Research Article, J Plant Physiol Pathol Vol: 7 Issue: 1
Promotive Effect of Ascorbic Acid, Gallic Acid, Selenium and Nano-Selenium on Seed Germination, Seedling Growth and Some Hydrolytic Enzymes Activity of Cowpea (Vigna unguiculata) Seedling
Ibrahim Mohamed Zeid, Fatma Abd El Lateef Gharib, Safia Mohamed Ghazi and Eman Zakaria Ahmed*
Department of Botany and Microbiology, Faculty of Science, Helwan University, Cairo, Egypt
*Corresponding Author : Eman Zakaria Ahmed
Department of Botany and Microbiology, Faculty of Science, Helwan University, Cairo, Egypt
Tel: +20 1145103804
E-mail: [email protected]
Received: March 01, 2018 Accepted: March 13, 2019 Published: March 22, 2019
Citation: Zeid IM, Gharib FAEL, Ghazi SM, Ahmed EZ (2019) Promotive Effect of Ascorbic Acid, Gallic Acid, Selenium and Nano-Selenium on Seed Germination, Seedling Growth and Some Hydrolytic Enzymes Activity of Cowpea (Vigna unguiculata) Seedling. J Plant Physiol Pathol 7:1. doi: 10.4172/2329-955X.1000193
This study aimed to improve cowpea seed germination and seedling growth by using some antioxidant compounds naturally produced or up taken by plants. The effect of soaking cowpea (Vigna unguiculata) for 2 h in aerated solution of different antioxidants; ascorbic acid (AsA) and gallic acid (GA), each at 0.0, 50, 100, 150, 200, 250 and 300 ppm, sodium selenate (NaSeO4) and nanoselenium (SeNPs ≈ 33.4 nm) chemically prepared, each at 0.0, 6.25, 12.5, 25, 50 and 100 μM on seedling length and activities of some hydrolytic enzymes were tested. Seeds of treated and untreated cowpea were germinated at 25°C ± 0.5 under dark controlled conditions for 4 days. Results showed that AsA, GA, NaSeO4 and SeNPs at low concentrations significantly improved plumule and radicale lengths compared with their corresponding controls. Moreover, AsA and GA up to 150 ppm, NaSeO4 and SeNPs up to 25 μM significantly increased the activities of α-amylase, β-amylase and protease enzymes as well as the contents of total soluble sugars and total soluble proteins. The results indicate the successful use of ascorbic acid and gallic acid up to 150 ppm as well as sodium selenate and nanoselenium up to 25 μM in enhancing seedling growth and hydrolytic enzymatic activity in cowpea germinated seeds.Further promising results on the effect of these compounds on vegetative growth and yield can be concluded from the previous results on germination and seedling growth.
Keywords: Phaseolus vulgaris; Ascorbic acid; Gallic acid; Sodium selenate; Nanoselenium; Enzymes activities
Cowpea (Family: Fabaceae) is an annual legume, internationally known as lubia, niebe coupe or frijol . Cowpea(Vigna unguiculata L.) seeds contains about 64 percent carbohydrate and 25 percent protein rich in lysine and tryptophan, compared to cereals as wheat and hordeum grains . Cowpea use as a grain crop and for animal fodder [3,4]. Cowpeas enhance soil fertility by nodules formation . Recently, cowpea has gained more attention from consumers and researchers worldwide as a result of its exerted health beneficial properties, including anti-diabetic, anti-cancer, anti-hyperlipidemic, anti-inflammatory and anti-hypertensive properties .
Antioxidants are compounds or molecules that have the ability to scavenge reactive oxygen species (ROS). The formation of ROS occurs naturally as a by-product of metabolism. Also, environmental stresses are known to increase ROS to toxic levels which cause damage to the biomolecule of the cell or the building blocks like DNA, proteins and lipids . The antioxidant defence system of the plant comprises enzymatic and various non-enzymatic substances which act as various scavengers for different types of ROS. In many crop plants, natural accumulation of osmo-protectants and other organic compounds is very low and this deficiency can be overcome by their exogenous application  Of the non-enzymatic antioxidants, vitamins, phenolic compounds, selenium and nanoselenium are important due to their promotive role in plant growth and development in addition to their antioxidant capacity [9-12], so application of non-enzymatic antioxidants may help in improving the activity of antioxidant system in plants, for that ascorbic acid, Gallic acid, selenium and Se nanoparticles are selected as a part of the normal antioxidant system in plants.
