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12ХИМИЧЕСКИЕ НАУКИ УДК 57.042
THE IMPACT OF NANOPARTICLES ON THE CONTENT OF CHLOROPHYLL PIGMENTS AND ACTIVITY OF FERMENTS IN THE COTTON SEEDLINGS LEAVES CULTIVATED IN THE SOIL FROM MUGAN REGIONS
FARIDE HASANOVA
Department of Bioecology of BSU, Baku, Azerbaijan
ISMAT AHMADOV
Associate Professor, Department of Chmeical Physics, Baku State University, Baku, Azerbaijan
Abstract.The role of nanotechnology in solving environmental problems is increasing, and there is a need for additional research in this area. In these experiments, the effects of aluminum nanoparticles on chlorophyll pigment in cotton seeds Ganja-110 and the activity of enzymes ascorbate oxidase, polyphenol peroxidase and quaiacol peroxidase in soil samples taken from different regions of the Mugan zone were investigated. It was found that the amount of chlorophyll pigments a and b in the leaves of cotton seeds treated with Al nanoparticles in fertile soil (0.44 dS / m) decreases during salt stress in saline soils (2.32 dS / m) increases. The activity of the enzyme ascorbate oxidase increases in saline soil, the activity of the enzymes polyphenol peroxidase and quaicolol is relatively reduced.
Keywords: nanoparticles, cotton, salty soil, chlorophyll, enzyme activity
Introduction. Most lowland regions of Azerbaijan have favorable conditions for cotton growing. The most important of these areas is the Mugan Plain. It is clear from historical sources that cotton growing was widespread in the Mil-Mugan and Shirvan plains since the 18th century. Later, cotton growing began to be applied in Ganja, Goychay, Agdash and other regions, as well as in Nakhchivan. At that time, the Mugan-Salyan zone produced 32.8% of the country's cotton. The fact that the Mugan plain in Azerbaijan has always had favorable conditions for cotton growing has led to a high share in its production. This is due to the favorable soil and climatic conditions in the region. The Mugan zone, located at the confluence of the Kur and Araz rivers, has recently been severely affected by salinization. An important environmental problem, such as soil salinization, has a serious impact on the productivity of the Mugan Plain. Therefore, the cultivation of agricultural crops and achieving high productivity in this zone is one of the promising issues. Most modern cotton varieties are salt-resistant. In general, the cotton plant is classified as the most salt-tolerant plant among agricultural crops. Even when the amount of salts in the soil is high, the cotton plant can grow and produce normally. However, when the amount of salt is too high, the growth of cotton is slowed down, productivity is low, fiber quality is low [13,22]. However, the salinity tolerance of cotton is limited and much research is still needed to increase its salt tolerance [4,27]. There are a number of ways to increase the salinity tolerance of cotton. This is, first of all, a way to genetically improve varieties. Another way is to increase the salt resistance of almond seeds or seedlings by chemical, biological or physical methods. For the normal development of cotton in saline soils, it is possible to reduce the amount of salts in the soil by washing the soil or applying agromeliorative methods. Although agromeliorative methods have certain effects, they are not economically viable. In the cotton plant, as in all plants, the initial stage of germination, the flowering phase is very sensitive to salinity, as well as other stressors. It is not a problem for plants that pass these stages normally to produce in saline soils [10]. During salt stress, germination rate in all plants, including cotton, growth and development of plants is slowed down, the amount of pigments, chlorophyll and carotenoids in
the leaves decreases, the activity of important physiological processes - photosynthesis, respiration, enzymes activity decreases [7,11,16] . Veiping Chen and colleagues (2010) found in their experiments that the cotton plant is more sensitive to salt stress in the germination phase and in the early stages of development than in the later stages of growth [32]. At high salinity, the growth rate of cotton plants decreases, even the seedlings can be destroyed, the dry weight of the roots and stems is much less, the leaves lose their color and darken, the flowering period is shortened [15]. In highly saline soils, the number of productive branches of the cotton plant is small, and its development is slowed down [5]. The results of experiments with four varieties of cotton (Gossypium hirsutum L.) growing in altitude zones showed that in saline soils the height of their seedlings, root length, number of leaves, leaf area, chlorophyll a and b content, osmotic potential, dry biomass and dry mass of roots significantly decreases [5,11,14,17,25]. The effects of salt stress varied depending on the variety of cotton [8]. It is well known that the amount of chlorophyll under the influence of environmental stressors, including salt stress, depends on the genotype of cotton. There is a positive correlation between antioxidant and salt resistance in cotton [23]. However, the activity of antioxidant enzymes after stress removal, which reduces the damage that occurs during oxidation, plays an