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14БИОЛОГИЧЕСКИЕ НАУКИ
THE STUDY OF ISOENZYME CONTENT OF SUPEROXIDE DISMUTASE IN WHEAT PLANTS UNDER DROUGHT AND REHYDRATION
LALA MUSEYiB AYDiNLi
PhD in biology, ANAS IMBB, Baku, Azerbaijan
ANELLA GASiM GARiBOVA
Master's student, BSU, Baku, Azerbaijan
IRADA MAMMAD HUSEYNOVA
Academician, ANAS IMBB, Baku, Azerbaijan
Abstract. Relative water content (RWC) of leaves, lipid peroxidation degree (MDA), and isoenzyme content of superoxide dismutase (SOD) have been studied in bread wheat (Triticum aestivum L.) genotypes with contrasting drought tolerance (Gobustan - drought-tolerant, Tale 38 -drought-sensitive) under conditions of water deficiency and after re-watering. Under drought, the amount of MDA, which is an indicator of stress, increased sharply in the leaves of both genotypes. In the process of rehydration, this indicator was almost completely restored in the drought-tolerant wheat genotype. This reflects the more dynamic self-restoration ability of the genotype. Eight isoenzymes of superoxide dismutase were observed under both normal watering and drought conditions. These isoenzymes respond to drought and re-watering by varying their intensities.
Keywords: Triticum aestivum L., drought, rehydration, superoxide dismutase
Introduction
The study of the mechanisms of adaptation of plants to various external abiotic stress factors is one of the important problems of modern biology. Drought, one of the abiotic stress factors, directly affects plant productivity and product quality [1]. At present, this problem is being studied more actively. The growing population of the world, and the declining and gradual decommissioning of arable lands raise the issue of food security. The effect of any stressor is known to cause the formation of reactive oxygen species (O2~, OH, 1O2, H2O2), which damage vital macromolecules (DNA, RNA, proteins, lipids, etc.) of the cell. As a result, damage occurs at the cellular level. Environmental stresses such as drought, salinization, decreased temperature, heavy metals, UV radiation, and pathological microorganisms disrupt cellular homeostasis and increase the generation of ROS in cells [2]. High concentrations of ROS are dangerous for plants. When the level of ROS exceeds the potential of the cell's self-defense mechanisms, the cell is exposed to oxidative stress. Among the defense mechanisms operating in plants, the main place is occupied by the antioxidant defense system, which eliminates the reactive oxygen species. Excessively accumulated ROS are utilized by an antioxidant defense system consisting of enzymatic and non-enzymatic components. Therefore, the activity of antioxidant enzymes increases during oxidative stress in most plants. The ability of antioxidant enzymes to maintain high activities in tolerant genotypes and respond by reducing the effects of oxidative damage depends on the genetic potential of each genotype. The plant's response to stress depends on the intensity, duration, and type of stress [3].
Wheat, which is of strategic importance, is also affected by a number of extreme factors, including drought, soil dryness, salinity, UV radiation, etc. during its development. Wheat ranks first among agricultural crops in terms of production volume and bread supply. From this point of view, both fundamental and applied research work with wheat is of great importance in solving the food problem [4]. Monitoring of changes in morphophysiological and biochemical properties of the plant after
drought and re-watering is important for the creation of drought-tolerant wheat genotypes adaptable to different environmental conditions.
Materials and methods
Some physiological and biochemical changes occurring in bread wheat (Triticum aestivum L.) genotypes (Gobustan - drought-tolerant, Tale 38 - drought-sensitive) with contrasting drought tolerance have been studied under water deficiency conditions and during restoration processes after rehydration. The relative water content (RWC) of leaves was measured using a method proposed by Turner [5]. The degree of lipid peroxidation was determined spectrophotometrically based on the dynamics of the accumulation of malondialdehyde (MDA) [6]. The isoenzyme composition of superoxide dismutase was determined at a constant electric current (30 mA) at 40C for 3 hours using the native PAAG electrophoresis method [7]. 0.5 g of leaf sample was crushed in liquid nitrogen and homogenized in 100 mM Na-phosphate (pH 7.8) buffer solution containing 1 mM EDTA, 2 mM PMSF, 1% PVP, and 0.1% Triton X-100, at 4°C for 20 minutes at 15000 g. The obtained supernatant was used to study the isoenzyme content of SOD. A solution containing 4 mg of riboflavin, 4 mg of Na-EDTA, 20 mg of NBT, and 0.1 M Tris HCl (pH 7.4) buffer was used to stain the gel. After incubation for 40 minutes in the dark, the gel was incubated for 15 minutes in high-intensity light until light-colored spots appeared on a purple-blue background.
Results and Discussion
Significant changes in the relative water content of wheat leaves were observed during drought and re-watering. The relative water content in the leaves of the Gobustan genotype grown under field conditions decreased in the drought-exposed variant compared to the control variant. Then, 3 and 7 days after re-watering, this indicator increased and approached the watered variant (Table 1). After 7 days of re-watering, the difference in the relative water content between the control and stress-exposed plants was only 4%.
