The aim of this research was to investigate the influence of different concentrations of salt (NaCl) on the pigments and oxidative processes in the plant leaves. The object of research: Marsh Pennywort (Hydrocotyle vulgaris) leafs. Have been applied in practice the common information on the main processes associated with adaptation of plant cells, the effects of stressors on the factors that affect the homeostasis of the plant, the stability of its development, the ability to adapt and survive in different environments and under different environmental factors.
Key words: Hydrocotyle vulgaris, allozyme, Marsh Pennywort, morphological characteristics, protein electrophoresis, spectrophotometry, NaCl.
Urban development can cause environmental problems associated with local increase of negative human impacts, including increased use of salt antifreeze in the winter on the ecological state of urban soils. In turn, soil salinity can lead to negative effects on the root system of plants, cause deterioration of plant growth and development, and prolonged exposure to high concentrations of sodium chloride, can lead to irreversible physiological changes of plants and loss of the whole plant. A similar situation can be observed with plants germinating on the coast and in coastal areas, especially in times of flooding. Thus, the development of a system for studying variations in responses of various plants on the stress factor, can give an answer on how quickly plants can adapt to the changing environment and to identify patterns, how quickly the plant is able to respond to external stress factors. The model experiments showed that the typical study of soil salinity (high concentrations of sodium chloride), the plants are observed changes in photosynthetic activity, changes in absorption spectrum of pigments and fluorescence emission spectrum of Hydrocotyle vulgaris leaves, growth retardation leaf plates, heavy fading and discoloration of leaves [9, 310].
Also was showed an increasing of ROS in Hydrocotyle vulgaris leaves after the gradual increasing of sodium chloride concentration in soil. Reactive oxygen species cause not only negative changes, such as, damage cellular organelles, cell membranes, etc., but also serve as secondary messengers and inducers of defence responses of cells. The impact of various adverse factors: high concentrations of salt, drought, low and high temperature, the effect of heavy metals and many others, can lead to an increased production of reactive oxygen species [4, 263].
The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state. However, more severe oxidative stress can cause cell death and even moderate oxidation can trigger apoptosis, while more intense stresses may cause necrosis. Understanding these issues is essential to address the ecological and physiological problems that are priorities for plant physiology [2, 407].
MATERIAL AND METHODS
For a series of experiments was grown plant Hydrocotyle vulgaris in the laboratory in plastic containers in vitro. The depth of the container was 12 cm, Ph of the soil varied between ≈ 5,4 - 6,3 for feeding the plants in the soil was added to the complex fertilizer NPK VITO. Plants were grown in a climate chamber, with the division of the light period on day / night; relative humidity was 75-80%. Initially, the plant material was taken from plants that grew in natural conditions and has been introduced in the laboratory. In the laboratory, were grown genetically identical, cloned plants that were growing in separate plastic pots with drainage system.
Plants were grown in a climate chamber, with photoperiodicity 14 hours, the average temperature was ≈ 22 + / - 2 C. Watering place 3 times a week. Tap water was taken with an admixture of different salt concentrations or no added salt, depending on the experimental container.
Salt concentration in the Petri dish was: 0,0; 0,025; 0,05; 0,1; 0,5 1,0 M. For this study we used leaves of plants grown in a Petri dish with the addition of a solution Hoagland's NO.2 Basal salt mixture, hygroscopic 1.6g / l. And throughout the experiment were exposed in a climate chamber. All 6 samples were grown under identical conditions: temperature, humidity, illumination. Leaves were analyzed each week for 4 weeks. For analysis were taken on 6 sheets at each concentration. Scans were taken using a microscope to Carl Zeiss: Axioskop 40 Axioskop 40, processed using a computer program Ocean Optics. Analysis of pigments was carried out using a spectrophotometer SP8-400UV-VIS spectrophotometer. Were also analyzed changes in the number of active oxygen in the leaves of plants and to determine the activity of enzyme systems Catalase and Superoxide dismutases (SOD).
- Results and discussion
During the emission spectra were obtained by fluorescence leaves Hydrocotyle vulgaris, which were influenced by different salt concentrations, were calculated and statistically processed by the parameters of the fluorescence spectra of cells. A critical concentration of salt in a medium that negatively affect the growth of leaf plates, the formation of chlorophyll a, b, and the relationship between the formation of carotenoids and chlorophyll at different intensity of salt exposure. The change in chlorophyll a, b. observed a gradual decrease of chlorophyll as a function of increasing salt concentration in the Petri dish. However, while reducing the chlorophyll gradual increase in the amount of carotenoids, as a function of increasing salt concentration in the culture medium.
A fluorescence cell reflects changes in all its components in the form of fluorescent emission spectra. Using a spectrometer in combination with a fluorescent microscope made it possible to observe the quantitative change in the fluorescence spectra of the whole body of the plant, which in turn reflect the change processes in the plant as a whole. Fluorescence method is more sensitive in the early stages of determining the changes in cells and body as a whole. [6, 55]
We observed the appearance of new peaks, while observed the destruction of chlorophyll, according to our assumptions, this peak corresponds to the fluorescence of nicotine amide adenine dinucleotide phosphate (NADPH, NADP) - which is included in the protection of chloroplasts under various stresses, including oxidative and NaCl salt, it is also possible that the emergence of the second peak is the formation of phenolic compounds, which may also participate in defense reactions when exposed to various stressors in general. First, the high peak of fluorescence corresponds to peaks of chlorophyll fluorescence, which gradually decreased, whereas at high concentrations of NaCl in the nutrient solution, the decline was more intense [13, 615].
After 2 weeks of exposure the plants in a climate chamber with the addition of saline solution into the soil (concentration of 0,1 and 0,5 M), significant changes in the activity of the enzyme system Catalase and SOD compared with the control was observed. At this stage the toxic effects of NaCl salt has an impact on the root system of plants [12, 1105]. Measure the dynamic changes in formation of active oxygen in the samples of leaves grown in Petri dishes showed that the number of active oxygen increased in the samples grown on the substrate with a high concentration of NaCl.
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