important role in the recovery of processes after salt stress. However, the nature of these physiological processes in cotton has not yet been clarified and requires new research [9]. In experiments with genetically improved Ipt cotton, the effects of CuO nanoparticles on the height of the stem of the cotton plant, the length of the roots, the density of the root absorbers and the biomass were studied. At low concentrations of CuO nanoparticles, there is no significant change in the mineral nutrition and in the development of sprouts and roots of cotton . However, CuO nanoparticles have been found to have a positive effect on the absorption of Fe and Na elements, inhibiting the products of some phytohormones (IAA, ABA, GA and tZR). Due to the action of CuO nanoparticles, the concentration of iPA in Ipt cotton, which prevents the aging of plants, is significantly increased. As the concentration of CuO nanoparticles increases, the amount of Cu in the root of the seedlings increases. TEM analysis of samples taken from roots and leaves shows that CuO nanoparticles can accumulate in the roots and leaves of plants [21. In another study, the phytotoxic effects of CeO2 nanoparticles on a Bt-transgenic cotton plant were investigated. It became clear from their experiments that CeO2 nanoparticles spread from the root of the cotton plant to the stem and then to the leaves, where they enter the leaf cells and are adsorbed on the surface of the chloroplasts. In this case, the destruction of chloroplasts is observed. On the other hand, nanoparticles reduce the amount of important elements such as Zn, Mg, Fe and P, IAA and ABA growth hormones in xylem juice. CeO2 nanoparticles do not significantly affect the activity of peroxidase (POD in stem cells and superoxide dismutase enzymes in leaf cells), which can significantly alter the activity of the enzyme catalase (CAT), as these nanoparticles are present in both stem and leaf cells of Bt-transgenic cotton. At concentrations of 100 mg / l, the activity of the catalase enzyme is significantly different from the control variant. The results showed that the amount of protein in ordinary and Bt-transgenic cotton is different in both stem and leaf cells when processed with CeO2 nanoparticles at a concentration of 100 mg / l. r [20]. The toxic effect of another nanoparticle, SiO2, is still widely studied in Bt-transgenic cotton. The development of Bt-transgenic cotton at 0, 10, 100, 500 and 2000 mg / l at different concentrations of SiO2 nanoparticles was observed for 3 weeks. Dry mass, mineral nutrition, bioeffects at different stages of its development have been studied in detail under the influence of this nanoparticle. The movement of nanoparticles and their localization in plant organs were studied by TEM analysis. SiO2 nanoparticles have been shown to reduce the height, root size and biomass of cotton plants. It affects the amount of Cu, Mg and Na elements in seedlings and the concentration of growth hormone IAA. In addition, TEM analysis showed that SiO2 nanoparticles can spread from the roots to the stems and leaves through the xylem juice of the roots. The results of these experiments proved once again that SiO2 nanoparticles can accumulate in cotton plants and spread in its organs [20].
Research object and methods. Ganja-110 cotton variety was used in the experiments. This variety of cotton has been regionalized in Azerbaijan since 2009. Obtained by experimental
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mutagenesis. Seeds of Ganja-110 cotton variety were presented by the Institute of Genetic Resources of ANAS. Cotton seeds are cleaned of fibers before sowing. This variety of cotton is fast-growing, plant height - 90-110 cm, stalk - compact, pyramidal, monopodial branches - 1, stem - pale, green, weakly hairy, resistant to dormancy. Leaves - medium large, 3-5 slices, finger dark green, heart-shaped, flowers - medium large, yellow-cream color, no anthocyanin spots, pollen is yellow. Cones -large ovate, star-shaped, smooth, with brown spots, green. The product does not spill. The seeds are medium-sized, weighing 115-120 grams per 1000 seeds, ovate, dark green, moderately hairy [2]. The activity of enzymes was carried out by standard biochemical methods. To determine the activity of the ascorbate peroxidase enzyme was used ascorbic acid which absorbs light well at a wavelength of 265 nm, and the activity of the enzyme is determined by a decrease in the optical density of the sample. Thus, the degree of oxidation of ascorbic acid correlates with the amount of enzyme. Phosphate buffer (pH 7.3-7.4), 10-3M MgSO4 and 10-5M ascorbic acid solutions were used to measure the activity of the enzyme by spectroscopic method. In the three-leaf stage of cotton, 1 ± 0.001 g of leaves are taken from each variant, washed thoroughly in distilled water, and then carefully crushed in a mortar set. The obtained homogenate is dissolved in a 50 ml flask in a cooled phosphate buffer (25 ml). The extract is precipitated in a centrifuge (9000 rpm) for 10 minutes. The test tub is filled with 1 ml of MgSO4, 2 ml of phosphate buffer, 0.1 ml of filtrate and 0.7 ml of ascorbic acid. Another tub (control) is filled with the same amount of these components, but when ice is added 0.7 ml of H2O instead of ascorbic acid. For each tub, the measuring device takes a zero starting position. At the beginning of the measurement, the stopwatch is activated. The first measurement is performed after 30 seconds, then the measurement is performed after each minute and continues for 5-6 minutes. The activity of the enzyme (A) is determined by the following formula:
