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Journal of Stress Physiology & Biochemistry, Vol. 18, No. 3, 2022, pp. 42-52 ISSN 1997-0838
Original Text Copyright © 2022 by Ailenokhuoria and Olaiya
ORIGINAL ARTICLE
OPEN ACCESS
Comparative Effects of Salicylic and Giberellic Acid on Lipid Peroxidation and Antioxidant Potentials of Three Cultivars of Vigna unguiculata (L.) Walp under
Heavy Metal Toxicity
Bukola Victoria Ailenokhuoria 1*, Charles O. Olaiya 2
1 Agricultural Value Addition Programme , Institute of Agricultural Research and Training Obafemi Awolowo University P.M.B 5029 Moor Plantion Ibadan Nigeria.
2 Department of Biochemistry, University of Ibadan, Ibadan Nigeria.
*E-Mail: bukvic2008@yahoo. com
Received February 24, 2022
Cowpea is a commonest and affordable legume widely consumed in developing nation of the world. Salicylic (SA) and Gibberellic (GA) acids serve to play prominent roles in induction of growth and development in cowpea. However, heavy metal such as arsenic is one of the major pollutant commonly encountered and its exposure to plant often result in alteration in physiological and biochemical functions. Therefore the aim of this work was to examine the effects of Salicylic and Gibberellic acid on lipid peroxidation and antioxidant potentials of three cultivars of Vigna unguiculata under heavy metal toxicity. Seeds of three cowpea cultivars (Ife Brown, ITOK-768-18and ART 98-12) were sorted and each of the three cultivars were soaked in SA [0 control, (75, 150) mg/L} and GA [0 control, (75, 150) mg/L} respectively. The seeds (n=5) were planted in a completely randomised design in pots of soil containing each of 0, 250 and 500mg/L sodium arsenate. Vitamin C, otal flavonoid, phenolic, lipid peroxidation, superoxide dismutase, peroxidase, catalase activities in leaves were determined on day 90 using spectrophotometry The results show that in comparing treatment with the control among the three cultivars, superoxide dismutase, peroxidase and catalase, vitamin C, total flavonoid and phenolic were increased by (3, 4, 2, 12, 4 and 3 folds respectively), in Ife brown; (2, 5, 12, 12, 5 and 3 folds respectively), in ART 98-12; (4, 3, 14, 5 and 4 folds respectively), in ITOK-768-18 in the leaves of 250mg/L sodium arsenate-treated soil with SA (150mg/L). Lipid peroxidation were reduced by (5 and 3 folds respectively), in Ife brown and ITOK-768-18 in 250mg/L sodium arsenate-treated soil with SA (150mg/L) but was reduced by 3 folds in 250mg/L sodium arsenate-treated soil with SA (75mg/L) for ART98-12. Salicylic and gibberellic acids therefore reduced lipid peroxidation, enhanced antioxidant potentials of Vigna unguiculata to increase tolerance and promote growth during heavy metal toxicity.
Key words: SA, GA, heavy metal. arsenic, cowpea
The cowpea is an annual herbaceous legume which utilise sandy soil and low rainfall for its growth, therefore in the dry regions all over Africa and other countries it serves as vital crop. (Maredia et al., ,2000). Higher amount of proteins, energy, minerals and vitamins are obtained from the seed. Countries that cultivate cowpea as a main food crop uses cowpea as supplements to their cereal diet. It is referred to as meat for the poor beacause of greater levels of protein in the seed and leaves (Odenda et al., 2011).
