µg mL^-1, 10 µg mL^-1, 15 µg mL^-1, 20 µg mL^-1, and 25 µg mL^-1) for 7 days. For the control, seeds were exposed to distilled water. Three replicas were maintained for each treatment including control in optimal conditions (temperature 24±2 °C, humidity 62-69% and photoperiod 16 h) at growth chamber. Solutions of Cd concentration were given to the respective petriplates to maintain the concentration. Over a period of seven days, seed germination was observed, and additional growth indices, including the germination index (Gt/Dt), Dt number of days to germinate; Gt = the number of seeds germinated each day corresponding to Dt),²⁵ lengths of shoots, roots, and seedlings, as well as the fresh weight of seedlings and the seedling vigour index ; where,  were measured.

Physiological assessment

Extraction and estimation of photosynthetic pigments

Photosynthetic pigments, including Chlorophyll a (Chl-a), Chlorophyll b (Chl-b), and Total chlorophyll (Total Chl), were extracted using the method of Lichtenthaler²⁶ with some modifications. 0.3 g of each treatment’s leaves were homogenized with 80% (v/v) acetone in a pestle and mortar before being centrifuged at 5,000 rpm for 10 min. After centrifugation, the supernatant was collected to assess the pigment content spectrophotometrically at 645 nm and 663 nm, respectively, and calculated using the formulae²⁷ listed below: 

Determination of protein content under Cd stress

Proteins were extracted and estimated from the fresh seedlings. Fresh seedlings (100 mg) were collected at random from each treatment and homogenized in 5 mL of 0.1 M potassium phosphate buffer (pH 7.0) before being centrifuged at 10,000 rpm for 20 min. The protein content in the supernatant was determined using the method described by Lowry et al.²⁸ Absorbance readings were taken at 660 nm, and the protein concentration in the samples was calculated and reported as mg/g of fresh weight. A standard curve was generated using bovine serum albumin (BSA).

Detection and estimation of H₂O₂ and lipid peroxidation

Hydrogen peroxide (H₂O₂) was detected histochemically by using 3, 3ʹ-Diaminobenzidine (DAB).²⁹ The procedure described by Velikova et al,³⁰ with slight modifications, was utilized to determine the content of hydrogen peroxide in tissue samples. Fresh seedlings, each weighing 0.5 g, were homogenized with 3 mL of 0.1% trichloroacetic acid (TCA) while kept chilled in an ice bath. This mixture was then centrifuged at 12,000 g for 20 min. Subsequently, 1 mL of 1 M potassium iodide (KI) was added to a reaction mixture containing 0.5 mL of 10 mM phosphate buffer and 0.5 mL of the supernatant. The resulting solution was incubated in darkness for 30 min. The optical density (OD) of the supernatant was measured at 390 nm, and the hydrogen peroxide content in each sample was calculated and expressed as µM g^-1 FW.

The method of Shen et al³¹ with slight modifications was used to get the malondialdehyde (MDA) content of the tissue, produced by the thiobarbituric acid (TBA) reaction. Fresh leaves (0.5 g) from each treatment was homogenized with 10% TCA (5 mL) and centrifuged at 6,000 rpm for 10 min. The mixture containing aliquot (2 mL) of the supernatant and 0.6% TBA (2 mL) in 10% TCA was heated at boiling temperature and cooled in an ice bath. Again, samples were centrifuged and absorbance was read at 450, 532 and 600 nm by UV-VIS spectrophotometer (Analytical UV-2080T, Gujarat, India). MDA content as MDA (µmol L^-1) = was measured and expressed in µmol^-1 FW.

Biochemical assessment

Determination of proline content

About 0.5 g of fresh seedlings was ground in 10 mL of 3% aqueous sulphosalicylic acid and then subjected to centrifugation at 6,000 rpm for 15 min.³² Prior to cooling in an ice bath, a mixture consisting of 2 mL of the supernatant, 2 mL of glacial acetic acid, and acid ninhydrin reagent was boiled for 60 min. After cooling, 4 mL of toluene was added and mixed well for 20-30 sec. The chromophore was then cautiously moved to a new tube, and its absorbance was recorded at 520 nm using a toluene blank as a reference. In order to calculate the proline content, which was expressed as µmoles/g tissue, a standard curve was created using proline standards.

