EFFECTS OF DIFFERENT CONCENTRATIONS OF TITANIUM OXIDE NANOPARTICLES AND BULK ON GERMINATION CHARACTERISTICS AND ANTIOXIDANT ENZYMES ACTIVITY OF AVENA SATIVA L. SEEDS
DOI:10.30825/5.EJPAU.199.2021.24.1

Moein Iranmanesh, Abolfazl Beyraghdar, Homa Mahmoodzadeh
Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

ABSTRACT

The aim of this study is to compare the effects of different concentrations of nano and bulk titanium oxide on germination characteristics and the activity of antioxidant enzymes in Avena Sativa L. The titanium oxide nanoparticles diameter was determined about 40 nm using AFM, DLS, XRD analysis. In this study, based on a completely randomized design, seeds were placed in Petri dishes containing different concentrations of nano and bulk titanium oxide (50, 150, 250, 350 ppm). Three replications were considered for each concentration and the germination factors and antioxidant enzyme activity in the treated plant were compared with the control. The average data were compared using one-way ANOVA analysis and DUNCAN tests. According to the results, the following parameters of seedling vigor index, root length, dry weight, wet weight, guaiacol peroxidase enzyme, and polyphenol oxidase were statistically significant. The highest germination percentage, relative germination percentage, germination rate, weight germination index and shoot length were observed at 250 ppm bulk titanium oxide. In addition, the highest average germination time, seedling vigor index, optimum seedling index, and root length were observed in 50 ppm bulk titanium oxide treatment and the highest shoot length was observed in 250 ppm nano-titanium oxide treatment. In the enzyme activity, the highest activity of guaiacol peroxidase enzyme was observed in 150 ppm bulk titanium oxide treatment and the highest Polyphenol oxidase enzyme activity was observed in bulk titanium oxide 350 ppm treatment.

Key words: Nano titanium oxide, bulk titanium oxide, germination factors, antioxidant enzymes, Avena sativa L.

INTRODUCTION

Nanoparticles are tiny particles that are one, two or three dimensions and they are 100 nanometers or smaller in size. In recent years, their use and role in various fields have increased dramatically due to their complex physical and chemical properties. Although nanoparticles have many useful uses and properties, their release into the environment has a potential risk to human health and life. Interaction of nanoparticles on biological processes has always been one of the issues of interest to scientists and researchers. Thus, the researches on the toxicity of nanomaterials are increasing. However, the effect of nanomaterials on living organisms has not been fully investigated yet. However, in a study published in 2007 showed that the toxicity of the nanoparticles was due to the increase in oxygen species and oxidative charge [24]. In recent years, Nanotechnology, the science of working with the smallest particles has raised hopes to boost efficiency in agricultural systems by addressing problems left unaddressed in conventional ways [16]. It is found that the absorption rate of titanium oxide nanoparticles, bulk titanium and also the effect of different nanoparticles and bulk particle on growth and metabolic processes in plants are different. Therefore, few studies have reported the positive and negative effects of nanoparticles on higher plants. The positive effects of some nanoparticles such as titanium have been proven in relation to some plants. Plants are the most commonly used to investigate the effects of nanoparticles [11]. The application of titanium nanoparticles has recently received much attention from plant physiologists due to their prominent features [14]. Titanium as a useful element also stimulates the growth of plants and can increase the absorption of some elements such as nitrogen, phosphorus, calcium, magnesium, iron, manganese and zinc [36]. Because the titanium oxide nanoparticles are very small, this increases their contact area with the material and makes them more effective [35]. Their application in nutrient solution or sprayed on the leaves of plants increases biomass and growth of various plant species [9]. For instance, the application of titanium dioxide nanoparticles as treatment in spinach seeds has improved germination and growth in this plant [31, 44]. Avena sativa L. is a herbaceous and one-year-old plant from species of cereal grain (Poaceae). For the first time, oats were planted in Europe about a thousand years BC. The seeds of this plant have many medicinal properties, including booster, diuretic, laxative, painkiller, wound healer, nerve tonic, stimulant and disinfectant, tonic for heart and blood, cholesterol-lowering, Hypotensive, helpful in balancing blood sugar and insulin, anti-cancer, antibiotic and gonadotropic properties, reducing thyroid function and also in the treatment of respiratory tract infections, allergies, Vitamin C deficiency, Antioxidant, Digestive Disorders, Anti-diarrhea, Cerebral Palsy, Central Nervous System Stimulation, Anti-Bleeding, Rheumatism, Depression and Stress, Atherosclerosis and helpful in fighting premature aging [34, 23]. Biological and non-biological stresses result in the formation of reactive oxygen species (ROS). Production of reactive oxygen species causes peroxidation of membrane lipids, degradation of proteins and nucleic acids. Plants have different mechanisms to reduce the deleterious effects of reactive oxygen species [19]. One of these mechanisms is the antioxidant defense system, which is divided into two groups: 1) Non-enzymatic antioxidants; 2) Enzymatic antioxidants. Non-enzymatic antioxidants are the following: Beta-carotene, ascorbic acid, alpha-tocopherol and Restored glutathione and the enzymatic antioxidants consist of gliagol peroxidase, ascorbate peroxidase, catalase, polyphenol oxidase and glutathione reductase [32, 43]. In general, antioxidant systems play an important role in protecting membranes and plant organs from reactive oxygen. Catalase is a tetrameric protein with four chains each having 500 amino acids. The suitable pH for the activity of this enzyme varies from 4 to 11 depending on the plant species. Its optimum temperature also varies depending on the plant species [43]. Catalase is one of the important enzymes that work to remove H₂O₂ in peroxisomes. Guaiacol peroxidase is one of the major enzymes in the peroxidase group that oxidizes guaiacol and exhibits the highest activity around 30°C [15]. The enzyme complex of polyphenol oxidase plays a role in the conversion of some phenolic compounds to quinones and also plays an important role in the respiratory transport chain and has been reported as an important member of the seed and seedling oxidation system [22].