Ascorbic acid (Vitamin C) is a major antioxidant molecules required by plant for growth and regarded as one of the most effective growth regulators. Ascorbic acid is a co-factor for many enzymes and plays multiple roles in many physiological processes such as protein and nucleic acid contents in Solatium melongena , membrane permeability in Vigna catjang , photosynthesis , ion intake to roots , respiration , seed germination of Agropyron elongatum , Spergularia marina seedling growth  and activities of peroxidase and superoxide dismutase enzymes in Saccharum Spp .
Gallic acid (GA; 3,4,5-triphydroxyl-benzoic acid) is a strong antioxidant and one of phenolic compounds widely distributed in various plants, [21,22]. Phenolics have been considered as great antioxidants and proved to be more effective than vitamin C, E and carotenoids . Gallic acid protects plants against lipid peroxidation and reduces cell membrane damage by less production of H2O2 and increasing the activities of H2O2-scavenging enzymes .
Selenium is an indispensable trace element found naturally in both organic and inorganic (Selenite (Se4+), selenate (Se6+) and selenide (Se2-)) forms . Plants mainly uptake Se from soil solution in the form of selenate (SeO42-). In general, Se translocation from root to shoot is lower in plants fed with SeO32- than in those fed with SeO42- . Selenium is taken up in advertently via sulfate transporters and metabolized via the S assimilation pathway . In this pathway, selenate is reduced to selenite (SeO32-), which can undergo further reduction to selenide (Se2-). This may be incorporated into the organic forms, selenocysteine (SeCys), selenocystathionine (SeCysth), and selenomethionine (SeMet), chemical reduction from SeO42- to SeO32- and further SeCys likely occurs at chloroplasts in leaves, while the production of SeMet and methylation of SeMet likely takes place in the cytosol . In a protein chain, cysteine residues play an important role in protein structure and function, and helps in formation of disulfide bridges, enzyme catalysis, and metal binding site .
Recently, selenium nanoparticles (SeNPs), have aroused worldwide attention due to their distinguished properties and excellent biological activities . They are able to scavenge free radicals in vitro  and stimulate organogenesis . Nano-Se has a higher efficiency in up regulating selenoenzymes and exhibits less toxicity compared with other Se compounds such as selenite , selenomethionine and Se-methylselenocysteine . A new approach to fertilization of plants is the use of selenium nanomaterials .
Application of antioxidants compounds have been successfully used to improve the activity of antioxidant system in plants leading to increase in plant growth and yield. Ascorbic acid effectively improved plant growth and development by altering oxidative defense system, phytohormone signaling, cell expansion and ion transports under stress or non-stress conditions [36,37]. Also, AsA increased germination percentage, length of shoot and root and seedling total dry weight in sunflower and rape seed  and stimulated coleoptile percentage and radicle length of tomato, pea and barley seeds [39-41]. Na2SeO3 at low concentration promoted seed germination and seedling growth of wheat, while high concentration reduced germination rate, vigor and α-amylase activity . Optimal Se supply increased antioxidant capacity of wheat (Triticum aestivum L.) seedlings, reduced production of free radicals, membrane lipid peroxidation, and promoted biomass accumulation under cold stress . In addition, Low concentration of Nano-Se and Se, improved tomato growth parameters and chlorophyll content under high and low temperature stress .
However, studies on the effects of antioxidants compounds (AsA, GA, NaSeO4 and SeNPs) in plants are still limited. Therefore, it is important to increase our knowledge about their mechanisms before implementing large-scale agricultural utilization of these antioxidants compounds.
Thus, the present study aims to investigate the effect of soaking cowpea (Vigna unguiculata) seeds in different concentrations of varying classes of antioxidants; ascorbic acid (vitamin), gallic acid (phenolic compound), sodium selenate (a structural element of some antioxidant enzymes) and nanoselenium (SeNPs ≈ 33.4 nm) on seedlings length, total soluble sugars, total soluble proteins and activities of some hydrolytic enzymes in seedlings.
Materials and Methods
All biochemical used in this study were of high purity, purchased from Sigma-Aldrich Chemical Co., Germany and Merck (Rio de Janeiro, RJ, Brazil). Distilled and deionized water were used in all experimental work.