Arsenic is an ordinary element which acts like a metal. It exist in our environment, occurring in various different forms such as inorganic or organic, with highest toxicity found in inorganic form. Arsenic exist in all the natural environment and great amount in the atmosphere. Natural sources, such as volcanoes form three quarter of arsenic in the atmosphere while the remaining are from synthetic sources. Manufacturing practices such as mining, smelting and coal-fired power plants are responsible for the arsenic presence in air, water and soils all of which account for the environmental contamination. Arsenic can occur in four oxidation states namely: +3 (arsine), 0 (arsenic metal), -3 (arsenite), and +5 (arsenate). Arsenite {As(III)} prevailed under reducing condition, while arsenate {As(V)} is more stable in oxidizing condition. Solubility in water does not take place in elemental arsenic (Jana and Choudhari, 1982). Based on the incidence of occurrence, toxicity, and the likelihood of contact with humans, arsenic has been regarded as the most harmful substance (ATSDR, 2003). Arsenic-induced reactive oxygen species (ROS) causes oxidative stress and inactivation of antioxidant enzymes necessary to protect crops against injury mediated by reactive oxygen species. The effects includes: rise in lipid peroxidation, wilting, curling, necrosis of leaf blade, reduced growth and productivity and so on (Chandrakar et al., 2016).
Due to the fact that cowpea harbor pest both at pre and harvest stage of its production application of insecticide and pesticide is paramount. Also cowpea is being cultivated in Northern part of Nigeria where irrigation serve as substitute to rainfall, part of irrigated water may be contaminated with heavy metals
predominantly arsenic based on the frequency of its occurrence.Amelioration of heavy metal toxicity to increase crop productivity has therefore been of major concern. The modulation of biological and cellular molecules by plant bioregulators (Salicylic and Gibberellic acids) for defense against ROS-induced damage may therefore serve as approach to mitigate arsenic toxicity. Salicylic and Gibberellic acids are plant bioregulators that normally partake in the control of biochemical processes, demonstrating defense against arsenic-mediated injuries in plants because ROS produced by arsenic toxicity cause hormonal imbalance in plants. Arpita (2014) found out that there is reduction in growth inhibition through pre-treatment of the seedling of mungbean with phytohormones (GA 3, IAA, Kinetin and macronutrients (KH2PO4, NaH2PO4.2H2O,
K2SO4.CaCl2.2H2O, MgSO4.7H2O) under sodium arsenate toxicity. Ailenokhuoria and Omolekan (2019) has also reported on evaluation of heavy metals component of our locally consumed cowpea in major markets in Ibadan and arsenic content were found to be above tolerable standard stipulated by FAO. Hence the present study on. Comparative effects of salicylic and gibberellic acid on Lipid peroxidation and antioxidant potentials of three cultivars of Vigna unguiculata (L.) Walp under heavy metal toxicity.
MATERIALS AND METHODS
The present work was done in a screen at Institute of Agricultural Research and Training (I.A.R&T) Ibadan Oyo State, Nigeria. Five kilogrammes of pot were filled with soil and were arranged in triplicate in a complete randomised design with 15 treatments for each of the three cultivars of cowpea.
Preparation of 250 mg/L sodium arsenate
Sodium arsenate (1.45g) salt was dissolve in a small volume of distilled water and then transferred to a 1 liter volumetric flask and completed to the mark with distilled water to make 250mg/L
Preparation of 500 mg/L sodium arsenate
Sodium arsenate (2.90g) salt was dissolve in a small volume of distilled water and then transferred to a 1 liter volumetric flask and completed to the mark with distilled water to make 500mg/L
Application of sodium arsenate to the soil
Five hundred millilitres of aqueous sodium arsenate prepared above was applied to each 5 kg of soil in the pot. It was left for 14 days for proper equilibration of soil and sodium arsenate.
Plant materials
Three cultivars of cowpea seeds were obtained from Institute of Agricultural Research Training [(Ife Brown (Red coat) and ART 98-12 (white coat)] and International Institute of Agricultural Research & Training [ ITO7K-568-18 (Red coat)].
Preparation of bioregulators
Bioregulators (Salicylic and Gibberellic acids) were prepared by using procedure of (Coolbear and Heydecker 1997).