MATERIAL AND METHOD

Plant Material
Avena sativa L. seeds were prepared from Pakan Bazr Esfahan. Titanium oxide nanoparticles were prepared by Nano Pars Lima Co. The titanium nano oxide specification is as described in Table 1. In order to study the effect of different titanium nanoparticles and bulk titanium oxide treatments on germination and seedling growth of Avena sativa L., three replications were performed at 26°C and pH7 for each concentration. Concentrations consisted of four nano and bulk concentrations in ppm (50–150–250–350) and distilled water was used for control treatment. After sterilizing the seeds with 7% sodium hypochlorite and rinsing with distilled water, 20 barley oats seeds were placed on sterile filter paper per plate. After adding 5 ml of treatment solution to the plates, the prepared plates were incubated at 26°C. The number of germinated seeds was counted daily. After a 7-day period, root length, shoot length, wet weight, and dry weight were measured. For dry weight the specimens were placed in an aluminum foil at 80°C for 72 hours and then weighed with the same scale. The stages of this experiment were carried out in the Plant Physiology Research Laboratory of Mashhad Islamic Azad University.

Scanning Tunneling Microscope Studies (STM) 
An STM (Scanning Tunneling Microscope) is a device used to examine the structure and some properties of partially conductive and biologically conductive surfaces, as well as non-conductive thin films deposited on the conductive substrate in nanometer dimensions. No light waves or any other kind of lens is used and there is no lens in it. This microscope examines the surface structure of the sample. This microscope determines the size of the particle and its topography. A small amount of titanium dioxide nanoparticles (0.01 g) is poured into the test tube containing 15–20cc ethanol and then placed in an ultrasonic tube to disperse the particle in ethanol. Every 30 minutes the tube is removed and observed. When particles separated by ethanol were floated in solution, select a sample from the top of the test tube and drop one or two drops on a Sampler Holder to dry. The sampler is then coated with thin gold nanometer particles to allow flow in the STM. The coating was done by Sputtering apparatus under vacuum condition with argon for 45 seconds and current of 1.5–2 amps. Finally, the sample was transferred to STM (Model NANA – SS-5) for photographing.