Synthesis of selenium nanoparticles
Selenium nanoparticles (SeNPs ≈ 33.4 nm) were synthesized by reducing Na2SeO4 (10 mM) in the presence of ascorbic acid (1.5%) w/v as a reducing and stabilizing agent. Selenium nanoparticles were characterized by UV–vis spectroscopy; Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), Dynamic Light Scattering (DLS) and Fourier transform infrared (FT-IR) spectroscopy.
Plant materials and treatments
A homogenous lot of cowpea seeds variety (Aha 1) was supplied by Agriculture Research Center, Giza, Egypt. Seeds of cowpea were surface sterilized with 2.5% sodium hypochlorite solution for three minutes and then washed thoroughly with distilled water. Afterwards, seeds were equally divided into four batches and each batch was subdivided into groups (100 seeds each) to be soaked in the different working solutions. Soaking of seeds was carried out by putting a constant number of seeds of each batch for 2 h in glass containers, each containing a constant amount (100 ml) of the treatment solutions (AsA, GA, NaSeO4 and SeNPs) at concentrations of 50, 100, 150, 200, 250 and 300 ppm, for AsA and GA, and at concentrations of 6.25, 12.5, 25, 50 and 100 µM, for NaSeO4 and SeNPs. In addition, distilled water was used as a control. Soaking was carried out in a controlled cabinet (germinator). Afterwards, the seeds of the control and each treatment (5 replicates each contains 10 seed) were washed thoroughly with distilled water, and then allowed to germinate in sterilized petridishes containing filter paper wetted with about 5 ml of distilled water for 4 days. Germination experiments were carried out at 25°C ± 0.5 under dark conditions.
Seedling length of cowpea was calculated. At least 10 randomly choice 4-day-old seedlings were taken from each treatment and the control for measurements of seedling length (plumule and radicle length (cm)). The collected fresh seedling was used for determination of total soluble sugars, total soluble proteins or assaying enzymatic activities.
Hydrolytic enzymes (α-amylase, β-amylase, protease enzymes)
α-amylase, β-amylase and protease activities were assayed in 4-day-old seedlings of cowpea presoaked for 2 h in different antioxidants; AsA and GA solutions, each at 0.0, 50, 100, 150, 200, 250 and 300 ppm, NaSeO4 and SeNPs solutions, each at 0.0, 6.25, 12.5, 25, 50 and 100 µM.
One gram of fresh cowpea seedlings were ground in a pre-chilled mortar and pestle with 5 ml double distilled water till a paste was formed, and the extracts were centrifuged at 4000 rpm and 4°C for 20 minutes. The obtained clear supernatants solutions were used for various enzymatic assays.
α-amylase (EC 3. 2.11) and β-amylase (EC 220.127.116.11) enzymes
α-amylase and β-amylase were assayed according to Bergmeyer . The assayed mixture contained either 0.5 ml of starch in phosphate buffer pH 7.0 for α-amylase or 1.0 ml of starch in acetate buffer pH 4.8 for β-amylase, 0.5 ml double distilled water and 0.5 ml enzyme extract. After incubation for 10 minutes at 25°C, 1 ml of the color reagent (1% dinitrosalisylic acid) was added to the mixture and boiled in water bath for 10 minutes, then cooled in ice bath. The mixture was completed to 10 ml with double distilled water and the color density was measured at 546 nm using Cecil CE 1010 spectrophotometer.
Protease enzyme (EC 18.104.22.168)
Protease was assayed by the method described by Bergmeyer . The assaying mixture contained 1 ml of casein (1%) in phosphate buffer pH 7.5 and 1 ml of enzyme extract. After incubation period for 1 hour at 37°C, two ml of trichloroacetic acid (10%) was added to terminate the reaction and then the mixture was centrifuged for 20 minutes at 4000 rpm and 4°C. The amino acids content in the supernatant was estimated according to  to calculate the activity of protease enzyme in relation to produced amino acid from casein hydrolysis. The procedure of Lowry et al.  was followed. Briefly, 1 ml cowpea extract was mixed with 5 ml freshly mixed solution (50:1 v/v) of 2% sodium carbonate in 4% sodium hydroxide and 0.5% copper sulphate in 1% sodium tartarate. The mixture stood 10 minutes before addition of 0.5 ml Folin and made up to a definite volume. The optical density of the mixture was measured spectrophotometrically at 750 nm after 30 minutes.
Total soluble sugars and total soluble proteins
Extraction: A weight of 0.1 g of cowpea seedling was ground in 5 ml ethanol 70%, after centrifugation at 6000 rpm for 15 minutes, the supernatant was completed to 15 ml by distilled water.