Extraction and determination of antioxidant enzyme activities

To measure the antioxidant enzyme activity, precisely 0.1 g of leaves were weighed and homogenized with 5 mL of potassium phosphate buffer (pH 7.0). After that, the homogenate was centrifuged for 20 min at 10,000 rpm. Enzyme activities were determined by using the supernatant. Methods of Siddiqui et al³³ with slight modifications was used to determine the CAT activity. The reaction mixture was made which consisted of phosphate buffer (2.6 mL), enzyme extract (0.1 mL) and 0.1 mL of H₂O₂ (1%). Blank sample was prepared by replacing the enzyme extract with phosphate buffer to the reaction mixture. The OD value was taken at 240 nm at 30 sec intervals till 2 min. The CAT activity was calculated in nmol H₂O₂ min^-1, using an extinction coefficient of 39.4 mM cm^-1, and expressed as min^-1g^-1 FW. The method of Zhang³⁴ with slight modifications, was used to assess changes in peroxidase activity. The reaction mixture was composed of 4.7 mL of 0.1 M potassium phosphate buffer, 0.1 mL of 20 mM H2O2, 0.1 mL of 27 mM guaiacol, and 0.1 mL of enzyme extract. Absorbance at 470 nm was monitored every 30 seconds for a duration of 3 min. The activity was calculated with an extinction coefficient of 25.5 mM cm^-1 and expressed as min^-1g^-1 FW.

Statistical analysis

Data collected from six different treatments, including control and concentrations of 5, 10, 15, 20, and 25 µg mL^-1 of Cd, were subjected to statistical analysis through analysis of variance (ANOVA) using Statistix 8.1, with each treatment replicated three times. Multiple comparisons were performed to compare the mean values between treatments (p<0.05) using Fischer least significant difference (LSD) test. Pearson correlation analysis was carried out to find the exact correlation coefficient values among the morpho-physiological and biochemical parameters studied under stress.

Results

Cadmium stress on plant growth

Results exhibited that all the morphological traits were greatly affected by Cd stress. Root length, shoot length, germination index, seedling length, seedling vigour index and fresh weight were sharply decreased under stress. Table 1 shows how morphological parameters are affected by Cd stress at different concentrations. The germination index did not significantly change in the current study at the lower concentrations of Cd (5, 10, and 15 µg mL^-1), but it significantly decreased in seed treated with Cd at 20 µg mL^-1 and 25 µg mL^-1 (Table 1). Plants exhibited a significant reduction in root length (26.81-82.1%), shoot length (25.79-67.75%), and fresh weight (11.93-37.61%) at various levels of Cd stress (5 – 25 µg mL^-1) compared to the control (Table 1). Similarly, compared to the control, plants treated with 20 µg mL^-1 Cd, showed greater decrease in seedling length and seedling vigour index by 67.75% and 67.45% respectively. Similar results were also recorded at 25 µg mL^-1 Cd for seedling length and seedling vigour index. Even fresh weight was also recorded Lowest at 25 µg mL^-1 Cd.

Cadmium stress on photosynthetic traits

Cd stress significantly reduced the accumulation of chlorophyll content in all the treated plants compared to control. Maximum chlorophyll content was observed in control (Chl-a= 0.403 mg g^-1FW, Chl-b= 0.185 mg g^-1FW and Total chlorophyll= 0.587 mg g^-1FW) but sharply declined in all the Cd-treated plants. There was a successive decline in all the treated plants from lower to higher concentration. The reduction was maximum at 25 µg mL^-1 of Cd (50.62% of Ch-a, 53.51% of Ch-b and 51.62% of Total chlorophyll).

Protein, Proline, ROS and lipid peroxidation under cadmium stress

All of the treated plants showed a progressive increase in proline content when exposed to higher Cd concentrations. Proline content increased by 172.4% at 25 µg mL^-1 of Cd, while it was lowest in the control. As demonstrated in Fig. 2C, all treated plants exhibited an increasing trend in protein content up to 20 µg mL^-1 of Cd, after which a significant decline was observed. However, no significant difference was observed in protein content between 10 µg mL^-1 of Cd and 15 µg mL^-1 of Cd. H₂O₂ was found to be present and to accumulate in all the Cd-treated plants. The appearance of distinguished brown precipitates in the leaf in stressed plants marked the accumulation of H₂O₂ (Fig. 1C). All the treated plants showed significant accumulation of H₂O₂ compared to control (Fig. 1B) with maximum at 25 µg mL^-1 of Cd. In order to determine the levels of lipid peroxidation, MDA content in all the treated plants was measured. Study found significant elevation (p˂0.05) of MDA content in stressed plants when compared with control. No significant enhancement was reported among the Cd-treated plants at 15, 20 and 25 µg mL^-1 of Cd (Fig. 1A). However, they all had an 80% enhancement in MDA content compared to untreated plants (control).