Di is the optical density of the solution at the beginning of the experiment (in the first measurement), D2 is the optical density of the solution at the end of the experiment, t1 and t2 are the time at the beginning and end of the experiment, H is the mass of the sample (g), V is the mass of enzyme extract (ml), required volume for experiments (ml). For determining the activity of the enzyme ascorbate peroxidase was also used to determine the activity of polyphenol oxidase and quiacol peroxidase enzymes by spectrophotometric method. In these experiments, 0.5 g of leaf material and 10 ml of 0.06 M phosphate buffer (pH 7.2) were used. 0.02% polyphenol oxidase and diethylparaphenylenediamine solutions were used as determinants [1]. Fluorescence spectra were recorded on a spectrophotometer (Cary Eclipse, Varian, Austrian). Cary Eclipse is an optical device for measuring fluorescence, phosphorescence, chemical and bioluminescence modes, and for measuring kinetic processes. The device uses a xenon lamp as a light source. The fluorometer can record the light rays emitted by the samples in 4 modes. The high scanning speed of the device (24,000 mm / inch) allows you to get the full spectrum in 3 minutes. The excitation and emission spectra are in the range of 200-900 nm for leaves.
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Figure 1. Germination of cotton seeds in vegetation containers (seeds with ordinary and nanoparticles on the right), seedlings on the left with A-variant not treated with nanoparticles, and B-variant with seeds treated with Al nanoparticles.
Determination of salinity and pH of soil samples was carried out in the laboratory of mineral fertilizers of the Institute of Soil Science and Agrochemistry of ANAS by the method of electrical conductivity (EC meter) [3]. Soil samples were taken from different regions of the Mugan Plain. Cotton seeds were planted in vegetation containers in soil samples with different salinity. The seeds of Ganja-110 cotton variety in 4 variants with 10 seeds in each vegetation pot were grown in a laboratory phytatron (plant growing chamber) with automatic temperature, light and humidity control.
Results of experiments. Investigation of the kinetics of changes in chlorophyll pigment in leaves by fluorescence spectra. The salinity of the soil samples used in the experiments was 2.32 dS / m for soil sampl I, 0.44 dS / m for soil sampl II, 0.56 dS / m for soil sampl V and 1.10 dS / m for soil sampl VI. In all samples, the pH ranged from 6.5 to 7. Cotton seeds were pre-treated (coated) with Al nanoparticles and in the control variant were planted in unprocessed form in vegetation containers. In the 3 leaf stages of cotton seedlings, 3 mm wide and 1 cm long sections were taken from the leaves and kept in the dark for 1 hour. Fluorescence spectra were recorded. The main purpose of the experiments was to determine the effect of Al nanoparticles on the chlorophyll (Chla and Chlb) content of cotton seedlings growing in saline and fertile soils. In vivo, two maxima are observed in the fluorescence spectra of chlorophyll at room temperature: the wavelength in the red region is 689 nm, which is irradiated by Photosystem II (PSII), and the second is 720-740 nm in the far red region irradiated by Photosystem I (PSI). In the experiments, the change in the ratio of F689 / F740 was taken as an indicator of changes in the amount of chlorophyll. It was found that the change in the amount of chlorophyll is inversely proportional to the change in the ratio of F685 / F740 [12,18]. By drawing fluorescence spectra and calculating the ratio of these maxima, it is possible to study the kinetics of changes in the amount of chlorophyll in the leaves of plants of one or another stress factor. Therefore, fluorescence spectra of seedlings obtained from seeds treated with Al nanoparticles were compared in both saline and fertile soils. Figure 2 shows the fluorescence spectrum (curve 2) of seeds treated with Al nanoparticles (pot I) in soil sample I and the fluorescence spectrum (curve1 ) of seedlings treated with Al nanoparticles in seed soil sample I (pot 1). Figure 3 shows the fluorescence spectra of plants grown in soil sample II, figure 4 in soil sample V, and Figure 5 in soil sample VI. The results of the experiments showed that in the soil sample with salinity (soil I), the intensity of the maximums in the seedlings of seeds treated with Al nanoparticles was slightly reduced compared to the control variant. The ratio of the maximum fluorescence spectra was F683/732 = 1.58, and in the sprouts of seeds not treated with nanoparticles was F683/732 = 1.64. A decrease in this ratio indicates that Al nanoparticles have increased the amount of chlorophyll in the saline soil sample. In the relatively low salinity soil sample (soil VI), F684/732 = 1.78 in the seedlings treated with Al nanoparticles and F684 / 732 = 1.84 in the control variant.