Total soluble sugars: Total sugars were determined using anthrone technique as described by Umbriet et al. . 6 ml anthrone solution (2 g/l H2SO4 95%) were added to 3 ml sample and maintained on a boiling water-bath for 3 min. After cooling, the developed color was measured spectrophotometrically at 620 nm.
Total soluble proteins: The procedure of Lowry et al.  was followed. Briefly, 1 ml cowpea extract was mixed with 5 ml freshly mixed solution (50:1 v/v) of 2% sodium carbonate in 4% sodium hydroxide and 0.5% copper sulphate in 1% sodium tartarate. The mixture stood 10 minutes before addition of 0.5 ml Folin and made up to a definite volume. The optical density of the mixture was measured spectrophotometrically at 750 nm after 30 minutes.
Statistical analysis: The data was expressed as mean of five replicates for seedling length and as mean of triplicate values for the enzymes activity, total soluble sugars and total soluble proteins. Statistical analysis was performed using one way analysis of variance ANOVA followed by Duncan’s Multiple Comparison Test using IBM Statistical Product and Service Solutions, SPSS Statistics for Windows, Version 21 at P<0.05 that was denoted as being statistically significant for the means compared, using least significant difference (LSD at 5% level).
Data presented in Figures 1 and 2 show that plumule and radicle lengths were significantly increased, as compared to corresponding controls, in response to soaking in either ascorbic acid or gallic acid up to 200 ppm, soaking in either sodium selenate or nanoselenium up to 25 µM. The best results were obtained in response to soaking seeds in solutions at 100 ppm of AsA or 150 ppm of GA or 6.25 µM of either NaSeO4 or SeNPs. However, the seedling length of cowpea was positively affected by increasing AsA or GA up to 250 ppm and NaSeO4 or SeNPs concentrations up to 25 µM, and then these values were progressively decreased at higher concentrations than control. Furthermore, SeNPs was more efficient than NaSeO4 at comparable concentrations in increasing plumule and radicle lengths of cowpea.
Figure 1: Seedlings growth of cowpea (Vigna unguiculata L.) as affected by soaking for 2 h in ascorbic acid (AsA), gallic acid (GA), each at 0.0, 100, 150, 200, 250 and 300 ppm), or sodium selenate (Na2SeO4), Nano selenium (SeNPs), each at 0.0, 6.25, 12.5, 25, 50 and 100 µM followed by 4 days germination at 25ºC ± 0.5 in dark.
Figure 2: Effect of ascorbic acid (AsA), gallic acid (GA) each at 0.0, 100, 150, 200, 250 and 300 ppm, sodium selenate (Na2SeO4), and Nano selenium (SeNPs), each at 6.25, 12.5, 25, 50 and 100 µM on plumule and radicle length of 4 days-old seedlings of cowpea (Vigna unguiculata L.). The data are the mean of five replicates. Vertical bars represent LSD at 5%.
Activities of α-amylase, β-amylase and protease enzymes
Data presented in Table 1 shows that soaking treatments of cowpea seeds for 2 h in AsA up to 150 pp, GA up to 250 pp, NaSeO4 up to 25 µM or SeNPs up to 50 µM solutions increased activities of α-amylase, β-amylase and protease in the 4-day-old seedlings of cowpea, as compared to corresponding controls and then these values were progressively decreased at higher concentrations than control.