Effect of cadmium on catalase and peroxidase activity

In comparison to control, plants treated to varying concentrations of Cd showed a notable increase in CAT and POD activity. Cadmium stimulated the enhancement in antioxidant enzyme activity with highest at 25 µg mL^-1 Cd with catalase exhibiting 725.2 % enhancement whereas, peroxidase showed 1704.6% enhancement (Fig. 2A and B). In comparison, the control, which did not receive any treatment, exhibited the lowest levels of enzyme activity.

Relationship between plant growth and physiological traits under cadmium stress through correlation analysis

Pearson’s correlation analysis revealed the existence of positive and/or negative correlation among growth, physiological and biochemical traits of all the treated plants. There was a notable correlation between the germination index (GI) and several parameters: fresh weight (r=0.89), shoot length (r=0.80), seedling length (r=0.85), seedling vigor index (r=0.85), chlorophyll a (r=0.90), total chlorophyll (r=0.89), and chlorophyll b (r=0.83). However, the correlation with root length was comparatively weaker (r=0.78). This pattern suggests that cadmium toxicity significantly influences these parameters. Additionally, POD, CAT, MDA, and H₂O₂ were found to have strong negative correlations with all morpho-physiological parameters (Fig. 3).

Discussion

Cadmium is one of the most toxic and non-essential heavy metals with severe impact on both plants and animals.³⁵ Cd-induced toxicity brings about various morpho-physiological and biochemical changes in the plants, which cost the plants heavily causing a decrease in seed germination, poor seedling growth, reduction in biomass, inhibition of photosynthetic activity and enhanced ROS generation.^36,37 Several studies have reported the morphological and physiological impacts of Cd stress on fenugreek following substantial Cd deposition.^38,39 Under environmentally controlled conditions, exposure of plants to different concentrations of Cd produced a substantial effect on the growth performances of fenugreek, including germination, root length, shoot length, and fresh weight, as compared to the plants growing in normal (control) condition. Although all the treated fenugreek seeds initially didn’t show any challenges from Cd stress and continued to germinate, other growth parameters were substantially reduced. The results are consistent with the findings of Meng et al,³⁶ where lower concentrations of Cd had no significant effect on germination in Brassica napus, while higher concentrations reduced the other growth indices of the treated plants. As Cd levels increased particularly at relatively higher Cd concentrations, the germination index decreased along with other morphological parameters such as root length, shoot length and fresh weight of the seedlings.

 Table 1: Effect of different concentrations of cadmium (Cd) on Root length, Shoot length, Germination index, Seedling length, Seedling vigour index, Fresh weight, chlorophyll a (Chl-a), chlorophyll b (Chl-b), and total chlorophyll in fenugreek

Treatment	Root length (cm)	Shoot length (cm)	Germination index	Seedling length (cm)	Seedling vigour index	Fresh weight (g)	Chl-a  (mg g-1FW)	Chl-b (mg g-1FW)	Total chlorophyll (mg g-1FW)
Control	0.933 ±0.115a	3.200 ±0.361a	38.893 ±0.000a	4.133 ±0.351a	413.33 ±35.119a	1.09 ±0.02a	0.403 ±0.0002a	0.185 ±0.0002a	0.587 ±0.0003a
5µg ml-1	0.733 ±0.252ab	2.333 ±0.321b	37.726 ±0.289b	3.067 ±0.569b	306.667 ±56.862b	0.96 ±0.06b	0.375 ±0.0005b	0.158 ±0.0002b	0.533 ±0.0005b
10µg ml-1	0.633 ±0.208ab	1.733 ±0.115c	37.06 ±0.577b	2.367 ±0.208bc	236.667 ±20.817bc	0.9 ±0.01c	0.297 ± 0.0002 c	0.112 ±0.0001c	0.410 ±0.0002c
15µg ml-1	0.567 ±0.153b	1.567 ±0.231cd	36.893 ±0.866b	2.133 ±0.252c	213.333 ±25.166c	0.82 ±0.03d	0.287 ±0.0001d	0.112 ±0.0014c	0.400 ±0.0001d
20µg ml-1	0.467 ±0.252bc	1.300 ±0.283d	34.226 ±0.289c	1.333 ±0.551d	133.333 ±55.076d	0.77 ±0.03d	0.268 ±0.0002e	0.104 ±0.0010d	0.373 ±0.0002e
25µg ml-1	0.167 ±0.058c	1.167 ±0.379d	32.56 ±0.764d	1.333 ±0.321d	133.333 ±32.146d	0.68 ±0.02e	0.199 ±0.0003f	0.086 ±0.0001e	0.284 ±0.0004f

