The Impact of UV Irradiation of Winter Wheat Seeds on Enzymatic Activity in the Germination Period

Wheat has always been and still remains the main food crop of the Rostov region (the southern part of Russia) (Samofalova et al., 2015). The introduction of innovative environmentally friendly methods of pre-planting seed treatment in crop production is the key to the effectiveness and competitiveness of agricultural crop production (Shakirova M.F., 2001, Willekens H. et al., 1994).
       
The application of various types of stimulating treatments can change the rate of bio-chemical processes, growth and morphogenesis (Medvedev S.S. 2013).
       
The physiological processes of germinating seeds proceed in the presence of amylolytic enzymes, which become active when swelling. The enzymatic activity which accelerates seed germination, is an adaptation mechanism to stresses caused by exposure to UV irradiation sources (Alekhina, N.D., 2005).
       
Amylase participated in starch splitting into maltose, which was split into glucose and simultaneously formed sucrose was used by growing sprouts (Kretovich, 1971).
       
Catalase is a heme tetrameric enzyme that catalyzes the conversion of hydro-gen peroxide to water. The catalase reaction to stress caused by high-intensity light can be various and depends on the duration of the stress factor and the type of plant (Chupakhina G.N. et al., 2011).
       
Peroxidase involved in plant respiration along with catalase prevents the accumulation of hydrogen peroxide in cells, performs a protective function and inhibits pathogenesis and stress factors. (Rogozhin V.V. et al., 1996).
       
The objective of the current study was to study the effect of UV irradiation on enzymatic activity in germinating winter wheat seeds and to choose UV irradiation modes that are optimal for stimulating germination.
The current study’s tasks were:

• To study of the effects of UV irradiation of different durations on enzymatic activity in germinating winter wheat seeds;
• To identify optimal UV irradiation modes for stimulating seed germination.

The studies were carried out in 2017-2019. The seeds of the wheat variety ‘Rostovchanka 5’ (Triticum aestivum L.) were the object of the study. The variety was developed in the Agricultural Research Center ‘Donskoy’ by crossing of the varieties ‘Donskaya Yubileynaya’ and ‘Darunok’. The variety has been included in the State List of Breeding Achievements approved for use in the North Caucasus region. This semi-dwarf variety belongs to the group of lutescens with 1000-kernel weight of 35-44 g. The kernels of the variety are large and vitreous with average yield of 49.7 hwt/ha. It’s a middle-early variety with the growing duration of 228-286 days. The variety is resistant to lodging, drought, smut and powdery mildew, partially tolerant to brown rust. The variety has good baking qualities.
       
UV irradiation of seeds was effective when the seeds of the 2.6 mm fraction were pre-soaked in distilled water, after which the seeds were UV-irradiated and germinated in a thermostat at 20°C in four fold sequences of 100 grains, with daily moistening. To maintain humidity in the thermostat (at a level of 90-95% relative humidity), a tray with water was placed at the bottom of the thermostat. (Meyer et al., 1982). This year laboratory experiments were carried out in February - March 2019, at the Department of Biotechnology and Chemistry, FSBEI “K.G. Razumovsky Moscow State University of technologies and management”. The swollen seeds were UV-irradiated with BNPO2-30-001UZ,5 with an UV irradiation intensity of 30 W/m². The seed were exposed to UV irradiation for 1, 3, 5 and 7 minutes at a distance of 25 cm from the UV irradiation source. The purpose of seed treatment was to stimulate the enzymatic activity.
       
The total amylase activity was determined by isolating them with a NaCl solution, followed by incubation with a standard starch solution for a certain duration, followed by colorimetry of non-hydrolyzed starch. Enzymatic activity was given in mg of hydrolyzed starch per 1 ml of enzymatic extract. Catalase activity was estimated by a gasometric method (Ermakov, 1987). The total peroxidase activity (TPA) was evaluated according to the Boyarkin method (Ermakov, 1987), based on the ability of benzidine to oxidize with hydrogen peroxide if peroxidase participated.

During seed swelling and germination, the nature and intensity of physiological processes in germinating seeds depended on enzymatic activity of grain and on environmental conditions (Gao et al., 2013). The process of seed germination was differentiated into four stages: swelling, emergence, sprout development due to heterotrophic nutrition and transition to autotrophic nutrition (Farrel et al., 2008). During swelling there were distinguished physical and biological stages. At the physical stage, through the cell mem-branes water entered the seed moistening the endosperm cells; at the second stage its enzymatic systems became active (Chirkova 2002). One of the enzymes actively involved in the process of seed germination was amylase (Masojć et al., 2009). The starch hydrolysis began under the effect of amylases, intensively formed from the moment of germination (Gelmanov et al., 1981). The amylase activity is usually studied on a certain day of seed germination. It is well-known that the maximum activity of total amylases during wheat seed germination usually occurs on the 3^rd – 5^th day (Gelmanov et al., 1981).
       