|μg g-1fwt sec-1|
|AsA 50 ppm||65.80b||36.47b||16.20a|
|AsA 100 ppm||66.93a||66.73a||16.45a|
|AsA 150 ppm||50.70c||34.17c||15.30b|
|AsA 200 ppm||41.78e||28.76e||14.21d|
|AsA 250 ppm||36.97f||26.26f||11.21e|
|AsA 300 ppm||25.59g||25.85g||7.60f|
|L.S.D at 0.05||1.13||0.41||0.30|
|GA 50 ppm||49.59d||51.10c||14.50b|
|GA 100 ppm||55.61b||52.40b||16.73a|
|GA 150 ppm||65.94a||64.70a||16.75a|
|GA 200 ppm||54.51c||46.59d||14.73b|
|GA 250 ppm||44.39e||34.27e||14.53b|
|GA 300 ppm||36.18g||26.15g||11.17c|
|L.S.D at 0.05||1.10||1.30||2.00|
|Na2SeO4 6.25 µM||68.83a||67.55a||16.80a|
|Na2SeO4 12.5 µM||66.13b||58.76b||16.78a|
|Na2SeO4 25 µM||55.21c||51.70c||16.44a|
|Na2SeO4 50 µM||41.86e||28.97e||14.30b|
|Na2SeO4 100 µM||28.96f||21.34f||8.50c|
|L.S.D at 0.05||1.40||7.06||1.93|
|SeNPs 6.25 µM||96.09a||67.84a||16.81a|
|SeNPs 12.5 µM||86.27b||60.11b||16.78a|
|SeNPs 25 µM||79.16c||57.31c||16.41b|
|SeNPs 50 µM||43.30d||44.19d||14.46c|
|SeNPs 100 µM||31.48f||25.69f||11.10d|
|L.S.D at 0.05||7.11||2.80||0.37|
Table 1: Effect of seed presoaking for 2 h in ascorbic acid and gallic acid, each at 0, 50, 100, 150, 200, 250 and 300 ppm, sodium selenate and nanoselenium (SeNPs ≈ 33.4 nm) solution, each at 6.25, 12.5, 25, 50 and 100 µM on the activities of ἁ-amylase, ß-amylase and Protease (µg g-1 f. wt sec-1) of 4-day-old seedlings of cowpea (Vigna ungiculata L.) and grown in dark at 25ºC ± 0.5. Each result is a mean of 3 replicates. Statistical analysis was carried out using Duncan test (different letters next to means indicate significance difference between treatments with letter a for the highest value).
The maximum activities of α-amylase, β-amylase and protease enzymes were obtained in response to soaking seeds in solutions at 100 ppm of AsA, 150 ppm of GA or 6.25 µM of either NaSeO4 or SeNPs.
Furthermore, SeNPs was more effective than NaSeO4 at comparable concentrations, in increasing the activities of α-amylase, β-amylase and protease enzymes of cowpea seedlings.
Total soluble sugars and total soluble proteins
The results in Table 2 show that soaking cowpea seeds in AsA up to 150 ppm, GA up to 200 ppm, Na2SeO4 and SeNPs up to 25 µM significantly increased total carbohydrates (TC) and total soluble proteins (TSP) content in cowpea seedlings and then these values were progressively decreased at higher concentrations than controls.
|Treatments||Soluble sugar||Soluble protein|
|mg g-1 fwt|
|AsA 50 ppm||26.61b||44.78b|
|AsA 100 ppm||27.30a||55.01a|
|AsA 150 ppm||26.12c||41.33c|
|AsA 200 ppm||20.32e||31.10e|
|AsA 250 ppm||17.50f||22.34f|
|AsA 300 ppm||16.94g||20.08g|
|L.S.D at 0.05.||0.49||1.45|
|GA 50 ppm||26.23b||38.25c|
|GA 100 ppm||26.86a||46.71b|
|GA 150 ppm||27.01a||62.11a|
|GA 200 ppm||25.22c||36.72d|
|GA 250 ppm||24.94d||26.01f|
|GA 300 ppm||16.98e||20.87g|
|L.S.D at 0.05||0.28||1.53|
|Na2SeO4 6.25 µM||28.62a||87.22a|
|Na2SeO4 12.5 µM||27.24b||77.64b|
|Na2SeO4 25 µM||26.05c||69.94c|
|Na2SeO4 50 µM||24.41e||32.00d|
|Na2SeO4 100 µM||17.38f||24.58e|
|L.S.D at 0.05||0.50||7.42|
|SeNPs 6.25 µM||29.12a||99.04a|
|SeNPs 12.5 µM||27.95b||96.40b|
|SeNPs 25 µM||26.31c||87.22c|
|SeNPs 50 µM||24.95d||35.32d|
|SeNPs 100 µM||18.09e||26.01f|
|L.S.D at 0.05||1.17||2.64|
Table 2: Changes in soluble sugars and soluble proteins (mg g-1 f. wt) of 4-day-old seedlings of cowpea (Vigna ungiculata L.) as affected by seed presoaking for 2 h in ascorbic acid and gallic acid, each at 0, 50, 100, 150, 200, 250 and300 ppm, sodium selenate and nanoselenium (SeNPs ≈ 33.4 nm) solution, each at at 6.25, 12.5, 25, 50 and100 µM and grown in dark at 25ºC ± 0.5. Each result is a mean of 3 replicates. Statistical analysis was carried out using Duncan test (different letters next to means indicate significance difference between treatments with letter a for the highest value).