The values are means ± SD (n=3). Different letters within columns are statistically different ((p<0.05) according to LSD test

Since Cd stress affects germination and germination index, consequently the other morpho-physiological parameters are negatively affected. In line with this,  Revathy and Shanker⁴⁰ and Zayneb et al³⁸ have also investigated that Cd stress negatively affected germination and other morpho-physiological traits such as seedling vigor index  and photosynthetic pigments. The decrease in these growth parameters in the treated plants may be attributed to the reduced uptake of required minerals and reduced photosynthetic activity under Cd stress.⁴¹ Photosynthetic pigments play an integral part in plant growth and development. The leaf photosynthesis is highly sensitive to Cd stress contributing negatively to the growth of plants and hence, Cd is great suppressor of photosynthesis.⁴² Therefore, reduction in the growth indices in current study may be due to the reduced production of photosynthetic pigments. Many have found the deleterious impact of Cd on photosynthetic pigments,^43,44 which is consistent with our findings. Cadmium also inhibits the enzymes essential for the biosynthesis of chlorophyll and this may also be the reason for the reduced photosynthetic efficacy of the treated plants and their reduced growth.^45,46,7 Reactive oxygen species (ROS) are primarily responsible for heavy metal toxicity in plants. ROS-led oxidative damage is largely responsible for physiological and biochemical changes and degradation of various macromolecules, enzyme activity and cellular structure.⁴⁷ When plants are subjected to cadmium stress, there is an overproduction of ROS, including H₂O₂, ( ), and  ^48,7 which adversely affects plant health. This excessive production of ROS hampers plant growth and inflicts significant damage on lipids, proteins, and DNA.⁴⁹ Altaf et al⁵⁰ found that oxidation of 3, 3′-diaminobenzidine (DAB) staining by H₂O₂ resulted in the formation of brown precipitate in leaf confirming the presence of H₂O₂. Increased production of ROS (H₂O₂) causes the destruction of membrane through lipid peroxidation. Lipid peroxidation, driven by ROS, is the most used parameter to estimate the degree of oxidative stress. Polyunsaturated fatty acids in membranes undergo oxidation due to accumulation of free radicals, which results in the production of MDA.⁵¹ To assess lipid peroxidation, the MDA content was analyzed in plants treated with Cd and in control plants. In this study, a notable increase in the MDA content of plants exposed to Cd treatment was observed. The treated plant exhibited a progressive rise in MDA content with highest at 25 µg mL^-1 Cd. The rise in MDA levels in the treated plants indicates that Cd induces the lipid peroxidation causing oxidative damage to the plants and also reveals that more the oxidative stress induced by Cd, more is the lipid peroxidation. These findings corroborate the work of  Sakhai et al,⁵² who discovered that increased concentration of Cd elevated the MDA level in fenugreek.

Figure 1: Changes in A) malondialdehyde (MDA) (µmol-1 FW) and B) hydrogen peroxide (H2O2) (µM g-1 FW) under different concentration of Cd (5µg mL-1, 10 µg mL-1, 15 µg mL-1, 20 µg mL-1, and 25 µg mL-1). C) appearance of brown precipitates in the leaves indicating the accumulation of H2O2 treated plants. Click here to view Figure

Many studies have reported the reduction in soluble protein in plants exposed to cadmium.^53,54 The reduced protein content at higher Cd concentrations revealed the Cd-induced oxidative stress was evident in fenugreek. These findings suggest that while lower concentrations of Cd do not adversely affect protein content, higher concentrations do, aligning with the work of Zhao et al⁹. Our results also align with those of John et al⁵⁵ and Aibibu et al.⁵⁶ The reduction in protein content at elevated concentrations might result from the overproduction of ROS, which aggressively degrades the protein.⁵⁷

Figure 2: Effect of Cd stress on A) Catalase (CAT) (mM min-1g-1 FW); B) Peroxidase (POD) (mM min-1g-1 FW); C) Protein (mg/FW) and D) proline content (µmol g-1 FW) in fenugreek under different concentration of Cd (5 µg mL-1, 10 µg mL-1, 15 µg mL-1, 20 µg mL-1, and 25 µg mL-1). Click here to view Figure