Table 1
RWC in leaves of bread wheat genotypes (Gobustan and Tale 38) cultivated under field conditions: W - watered, D - drought, R1 - samples taken 3 days after re-watering, R2 - samples taken 7 days after re-watering. Statistical analysis was performed based on the Student t-test: Differences in mean values are considered significant when the P-value <0.01 (**), <0.05 (*).
№ Genotypes F iel d conditions RWC (%)
1 W 79±3.16
2 Gobustan D 54±2.16**
3 R1 72±2.88**
4 R2 76±3.04**
5 W 80±3.21
6 Tale 38 D 59±2.36**
7 R1 72±2.88**
8 R2 72±2.88**
The relative water content in the leaves of the Tale 38 genotype also decreased in the drought-exposed variant compared to the control variant. Although it increased relatively after 3 days of re-watering, no change occurred after 7 days. Analyses showed that after re-watering, the Gobustan variety recovered almost completely, while in the Tale 38 variety this process was relatively weak. After 7 days of re-watering, the difference in the relative water content between the control and drought-exposed plants was 10%. The high self-restoration properties of Gobustan after re-watering can be explained by the relative stability of RWC in the leaves during drought and the less exposure
of the root system to osmotic effects due to deeper penetration into the soil [8].
Malondialdehyde is a key indicator of lipid peroxidation and plasma membrane damage in plant cells [9]. In the Gobustan genotype, which was cultivated in the field and exposed to drought, the amount of malondialdehyde increased 2-fold compared to the irrigated variant and amounted to 3.27 mM / g (Table 2). Drought-affected plants were re-watered, and a sharp decrease in MDA was observed in samples taken 3 days after irrigation. However, the MDA amount increased again in samples taken after 7 days.
Table 2
MDA amount in leaves of bread wheat genotypes (Gobustan and Tale 38) cultivated under field conditions: W - watered, D - drought, R1 - samples taken 3 days after re-watering, R2 - samples taken 7 days after re-watering. Statistical analysis was performed based on the Student t-test: Differences in mean values are considered significant when the P-value <0.01 (**), <0.05 (*).
№ Genotypes F iel d conditions MDA (mM/g FW)
1 W 1.67±0.12
2 Gobustan D 3.27±0.23**
3 R1 1.97±0.14**
4 R2 2.01±0.14**
5 W 0.87±0.06
6 Tale 38 D 2.63±0.18**
7 R1 2.32±0.16 ns
8 R2 2.35±0.16 ns
In the Tale 38 genotype, which is more sensitive to water deficiency, the amount of MDA in the stress variant was 3-fold higher (2.63 mM / g) than in the irrigated variant (0.87 mM / g). The amount of MDA relatively decreased (2.32 mM / g fresh weight) in the samples taken 3 days after re-watering, and remained stable (2.35 mM / g) in the samples taken 7 days after re-watering. Accumulation of membrane-damaging malondialdehyde in plants accelerates lipid peroxidation, but the trend of recovery processes after repeated irrigation shows the plasticity of the metabolism of the studied plants [10, 11]. In this regard, the amount of MDA accumulated in the leaves of wheat during the drought period and the plant's ability to recover after repeated irrigation can be used in the screening of tolerant varieties.
One of the early responses of the plant to abiotic stress is an increase in the generation of the reactive oxygen species - superoxide anion (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (*OH), etc. The activity of antioxidant enzymes, some of the main components of oxidative stress in plants grown in arid areas, is higher than in those grown in normally irrigated areas [12]. Spectrophotometric analysis revealed that the constitutional activity of the enzyme superoxide dismutase in the leaves of both genotypes was approximately the same during normal irrigation under field conditions. SOD catalyzes the conversion of the superoxide anion into H2O2 and O2. The enzyme activity increased 2fold in the drought-affected Gobustan genotype and 1.2-fold in Tale 38. Although rapid recovery was observed in Gobustan after 3 days of re-watering, the enzyme activity continued to increase in Tale 38. In Gobustan after 7 days of re-watering, the activity decreased and approached the watered variant but the activity in Tale 38 continued to increase. Electrophoretic analysis of the isoenzyme content of superoxide dismutase revealed 8 isoforms: 2 high molecular weight isoforms (SOD1 and SOD2), 1 medium molecular weight isoform (SOD3), and 5 low molecular weight isoforms (SOD4, SOD5, SOD6, SOD7, and SOD8) (Figure 1). Under drought conditions, the intensities of medium and low molecular weight isoforms increased slightly and were relatively restored after the elimination of the stress effect.
Gobustan Tale-38
_A__Л
С D R С D R
Mn-SOD Mn-SOD —►
Fe-SOD —►
Cu/Zn-SOD Cu/Zn-SOD Cu/Zn-SOD —►
Cu/Zn-SOD Cu/Zn-SOD
Figure 1. Isoenzyme content of superoxide dismutase in leaves of the Gobustan and Tale 38 genotypes. W- watered, D- drought, R-rehydration
Conclusion
Thus, the analysis revealed that the activity of the enzyme superoxide dismutase increased under water deficit compared to the irrigated variant in both genotypes, and after rehydration, the enzyme restored its activity more dynamically in the tolerant genotype. The Gobustan variety, which demonstrates high physiological performance, can be used as a starting material for the creation of stress-tolerant wheat varieties in practical breeding programs.
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