As a result of the experiment, there have been identified the features of amylase activity at various durations of UV irradiation. Fig 1 showed amylase activity change in winter wheat seeds on average for all variants of trial, when they germinated under optimal moisture.
 

       
The graph representing amylase activity change in germinating seeds and the maximum position makes it possible to draw a conclusion about the nature of the enzymatic activity change during five days of seed germination (Obrucheva 2003). The experimental curve indicates this process irregularity during seed germination under unchanged external conditions (Masojć et al., 2009.). Amylase activity increase was observed as early as at the end of the first day of wheat germination in all variants of the experiment. Analyzing the correspondence of amylase activity by days of germination to the sections of the curve, we can conclude that the fourth and fifth days after sowing are the key duration when amylase activity in the germinating seed reaches its maximum value, whereas after the level of enzyme activity decreases.
       
Amylase activity was observed as early as at the end of the first day of germination. By the second day the amylase activity increased by 19% in the variant with the three-minute UV treatment compared to the control, whereas by 23.5% in the variant with the five-minute UV treatment.
       
The maximum amylase activity in the variants with three- and five-minute UV treatment was observed on the fourth day from the beginning of germination and was higher than the amylase activity in the control by 58.6 and 64.1%, respectively.
       
During the fourth day, the amylase activity continued to increase, remaining high even on the fifth day of seed germination. As a result, it was found that in the variants with a three- and five-minute UV irradiation, the amylase activity was higher than in the control by 44 and 58.8%, respectively.
       
It can therefore be concluded that the modes of three- and five-minute seed UV irradiation heavily increase the amylase activation rate compared to other variants of the trials and the embryo uses endosperm reserves more actively. As a result, germinating power and laboratory germination increase with these UV irradiation modes.
       
Catalase involved in the process of respiration is contained in dormant seeds in much larger quantities than amylase (Apel et al., 2004). Catalase prevents the accumulation of hydrogen peroxide, which is formed during aerobic oxidation (Apel et al., 2004). Catalase has its optimum activity at pH 6.5.
       
In more acidic and alkaline environments, its activity decreases (Thirupathi Karuppanapandian et al., 2011). In the oxidized state, catalase can also act like peroxidase, catalyzing the oxidation of alcohols or aldehydes. Catalase activity rises in parallel with the intensity of germinating seeds respiration (Fig 2).
 

       
The study of the effect of pre-planting UV irradiation of seeds of the winter wheat variety ‘Rostovchanka 5’ on catalase activity in germinating seeds showed that the highest enzyme activity - up to maximum values - occurred during the first four days, when it increased 7.3– 7.5 times compared with the first day of germination, after which it decreased.
       
Catalase activity in germinating wheat seeds exceeded the control value in all variants of the trials. UV treatment of wheat seeds before germination increased catalase activity on the fourth day from the beginning of germination for the seeds treated for three and five minutes on 14.7 and 17.7%, respectively, compared to the control. The maximum catalase activity was registered in the seed with a five-minute exposure to UV irradiation. By the fifth day from the moment of germination, the activity decreased both in the control and in all variants of the trials.
       
The experimental curve we obtained during the current study was S-shaped, which indicates the unevenness of catalase activation during seed germination in a thermostat under 20°C and optimum moistening. Analyzing the correspondence of catalase activity to certain sections of the S-shaped curve by days of seed germination, it can be concluded that during the first or second day catalase begins to activate in all variants of the trials; during the second and third days there is a logarithmic increase in enzyme activity.  By the fourth, key day, the catalase activity in germinating seeds reaches a maximum, after which the level of enzyme activity decreases.
       
Based on the data obtained as a result of the trials, it can be concluded that an increase in catalase activity after seed UV treatment indicates an increase in the synthesis occurring in wheat sprouts resulted from seed UV treatment (Shakirova 2001).
       
Peroxidase is a part of the antioxidant system, the activity of which determines the level of plant resistance to various factors during ontogenesis (Gazaryan et al., 2006). Peroxidase, along with catalase, is involved in the detoxification of H₂O₂ (Liszkay et al., 2003).
 