The maximum values of TC and TSP were obtained in response to soaking seeds in solutions at 100 ppm of AsA, 150 ppm of GA or 6.25 µM of either Na2SeO4 or SeNPs with superiority of SeNPs.
In the present work, the main plumule and radicle lengths of 4-day-old cowpea seedling resulting from soaking for 2 hours in different concentrations of antioxidants solutions; AsA, GA, NaSeO4 and SeNPs were mostly significantly increased, relative to controls, from 50 to 200 ppm concentrations of ascorbic acid, from 50 to 250 ppm gallic acid and from 6.25 to 25 µM concentrations of either sodium selenate or nanoselenium. The best performance was induced by 100 ppm of AsA, 150 ppm of GA and 6.25 µM of either NaSeO4 or SeNPs (Figures 1 and 2). Regarding the stimulatory effects of the experimented four antioxidants, SeNPs was more efficient than NaSeO4 at comparable concentrations and other treatments, where SeNPs at 6.25 µM induced highest significant increase in the seedling length. In accordance, Se at low concentrations act as an antioxidant and stimulated ryegrass growth, whereas at higher concentrations it act as a pro-oxidant and reduced the yields [48,49]. Se at low levels can stimulate the plant growth, especially those of shoots by up-regulation of enzymes of carbohydrate metabolism thus providing energy substrates for enhanced growth . Ascorbic acid significantly increased germination percentage and improved seedling fresh and dry weights, shoot and root lengths in Safflower (Carthamus tinctorius L.) and Brassica species under salt stress [51,52], enhanced germination rate, plumule length and seedling fresh and dry biomass in Sunflower (Helianthus annuus L.) and cucumber (Cucumis sativus) under drought stress [53-55] improved germination process and seed vigor in Cumine (Cumin cyminum L.) under Saline Conditions . Gallic acid increased shoot and root length at 60 μg mL−1 in rice seedlings .
The enhanced seedling growth by seed soaking with four antioxidant compounds (AsA, GA, NaSeO4 and SeNPs) particularly at lower concentrations may be due to de novo synthesis of certain germination-promoting substances, promotion plant cell division, elevation effect of some hydrolytic enzymes (α-amylase, β-amylase and protease) that resulted in efficient utilization of seed reserves, stimulation of antioxidant activity, and increased abilities for absorbing and utilizing water, as has been concluded by Luet al.  in soybeans and Fujikura and Karssen , Farooq et al. , Abdul Khaliq et al.  who found that seeds priming enhanced de novo synthesis of germination-promoting substances, membrane re-organization, activity of hydrolytic enzymes and reduced leakage of metabolites. According to Maiti and Sengupta  ascorbic acid improved seed germination and cell growth by stimulating mitotic activity of embryo, providing stabilization of cell membranes  raising antioxidant enzyme activities , preventing lipid peroxidation and decreased malondialdehyde content . Ascorbic acid serves as a cofactor in the biosynthesis of many plant hormones, the endogenous level of AsA affect biosynthesis, the levels and the signaling of some hormones including gibberellic acid (GA3) . Ascorbic acid application significantly increased the level of GA3 and IAA, but decreased the level of ABA . Gibberellic acid plays an important role in regulating and accelerating seed germination . Endogenous phytohormones that are known to balance cell proliferation are modulated by nanoparticles [67,68]. Gallic acid is known to be scavengers of various oxygen species , improve water status, stabilizing cell membranes and maintaining cellular structural integrity  and increase the antioxidant capacity of plant . According to Abdul Khaliq et al.  seeds of two rice cultivars subjected to Se priming had higher activities of antioxidants, strong antioxidant defense and least lipid peroxidation through increased GSH-Px activity [71,72] as Se is present in the GSH-Px enzyme active site which participates in the reduction of toxic hydrogen peroxide and lipid peroxides . Contrarily, at an elevated antioxidant concentration the decrease in cowpea seedling growth and biochemical attributes observed may be attributed to elevated MDA levels and the activities of antioxidants in an attempt to cope with oxidative stress. Metabolic activities are expected to increase remarkably in cowpea seeds following their soaking with antioxidant compounds that may lead to higher activity of ROS as secondary products of mitochondrial respiration. Smrkolj et al.  found higher respiratory activity associated with low doses of Se in young pea (Pisum sativum L.) plants. There is strong evidence that free radicals and peroxides are abundantly produced within seeds during germination  and are cooperatively tackled by enzymatic reactions . The enhanced expression and activity of antioxidant enzymes has been proposed as part of seed strategy to cope with ROS produced during seed priming [61,76]. Se has been ascribed as a natural stimulant of antioxidant activity in plants .