650 700 750 000
Wavelength (nm)
Figure 2. Fluorescence spectrum of seedlings of treated seeds with Al nanoparticles (I pot) (curve 2) and fluorescence spectrum of seedlings of seeds not treated with Al nanoparticles (KI pot) (curve
1)
Wavelength (nm)
Figure 3. Fluorescence spectrum of seedlings of treated seeds with Al nanoparticles (II vessel) (curve 1) and fluorescence spectrum of seedlings of seeds not treated with Al nanoparticles (KII
container) (2 curves).
In this variant, the ratio of F684/732 is also reduced, which indicates that the Al nanoparticles also increased the amount of chlorophyll in the weakly saline soil sample. However, the ratio of F684 / 732 in soil samples II and soil V in soil samples that are not saline and considered fertile has increased. Thus, the ratio of F684/732 in soil KII was 1.8, in soil II 2.0, in soil KV 1.6, in soli V 1.88. The results showed that Al nanoparticles reduce the amount of chlorophyll in soil samples where salinization is not observed.
Figure 4. Fluorescence spectrum of seedlings of treated seeds with Al nanoparticles (V pot) (curve 2) and fluorescence spectrum of seedlings of seeds not treated with Al nanoparticles (KV pot)
(curve 1)
Studies show that chlorophyll in plants decreases during salt stress. For example, chlorophyll levels have even decreased by 52% with increasing concentrations of NaCl in different types of beans [29]. Turan et al. (2007) in P. vulgaris L [31] and Taffouo et al. (2010) observed in Vigna subterranean L [30]. In our experiments, when cotton seeds are treated with Al nanoparticles, the amount of chlorophyll in saline soils increases.
Figure 5. Fluorescence spectrum of seedlings treated seeds with Al nanoparticles (VI pot) (curve 2) and fluorescence spectrum of seedlings of seeds not treated with Al nanoparticles (KVI pot) (curve 1).
Determination of the activity of ascorbate peroxidase, polyphenol oxidase and quiacol peroxidase enzymes. In our experiments, the activity of 3 enzymes - ascorbate peroxidase, polyphenol oxidase and quaiacol peroxidase was also measured in the variants with fluorescence spectra. Ascorbate peroxidase is a key enzyme in the ascorbate-glutation cycle of H2O2 detoxification in chloroplasts [6]. This enzyme and ascorbate-glutation system plays an important role in the quenching of active oxygen radicals not only in the chloroplasts, but also in the cytoplasm, mitochondria and perexi somes [24].
Figure 6. Enzymes activity in the leaves of seeds of treated seedswith Al nanoparticles (TI, TII, TVI variants) and in the leaves of seedlings of seeds not treated with Al nanoparticles (TKI, TKII, TKVI).
It was found that the activity of these enzymes increases under the influence of environmental stressors [28]. In our experiments, when seeds are treated with Al nanoparticles in soils with relatively high salinity (2.32 dS / m), the activity of the ascorbate peroxidase enzyme in the leaves of seedlings decreases, and in relatively weakly saline soils it doubles (1.10 dS / m). The activity of the polyphenol oxidase enzyme decreases, albeit slightly, in all three of these soil samples. The activity of the enzyme quaiacol peroxidase also decreases in saline soils (2.32 dS / m), but increases significantly in relatively weakly saline soils (1.10 dS / m). The results of the experiments are shown in Figure 6. Thus, it became clear from the results of the experiments that when cotton seeds are treated (covered) with Al nanoparticles, they grow well in saline soils. Significant changes occur in its development and in the kinetics of physiological processes. One of them is the increase in the amount of chlorophyll a and b pigments in the leaves of cotton seedlings (mainly in the 3-leaf stage). One of the interesting results is the change in the activity of enzymes. Thus, under the influence of stress factors (in salt stress) the activity of basic enzymes, such as ascorbate peroxidase, increases, but when they are treated with Al nanoparticles, the activity of this enzyme decreases in cotton leaves grown in saline soils. This decrease is negligible in polyphenol oxidase, but noticeable in quaiacol peroxidase enzyme.
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