       
The activity of the antioxidant system, which includes peroxidase, determines the plant resistance to various environmental stress factors during ontogenesis (Bhattacharya et al., 1994). Peroxidase is a catalyst for the oxidation of various inorganic and organic compounds (Rogozhin et al., 1996). Peroxidase can display oxidase properties due to its broad substrate specificity (Pradedova et al., 2014).  Having antioxidant activity, peroxidase inhibits free radical oxidation of lipids (Noori et al., 2018). When the seeds leave a dormancy stage, peroxidase activity grows as their respiration intensifies (Liszkayet_al2003). Available experimental data suggest that peroxidase is associated with a number of metabolic transformations in cells with a wide spectrum of the enzymatic activity, ranging from pH 3 to pH 14 (Lebedeva et al., 1996).
       
Peroxidase activity was studied for nine days in dynamics, in germinating wheat seeds exposed to UV irradiation. As a result, it was found that the germination process is accompanied by enzyme activity increase over time. There were found significant differences in the level of total peroxidase activity in the seeds in different variants of trials.
       
A gradual increase in the total peroxidase activity in the control and other variants of trials during the first seven days reached a maximum by the eighth day. Among all the variants of trials, the variants with three-and five-minute exposure to UV irradiation showed the highest activation (Fig 3).
       
Fig 3 shows the average change in total peroxidase activity in winter wheat seeds for all variants of the experiment. The current analysis of the experimental curves has shown that seed swelling and germination is accompanied by an increase in the total peroxidase activity during the nine-day period of germination. Peroxidase activity in the germinating wheat seeds, soaked at 20°C, was of peculiar dynamics and depended on the duration of UV irradiation and the time of germination.
 

       
The current study has shown that peroxidase activity increases 2.8 times on average during the first seven days of seed swelling and germination compared to the beginning of germination. The maximum activity was on the eighth day and by the ninth day it decreased. On the eighth day of seed germination, peroxidase activity increased on 50% in seeds with three-minute UV treatment and on 55% in seeds with five-minute UV treatment compared to the control. The peak of peroxidase activity on the eighth day in these variants of the experiment was on 12.5 and 16.6% higher than in the variant with one-minute UV treatment and on 5.8 and 9.8% higher than in the variant with seven-minute UV treatment.
       
The experimental data on peroxidase activity have identified that three- and five-minute UV treatment modes increase the sensitivity of seeds to UV irradiation, as an external stress factor, whereas one-minute and seven-minute UV treatment cause less revealed reactions.
       
The obtained data of the current study have proved the modern concepts of living systems response to the external factors of different nature. It is known that changes in metabolism caused by stress factors are similar in all organisms (Apel et al., 2004). Some medium-intensity effects contribute to improving a living system sustainability due to non-specific adaptive transformations (Liszkay et al., 2003).
       
When the stress factor does not exceed the threshold value for an organism, its response tested for some kind of functional indicator over time usually includes successive phases of stimulation, partial inhibition with plateau and activation or complete suppression depending on the pressure magnitude (Gazaryan et al., 2006).
       
Objects both resistant to external factors and responsive to them undergo similar phase changes, but these changes are more extended in time, that is, they occur later at a higher dose of exposure (Apel et al., 2004).

The current study has shown that the use of artificial sources of UV irradiation, mercury-quartz lamps, such as BNPO 2-30-001U3.5 in crop production accelerates the seed sprouting and germinating power by activation of enzymatic activity. The use of UV irradiation for stimulation of germinating seeds is of great relevance and necessity, as it is an environmentally safe technology for pre-planting seed stimulation in agricultural production. The use of a BNPO 2-30-001U3.5 mercury-quartz lamp to stimulate physio-logical and biochemical processes in germinating seeds allowed us to identify different levels of enzymatic activity depending on the duration of exposure to UV irradiation. After soaking winter wheat seeds in distilled water, they were irradiated with a BNPO 2-30-001U 3.5 mercury-quartz lamp and during seed swelling and germination, the activity of amylase, catalase and peroxidase was determined. During the experiment, there were identified the optimal modes of seed UV irradiation. It is known that electro-physical stimulation of seeds is of great efficiency. The yield increase may be up to 10-20% (Ponomareva 2006). Therefore, these methods are widely used in agriculture. This requires new structural concepts to develop production mechanisms with electro-optical converters for pre-planting seed treatment (Ponomareva 2006).