Cadmium-induced oxidative stress causes the generation and accumulation of H₂O₂, which subsequently increases the activity of antioxidant enzymes.⁵⁸ Çatav et al⁵⁹ documented that enzyme activities, specifically CAT and POD, were elevated under Cd stress compared to the control. Our findings align with this, as we observed a marked increase in the antioxidant activities of CAT and POD when exposed to Cd stress. This rise in enzyme activity in plants subjected to Cd stress might be linked to the accumulation of H₂O₂. Reports suggest that CAT and POD act as protective enzymes for neutralizing the oxidative damage resulting from H₂O₂.^60,61 Although the activity of CAT and POD increases under Cd stress, current study witnessed significant growth inhibition in the treated plants. This suggests that CAT and POD enzymes may not be sufficient to decompose H₂O₂ to protect the plants from Cd-induced oxidative damage_. Furthermore, as reported by Zhang et al,⁶² increasing toxicity of Cd could also impair the effectiveness and activity of such a defense system. Proline, a water-soluble amino acid, is an important osmotic protective substance that plays a crucial role in protecting and maintaining the normal function of a cell and its parts.^63,9 It is one of the most common metabolic responses to environmental stresses which enables plants to survive under stressful conditions. Typically, this amino acid elevates in response to heightened stress, serving as an indicator of toxicity caused by heavy metals. In this study, the proline level rose as the Cd concentration increased, reaching a peak of  39.733 µmol g^-1 FW at 25 µg mL^-1 Cd (Fig. 2D). This indicates that higher Cd concentration induced fenugreek to produce more proline under stress. The Pearson correlation analysis indicates that all morpho-physiological parameters, including root length, shoot length, fresh weight, seedling length, seedling vigor index, and photosynthetic pigments, exhibit a strong positive correlation with one another. This suggests that the growth of plants is linked to photosynthesis driven by chlorophyll. However, they had strong and negative correlations with biochemical changes such as proline content, POD, CAT, peroxide H₂O₂, and MDA. This indicates that although, Cd had an overall impact on growth performance and biochemical changes in Cd-exposed plants, they have different relationship, affecting each other.

Figure 3: Pearson’s correlation between morpho-physiological and biochemical indices under Cd stress. Germination index (GI), Fresh weight (FW), Root length (RL), Shoot length (SL), Seedling length (Sdl), Seedling Vigour Index (SVI) chlorophyll a (Chl-a), chlorophyll b (Chl-b), Click here to view Figure

The negative correlation between morpho-physiological parameters and biochemical parameters indicates that increasing oxidative stress negatively affects germination, seedling growth and photosynthetic pigment accumulation in plants. Proline showed strong positive correlation with antioxidant enzymes (POD and CAT), MDA and H₂O₂ highlighting the coordinated activation of the defense system.

Conclusion

Exposure of fenugreek to cadmium stress caused the accumulation of H₂O₂ leading to increased proline, MDA content and activity of CAT and POD. Study investigated that increasing concentration of Cd, inhibited the germination index, root length, shoot length, seedling length, and fresh weight. These results were attributed to decline in photosynthetic pigments and reduction in soluble protein at higher concentration by Cd-induced oxidative stress. These findings help in greater understanding of fenugreek response to Cd stress at its early stage. Despite increased activity of POD and CAT in the treated plants, they suffered from plant growth inhibition which needs to be investigated in further studies.

Acknowledgment

Authors are thankful to the Department of Botany, Raiganj University for providing instrumentation facility required for the submitted work.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article

Conflict of Interest

The authors do not have any conflict of interest

Data availability

The authors affirm that data supporting the investigations of study are included in the article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required

Clinical Trial Registration

This research does not involve any clinical trials

Author Contributions

• Sumitra Chhetri: Conceptualization, Methodology, Writing – Original Draft;
• Siddhu Chhetri: Data Collection, Analysis, Writing – review & editing, Validation;
• Arka Pratim Chakraborty: Visualization, Data curation;
• Parimal Mandal: Analysis, Writing – review & editing;
• Zerald Tiru: Conceptualization, Supervision, Methodology, Writing – review & editing, Validation.

Permission to Reproduce Material

Not Applicable

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Abbreviations

POD – Peroxidase

CAT – Catalase

BSA – Bovine serum albumin

KI – Potassium iodide

MDA – Malondialdehyde

TBA – Thiobarbituric acid

H₂O₂ – Hydrogen peroxide

O₂^∙- – Superoxide

¹O₂ – Singlet oxygen