Planting
Three different cultivars of cowpea (Ife brown, ART98-12 and ITO7K-568-18) were soaked in a plastic film with two concentration bioregulators (SA and GA) 75 and 150 mg/L respectively. Distilled water was used in place of bioregulators for other part of the seeds which represent the control. All were kept for 6hrs without light at room temperature. Thereafter, the solutions were decanted off, and seeds were rinsed twice with distilled water and air-dried for 1 hour. The seeds were sow under heavy metal stress, and allowed to germinate until maturity. The plants were harvested at maturity and were divided into leaf and seed for various physicochemical analysis: elemental composition, photosynthetic pigment, antioxidant enzyme activities, compatible solutes, phytochemicals, proximate analysis and growth parameters.
Determination of antioxidant enzymes
Sample preparation for enzyme assay
One gram of leaf were crushed in 10 ml solution containing 0.10M Potassium phosphate buffer, pH 7.5 and containing 0.50mM Ethylene diamine tetra acetic acid. The mixture formed were later centrifuged for 20 minutes at 1500 rpm and the filtrate were collected for enzymes assays.
Determination of catalase.
Procedure. Catalase activities was analysed based
on Raghu et al. (2014) technique. The result was expressed as unit/mgprotein.
Principle. The principle of the test above was on the disappearance in absorbance detected at 240 nm as catalase seperate hydrogen peroxide.
Calculation
Catalase activity = AWmin x ml of reaction x dilution
0.0435x ml of sample x mg protein/mL
= |jmH2O2/min/mg protein
Determination of superoxide dismutase.
Procedure. The SOD activity was determined by according to procedure of Shalini and Dubey (2003).
The result was expressed in unit/mg protein
Principle. Superoxide dismutase capability to obstruct the autoxidation of adrenaline at pH 0.99 by superoxide ability is the basis for this reaction. (Dhindsa, 1981).
Calculation
Rise in absorbance per minute= A 3 - A 0
2.5
Where A0 = absorbance after 0 seconds
A3 = absorbance after 150 seconds
% Inhibition = Rise in absorbance of substrate X 100 Rise in absorbance of blank
A unit of SOD activity is the amount of SOD necessary to produce 50% inhibition of the oxidation of adrenaline.
Determination of peroxidase
Procedure. Peroxidase activity was determine according to the method of (Raghu et al., 2014).
Principle. The method is by using pyrogallol as hydrogen donor. The rate of reaction is estimated by measurement of an increase in absorbance at 420 nm due to decomposition of hydrogen peroxide. One unit results to the decomposition of 1|M of H2O2 under the specified conditions (Raghu et al., 2014). The result was expressed in abs/min
Lipid peroxidation
Malondialdehyde (MDA) contents were estimated based on Dhinsa et al. (1981) technique with slight modification. The result was expressed in nmol/g/fw
Determination of phytochemicals
Phytochemicals (total flavonoid and phenolic) are regarded as secondary defencen on enzymic antioxidants and a great removal of singlet oxygen. Vitamin C plays role in eliminating Hydrogen peroxide produced from reactive oxygen species generated from arsenic toxicity. Salicylic acid act by increasing formation of specific phytochelatin to chelate with metalloids and then reduce arsenic toxicity.
Procedure
Determination of total phenolic
Total phenolic contents were estimated by use of folin- ciocalteau method (Singleton et al., 1999). The results were expressed as mg/GAE/g/fresh weight.
Determination of total flavonoid
The total flavonoid contents were determined by colorimetric method of Zhishen et al. (1999). Flavonoid content were expressed as mg/QUE/g/fresh weight.
Determination of Vitamin C
Vitamin C was determine according to the method of Sulladmath et al. (2012). The result was expressed as mg/g/fresh weight.
Statistical analysis
The data were analysed using Statistical analytical for science (SAS). Anova was used for separation of means. Duncan multiple range test was used to determine the level of 5% significant among the groups of treatment.