In the present studty soaking cowpea seeds in AsA up to 150 pp, GA up to 250 pp, NaSeO4 up to 25 µM or SeNPs up to 50 µM solutions increased activities of α-amylase, β-amylase and protease, in the 4-day-old seedlings of cowpea, as compared to corresponding controls and then these values were progressively decreased at higher concentrations than control. The maximum activities of α-amylase, β-amylase and protease enzymes were obtained in response to soaking seeds in solutions at 100 ppm of AsA, 150 ppm of GA or 6.25 µM of either NaSeO4 or SeNPs. Similarly, the activity of starch hydrolysing enzymes α- and β-amylases and sucrose hydrolysing enzyme invertase was stimulated significantly with 0.1 - 0.75 ppm selenium (as sodium selenate) associated with elevation of activities of sucrose synthesising enzymes; sucrose synthase and sucrose phosphate synthase in 10-day-old mungbean seedlings . In two fine rice cultivars (Super and Shaheen Basmati), the activity of α-amylase was significantly induced by priming with selenium (15–60 μmol L−1). Nevertheless, Se at 90 and 105 μmol L−1 had detrimental effect on α-amylase activity that was dropped even below than that of the control at 105 μmol L−1 . Pre-sowing treatment in AsA at 15 and 30 mg L−1 improved seedling growth, fresh and dry matter, protease activity and chlorophyll contents in pumpkin seedlings under salinity stress  exogenous AsA increased the dormancy breaking, germination, endogenous AsA, α-amylase activity and increases endogenous GA accumulation in Malussieversii seeds .
The results show that soaking cowpea seeds in AsA up to 150 ppm, GA up to 200 ppm, Na2SeO4 and SeNPs up to 25 µM significantly increased TC and TSP in cowpea seedlings and then these values were progressively decreased at higher concentrations than controls. The maximum values of TC and TSP were obtained in response to soaking seeds in solutions at 100 ppm of AsA, 150 ppm of GA or 6.25 µM of either Na2SeO4 or SeNPs with superiority of SeNPs. In Sugar cane (Saccharum officinarum L.), pre-sowing treatment with AsA at 0.25-1.0 mM significantly enhanced fresh weight and soluble protein . In two rice cultivars, Se-primed seeds recorded more soluble sugars than the control seeds, where a Se solution at 30–45 μmol L−1 recorded similar soluble sugars, while at 105 μmol L−1 soluble sugars were at par with the control seeds . Se markedly reduces the effects of heat stress on cell vitality, activity of caspase-3-like proteases, and the levels of some stress-related proteins . The enhanced TC and TSP in cowpea seedlings by seed soaking with four antioxidant compounds (AsA, GA, NaSeO4 and SeNPs) at lower concentrations and the progressive decrease at higher concentrations than controls can be explained according to  who found that low (non-toxic) Se concentrations up-regulate proteins involved in ROS detoxification and resistance to pathogens. The expression of the same proteins was down-regulated by high (toxic) Se concentrations. At high concentrations, Selenium replaces sulfur in amino acids leading to malformed selenoproteins formed due to the misincorporation of selenocysteine/seleno-methionine in place of cysteine/methionine in protein chain which are nonfunctional proteins and enzymes and contributes to its toxicity [83,84]. Seleno-amino acids incorporated into protein may result in S–S bonds being replaced by the less stable Se–Se bonds, leading to changes in biological activity of the protein [85,86].
The present study indicates that soaking cowpea seeds in solutions of different antioxidants; AsA, GA, NaSeO4 and SeNPs, particularly at lower concentrations significantly improved seedling length, activities of hydrolytic (α-amylase, β-amylase and protease) enzymes as well as the contents of total soluble sugars and total soluble proteins, compared to controls. Generally, SeNPs at 6.25 µM was the most effective treatment in enhancing seedling length and activities of enzymes followed by NaSeO4 at 6.25 µM then, AsA at 100 ppm and finally GA treatment at 150 ppm. However, field studies are needed to determine the possible role of different antioxidants in improving cowpea growth and yield under normal and different stresses conditions.
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