X-ray Diffraction Studies (XRD)
The X-ray Diffraction (X-Ray Diffraction), using the X-ray range between the gamma-ray and the ultraviolet wavelength region, can provide information on the structure, material, and elemental quantities. In this study, the crystal structure (anatase and rutile form) of titanium dioxide nanoparticles was determined by XRD. XRD studies were carried out by the Binaloud Minerals Research Company located in Mashhad. 

Enzyme extract
To extract the enzymes of guaiacol peroxidase and phenol peroxidase, first 0.1 g of leaf tissue was washed with distilled water with 1 ml of 0.8 mm potassium chloride solution and grind in a Porcelain mortar and mixed at a speed of 7000 rpm for 10 minutes using the following Cooling Centrifuge model (Vision Rs-1500 CFNII). In order to evaluate the activity of the enzyme, all the steps were performed in the ice container [25].

Measurement of Guaiacol Peroxidase Activity
In order to measure the activity of guaiacol peroxidase enzyme, all the enzyme extraction steps were carried out in ice-water, 3 ml of 0.2 mm phosphate buffer solution, 50 µl of guaiacol and 50 µl of 3% hydrogen peroxide were added to the enzyme extract and immediately the optical absorption changes at 436 nm using the spectrophotometer was recorded at 15 s intervals for 3 minutes [25].

Measurement of Polyphenoloxidase activity
The activity of polyphenol oxidase was measured by Raymond et al. 1993 method. The tubes were initially placed in a 40°C water bath. At first, to the numbers of samples, the tubes were initially placed in a 40°C water bath. Then, respectively, to each tube, 2.5 ml of 0.2 M phosphate buffer at pH 6.8 and 0.2 ml Pyrogallol 99%, 0.02 M was added. At the moment of enzyme observation, 0.2 ml of enzyme extract was added to each tube. Changes in absorbance of polyphenol oxidase over a period of 4 minutes were recorded at 430 nm using a spectrophotometer [37].

Catalase extraction
To prepare the enzymatic extract, the first pre-sterilized Porcelain mortar previously kept in the freezer was placed in a larger container containing crushed ice. Then, half a gram of leaf sample washed with distilled water and then dried with 5 ml of extraction solution containing 0.1 mM potassium phosphate-buffered at pH 7.5, mixed with half a mmol of EDTA, was grinned and passed through the Sterile cleaning. The purified solution was the enzyme extract. To extract the enzyme, the extract was poured into 1.5 ml tubes and placed in a refrigerated centrifuge at 4 ° C for 15 min at 15,000 rpm. The reaction volume of 3 ml containing 50 mM phosphate buffer, 15 ml molecular hydrogen peroxide and 0.1 ml of enzyme extract were prepared and the amount of light absorbed by the spectrophotometer was recorded at 240 nm every 5 seconds for 5 minutes to determine the enzymatic activity [20].

Statistical analysis
In this study, 12 germination and seedling parameters were determined including: germination percentage (GP%), relative germination percentage (RGP), germination rate (GR), mean germination time (MGT), germination index (GI), weight germination Index (WGI), seedling vigor index (SVI), optimum seedling Index (SOI), root length, shoot length, wet weight and dry weight. These parameters are measured by the following formulas [4–7, 13, 21, 40] were calculated