RESULTS AND DISCUSSION
The result in Figures 1-9 shows that SOD, POD and CAT activities of cowpea exposed to 250 and 500 mg/L arsenate significantly reduce with respect to the control plant respectively in all three cultivars, this was as a result of the fact that the rate of generation of oxidant surpasses the rate of production of antioxidants caused by higher level of arsenic. However, SA, GA (150, 75 mg/L) significantly increase the level of SOD, POD and CAT in all the three cultivars respectively. The enzymatic components associated with defense against reactive oxygen species generated by arsenic toxicity include SOD, catalase, peroxidase and ascorbate/ glutathione. Superoxide dismutation dissuades two
superoxide radicals (O2"- ) in water and O2. and it is an important constituent of the antioxidant protective system in plants. Catalase is one of the key enzymes involved in the elimination of toxic peroxides and is universally present as oxidoreductase that decomposes hydrogen peroxide in water and molecular oxygen. (Lin and Kan, 2000). Application of SA and GA was able to upregulate the activities of these enzymic antioxidants therefore able to mitigate against arsenic toxicity and protect plant from injury. Arpital (2014) had reported that pre-treatment of mungbean seedling with GA3, KH2PO4 and NaH2PO4.2H2O which helped in reduction of oxidative stress to some extent as observed by increasing seedling growth and lowered antioxidant enzyme actions and lessening proline accumulation. Chandra et al. (2011) had also reported the alleviating effects of Indole acetic acid on effect of toxic heavy metal (Pb, Cr ) on wheat plant due to stimulatory effect of antioxidant enzyme like Superoxide dismutase, Catalase and Glutathione reductase.
Moreover, the result in Figures 10-12 shows that Lipid peroxidation of cowpea exposed to 250 and 500 mg/L arsenate significantly increase with respect to the water treated control plant respectively in all three cultivars which is due to the oxidation of polyunsaturated fatty acid of the membrane which then results in Lipid-peroxidised products hence loss of function of membrane. However, SA, GA (150, 75 mg/L) significantly reduce the level of lipid peroxidation in all the three cultivars.
In addition, the result in Tables 1-3 shows that vitamin C, total flavonoid and phenolic of cowpea exposed to 250 and 500 mg/L arsenate significantly reduce with respect to control plant respectively in all three cultivars whereas SA, GA (150, 75 mg/L) significantly increase their level in all the three cultivars respectively. Vitamin C, total flavonoid and phenolic are non-enzymic antioxidant that act in addition to enzymic antioxidant to scavenge reactive oxygen species generated from arsenic toxicity. Vitamin C is a significant redox buffer that also serves as a cofactor for enzymes involved in photosynthesis, hormone production, and the regeneration of other antioxidants.. It is a critical substrate for the detoxification of reactive oxygen
species. It also serve as hydrogen peroxide eliminator while total flavonoid and phenolic act as phytochelatin to chelate the metal and renders them to non- toxic forms. Amit et al. (2015) also reported that pretreatment of rice
with salicylic acid also enhanced the level of Ascobate, GSH/GSSG and total non-protein thiol pon arsenate exposure in all treatments than corresponding arsenate exposed plants.
Table 1: Effects of SA and GA on Vitamin C, Total flavonoid and phenolic contents of cowpea(Ife brown) exposed to sodium arsenate
Arsenic (mg/L)
Bioregulators (mg/L) Vitamin C (mg/g/fw) Flavonoid (mg QE/g/fw) Phenolic (mg GAE/g/fw)
0 0 24.00 ± 0.57i 74.00±0.58d 92.33 ±1.45d
250 0 6.70 ± 0.15g 9.17± 0.44m 19.30 ± 0.15m
500 0 2.56 ± 0.23g 5.47±0.15n 14.46 ± 0.15n
0 GA3 150 31.66 ± 1.01e 94.00±0.58a 104.00± 0.58b
250 GA3I50 72.50 ± 1.32b 53.50±2.78f 69.16 ± 0.44g
500 GA3I50 42.17 ± 0.44d 38.10±0.12h 50.06 ± 0.62h
0 GA375 28.17 ± 0.44ef 78.17±0.44c 78.16 ±0.44e
250 GA375 63.50 ± 0.76c 35.43±0.54i 55.43±0.54i
500 GA375 38.50± 2.56d 25.60±0.31k 35.60±0.31l
0 SA 150 41.50± 0.58d 86.00±0.18b 136.00 ±0.58a
250 SA 150 83.67 ± 1.86b 46.33±0.88g 76.33±0.88f
500 SA150 41.83± 4.47e 30.27±0.37j 60.26 ± 0.37h
0 SA 75 33.00 ± 1.04e 65.17±0.44e 95.16±0.44c
250 SA 75 68.17 ± 0.93b 29.23 0.39j 59.23 ± 0.39h
500 SA 75 32.50± 1.443e 22.13±0.19l 48.13±0.185k
Means with different letter are significantly different from each other
Table 2: Effects of SA and GA on Vitamin C , Total flavonoid and phenolic contents of cowpea (ART 98-12) exposed to sodium arsenate
Arsenic (mg/L)
L) Bioregulators (mg/L) Vitamin C (mg/g/fw) Flavonoid (mgQE/g/fw) Phenolic (mgGAE/g/fw)
0 0 19.40 ± 0.21 de 55.43±0.54c 75.60 ±0.59c
250 0 10.93 ± 8.53 ef 6.37±0.32n 15.87 ±0.12l
500 0 3.67 ± 0.88f 3.90±0.06o 11.93 ± 0.08m
0 GA3 150 25.83 ± 2.92de 70.30±0.65a 90.30 ±0.65b
250 GA3150 48.83 ±9.44abc 41.00±0.58f 51.83 ± 0.44f
500 GA3150 26.00 ±10.26de 27.23±0.39i 37.23 ±0.39i
0 GA375 25.00 ±2.52 de 52.00±0.58d 62.00 ±0.57d
250 GA375 50.33 ±3.82 ab 34.00±0.58h 38.00 ±0.57i
500 GA375 30.33 ± 2.85d 16.10±0.21l 29.10 ±0.21k
0 SA 150 33.33 ± 4.42 cd 57.63±0.33b 107.30 ±0.45a
250 SA 150 53.17 ± 4.11 a 37.50±0.76g 107.30±0.45a
500 SA150 35.00 ± 2.84 bcd 22.00±0.58k 48.50 ± 0.29f
0 SA 75 30.83 ± 4.08 d 43.67±0.88e 63.17±0.60d
250 SA 75 52.50 ± 3.78 a 25.17±0.44i 45.17±0.44h
500 SA 75 31.17 ± 3.42d 14.00±0.58m 34.00±0.57j
Means with different letter are significantly different from each other
Table 3: Effects of SA and GA on Vitamin C, Total flavonoid and phenolic contents of cowpea (ART 98-12) exposed to sodium arsenate
Arsenic (mg/L)
Bioregulators (mg/L) Vitamin C (mg/g/FW) Flavonoid (mgQE/g/fw) Phenolic (mg GAE/g/fw)
0 0 23.27 ± 0.50bc 74.00±0.58c 72.33±1.45e
250 0 12.13 ± 9.43cd 9.17±0.44j 19.30± 0.15k
500 0 4.87 ± 0.94d 5.47±0.15k 14.47±0.15l
0 GA3I50 26.30 ± 2.65bc 95.00±0.58a 104.00±0.58a
250 GA3I50 51.67 ± 10.84a 53.50±2.78e 69.17 ±0.44f
500 GA3I50 26.00 ± 10.27bc 26.43±0.27i 50.07± 0.62 i
0 GA375 26.63 ± 1.73bc 78.17±0.44b 88.17±0.45 b
250 GA375 60.17 ± 2.46a 35.43±0.55g 55.43±0.55 h
500 GA375 24.83 ± 3.09bc 25.60±0.31i 48.00±0.58j
0 SA 150 33.00 ± 3.52b 96.00±0.58a 86.00±0.58 c
250 SA 150 67.00 ± 5.51a 46.33±0.88f 76.33±0.88d
500 SA150 34.43 ± 5.74b 30.27±0.37h 60.27 ±0.37g
0 SA 75 32.47 ± 3.30b 65.17±0.44d 85.17±0.44 c
250 SA 75 67.50 ± 3.75a 29.23±0.39h 59.23±0.39g
500 SA 75 34.00 ± 4.04b 52.13±0.19e 48.13±0.19 j
Means with different letter are significantly different from each other
Figure 1: SOD of cowpea (Ife brown) exposed to arsenic toxicity
SOD: Superoxide Dismutase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 2: SOD of cowpea (ART 98-12) exposed to arsenic toxicity