• Germination Percentage (GP) = GN / SN × 100
• GN = Total number of seeds germinated on the third day
• SN = the total number of seeds planted and treated
• 2- Relative Germination Percentage (RGP) = GP (treatment) / GP (control) × 100
• GP (treatment) = Germination percentage of treatment
• GP (treatment) = Percentage of germination control
• 3- Germination Rate (GR) =
• Si = the number of days from planting day
• Gi = Number of germinated seeds per day
• 4- Mean Germination Time (MGT) =
• Gi = Number of germinated seeds per day
• i = the number of days from planting day
• 5- Germination Index (GI) = (∑ (N-i) × Gi) ×100 / N × GN
• Gi = Number of germinated seeds in i day
• i = the number of days from planting day
• N = the duration of the experiment
• GN = Total number of seeds germinated on the last day
• 6- Weight Germination Index (WGI) = [N × n1+ (N-1) × n2+ (N-2) × n3+…+1 × n7] / N × N’
• N = the number of test days
• N’ = the total number of seeds in a plate
• n1, n2, n3 ... = Number of seed germinated on the first, second, third day...
• 7- Seedling Vigor Index (SVI) = GP × Seedling Length (Root+Shoot (cm)), [Kandil et al., 2015]
• 8- Seedling Optimum Index= GP) × Dry Weight
• 9- Response Index:
• If control (C) bigger than treatment (T):    RI = T/C-1
• If treatment (T) bigger than control (C):    RI = 1-C/T

Statistical analysis was performed using ANOVA at P ≤ 0.05.  Data were expressed as mean ± standard error (SE). Analysis of variance was performed by ANOVA and DUNCAN test. All statistical data were analyzed by SPSS software (29).

RESULTS

Results of Scanning Tunneling Microscope Studies (STM)
STM was performed to evaluate the size of titanium dioxide nanoparticles. According to the calculations, the average particle size was 20 nm (Figures 1–3).

Results of X-ray diffraction (XRD) studies
As mentioned before, titanium dioxide nanoparticles have three crystalline forms of anatase, rutile, and brookite. There are generally two or three crystalline forms in a single nanoparticle. But the percentages are different. X-ray diffraction (XRD) test was performed to determine this percentage. As shown in Figure 4, the crystalline phase of anatase is higher than that of rutile, in other words, the main phase of this nanoparticle is anatase and its second phase is rutile.

Results of germination test and Enzyme Activity
The effect of nano and bulk titanium oxide and on germination factors is shown in Table 1. According to the results, the effect of nano and bulk titanium oxide on following factors: germination percentage, germination rate, mean germination time, relative germination percentage, germination index, and weight germination index were not statistically significant. The highest germination percentage, relative germination percentage, germination rate, and seed germination index were observed in 250 ppm bulk titanium oxide treatment and the lowest was observed in 250 ppm nano-titanium oxide treatment. The highest and lowest mean germination time was observed in 50 ppm and 150 ppm bulk titanium oxide treatments, respectively. In addition, all nano-titanium oxide treatments showed the highest mean germination time than control. The highest germination index was observed in control and lowest in 50 ppm bulk titanium oxide treatment. The effect of nano-titanium oxide and bulk titanium oxide on the growth of oat seedlings plant is shown in Table 2. The optimal seedling index under the above treatment did not change significantly. The highest seedling vigor index and root length were observed in 50 ppm bulk titanium oxide treatment and the lowest in 150 ppm nano-titanium oxide treatment. Also, all nano-titanium oxide treatments showed the lowest seedling vigor index and root length compared to the control. The highest seedling vigor index was observed in 50 ppm bulk titanium oxide treatment and the lowest in 150 ppm nano-titanium oxide treatment. The highest and lowest shoot lengths were observed in 250 ppm and 150 ppm nano-titanium oxide treatments respectively and in all bulk titanium oxide treatments the lowest was observed (except of 350 ppm nano). The lowest seed wet weight was obtained in 150 ppm nano-titanium oxide treatment and the highest in control. Lowest dry weight in 350 ppm nano-titanium oxide treatment and also in all treatments [except 50 ppm nano-titanium oxide and bulk titanium oxide], the lowest dry weight compared to control was observed. Root length of oats were significantly different at the 0.05 probability level (Table 2). The effect of nano-titanium oxide and bulk titanium oxide on the guaiacol peroxidase, polyphenol oxidase and Catalase enzymes is shown in Table 3. The enzymes of guaiacol peroxidase and polyphenol oxidase showed significant differences at probability level of 0.05 (Table 3). The maximum activity of guaiacol peroxidase enzyme was observed in 150 ppm bulk titanium oxide treatment and lowest in control. Maximum Polyphenol oxidase activity was observed in 350ppm bulk titanium oxide treatment and lowest in control. Catalase enzyme had lower activity in all treatments than the control [lowest activity compared to control in 250 ppm nano-titanium oxide treatment that there were no significant differences between the individual experimental objects.