SOD: Superoxide Dismutase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 3: SOD of cowpea (ITOK-568-18) exposed to arsenic toxicity
SOD: Superoxide Dismutase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 4: POD of cowpea (Ife brown) exposed to arsenic toxicity
POD: Peroxidase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
arsenic concentrations(mg/L)
Figure 5: POD of cowpea (ART 98-12) exposed to arsenic toxicity
POD: Peroxidase, GA3 : Gibberellic acid , SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 6: POD of cowpea (ITOK-568-18) exposed to arsenic toxicity POD: Peroxidase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 7: Catalase of cowpea (Ife brown) exposed to arsenic toxicity GA3 : Gibberellic acid, SA : Salicylic acid
Means with the same letter are significantly different from each other
Figure 8: Catalase of cowpea (ART98-12) exposed to arsenic toxicity
SOD: Superoxide Dismutase, GA3 : Gibberellic acid, SA : Salicylic acid Means with the same letter are significantly different from each other
Figure 9: Catalase of cowpea (ITOK-568-18) exposed to arsenic toxicity GA3 : Gibberellic acid , SA : Salicylic acid
Means with the same letter are significantly different from each other
Figure 10: Lipid peroxidation of cowpea (Ife brown) exposed to arsenic toxicity GA3 : Gibberellic acid , SA : Salicylic acid
Means with the same letter are significantly different from each other
Figure 11: Lipid peroxidation of cowpea (ART 98-12) exposed to arsenic toxicity GA3 : Gibberellic acid , SA : Salicylic acid
Means with the same letter are significantly different from each other
Figure 12: Lipid peroxidation of cowpea (ITOK-568-18) exposed to arsenic toxicity Means with the same letter are significantly different from each other
CONCLUSIONS
Arsenic is a carcinogenic chemical that is found in our environment due to pollution from both natural and human source. However, continuous plant contamination with arsenic increases its movement along food chain which is' health hazard. Bioregulators such as salicylic and gibberellic acid have been shown to regulate some biochemical processes in plants and therefore serve as a potential modulator of antioxidant enzymes to mitigate arsenic toxicity. In the present study, three cultivars of cowpea seed pre-treated with salicylic acid and gibberrellic acid show varying modulatory effects against arsenic toxicity through increase in the level of both antioxidant enzymes (SOD, POD and catalase) and non enzymic antioxidant (vitamin C, flavonoid and phenolic) and therefore promote growth during heavy metal toxicity. Hence there is possibility of optimal growth and productivity during heavy metal stress in cowpea through application of salicylic and gibberellic acids and this would be of great value to agricultural producers.
ACKNOWLEDGEM ENTS
First author is grateful to the Institute of Agricultural Research & Training and International Institute of Tropical Agricultute both at Ibadan, Oyo State Nigeria for providing cowpea seed used for the work.
CONFLICTS OF INTEREST
The authors declare that they do not have any
conflict of interest for publishing this research.
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