The response index of different nano-titanium oxide and titanium oxide mass treatments on germination indexes are shown in Tables 4 and 5. RI values are in the range of +1 and -1, positive and zero values, respectively, indicate stimulation by treatment, and negative values indicate inhibition by treatment compared to control.

CONCLUSION

When the seed of a plant is placed in suitable conditions such as light, moisture, temperature, water, and nutrients, etc., it germinates and eventually becomes a complete plant, the change of any of the factors mentioned affects the seed and therefore affects the germination. The use of titanium nanoparticles has recently been favored strongly by plant physiologists due to a number of specific properties [14]. According to researchers, titanium has the potential to improve the plants' physiological activities [31, 44]. In this study, by adding nano-titanium oxide and bulk titanium oxide to the investigated environment, we investigated their effect on various germination factors. According to results, nano titanium oxide and bulk titanium oxide caused increase in some factors and respectively reduced some these factors and also in some cases had no significant effect on the factors and the amount was equal to the control. However, in most studies the results have shown that nano-titanium oxide does not have any significant effects on plant growth. For instance, a study by Seeger et al. In 2009 found that nano-titanium oxide at concentrations (0, 1, 10, 20, 50, 100 mg/l) produced no toxicity to the willow tree [38]. Also the results of several other studies showed that nano-titanium oxide had no on germination of barley [28], rice [3], lettuce, radish and cucumber [42], tomato [39]. Based on the results of research has done nano-titanium oxide has no effect on root length [12, 28]. But some results suggest otherwise. For example, our and Mushtaq's research [33], respectively, showed that nano-titanium oxide reduces the length of oat and lettuce roots. Yang and Watts [2005] also reported that nano-titanium oxide in the plants tested (radish, ryegrass, lettuce, maize, and cucumber) caused toxicity in the plant and inhibited root growth [43]. However, it should not be concluded quickly because different plant species may have different reactions to a single substance in the environment. For example, in an experiment nano-titanium oxide increased the length of corn seedling [8] or in other experiments, high concentrations increased seed germination and seedling growth of wheat compared to control and also at 1200 and 2000 ppm treatments increased seedling wet weight [26]. It has been concluded that nano-titanium oxide has a stimulating effect on wheat root at high concentrations and an inhibitory effect on wheat root at low concentrations. Another study has found that nano-titanium oxide at the appropriate concentration increases the germination properties and potency of five medicinal plants: Salvia mirzayanii, Alyssum homolocarpum, Sinapis Alba, Carum copticum, Nigella sativa [17]. Also, nano titanium oxide at concentrations above 1200 and 1500 ppm increased root and shoot length of rapeseed compared to control and at 1500 and 2000 ppm caused the lowest and highest seedling vigor index, respectively [27]. Plants have a number of enzymatic and non-enzymatic defense systems to remove and manage active oxygen species. Past studies showed that the reaction of antioxidant enzymes to metals is ambiguous and can vary between plant species and even between different tissues of a plant [29]. Catalase is an oxidoreductase that acts as an enzymatic defense system in removing reactive oxygen species [2]. For example, aluminum oxide nanoparticles reduced the activity of Catalase in the treated groups with aluminum nanoparticles compared to the control group [10]. Also in the treatment of Catharanthus roseus, the activity of Catalase antioxidant enzymes and guaiacol peroxidase was decreased at zero micromolar concentration of zinc nano oxide and then increased at 2, 4 and 10 micromolar concentrations [1]. The results also showed that the activity of antioxidant enzymes (catalase, superoxide dismutase, guaiacol oxidase, ascorbate oxidase, and peroxidase) was higher in the treatment of Salvia officinalis L. under the influence of titanium nano oxide [30].

REFERENCES

Received: 14.09.2020
Reviewed: 11.12.2020
Accepted: 9.02.2021

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