Complex ecological, physiological and genetic analysis of the response of Cucurbita genus representatives to salt stress

Abstract

Soil salinization is a characteristic feature of many natural ecosystems. It also covers about 20% of the arable land of the planet. Currently, salinization, the territory of which is constantly growing, is one of the powerful factors limiting the sustainable development and modernization of world agriculture. Therefore, studies aimed at elucidating the mechanisms of response and adaptation of plant organisms to salt stress are becoming more and more important. First, they should cover the leading agricultural crops, including members of the Cucurbitaceae family, which are widely cultivated in the world for their economic and ecological properties. Cucurbitaceae are not halophytes, most of them are sensitive to salt stress and usually show obvious inhibition of growth in conditions where the NaCl content in the soil solution reaches 0.1%. Taking into account the above, the question of clarifying the ecological and physiological aspects of the formation of salt tolerance in representatives of the Cucurbitaceae family and the actual genus Cucurbita and managing this trait on the basis of the use of both classical and modern scientific and technical means is gaining relevance. Taking into account the above, the issue of clarifying the ecological-physiological, genetic aspects of the response to salt stress of representatives of the Cucurbitaceae family and the actual genus Cucurbita, the formation of their salt resistance, becomes relevant and managing this trait on the basis of the use of both classical and modern scientific and technical means. The aim of the work was determined: to establish the mechanisms of response to salt stress and adaptation to it, which are implemented by representatives of the genus Cucurbita at different levels of the organization, as well as eco-physiological aspects of the formation of salt resistance, quantitative and qualitative characteristics of plants when using grafting technology. The work was performed on the basis of the application of theoretical (analysis, synthesis, explanation, generalization, a mathematical and statistical) and empirical (experiment, measurement, comparison) research methods. Experimental field and laboratory ecological-physiological and laboratory genetic studies were carried out, chemical, spectrometric, fluorescent, morphometric, and vitality analyses were applied, and among mathematical and statistical calculations, point estimation of statistical series and dispersion analysis were used. In studies aimed at establishing the effect of salt stress on the morphological characteristics, vitality, and degree of damage to pumpkin plants, as well as on the exchange of organic and inorganic compounds, the flow of leading physiological processes, the following experimental scheme was used: control (water, 0 mmol/L NaCl), low salt stress (60 mmol/L NaCl), high salt stress (120 mmol/L NaCl). Pumpkins of two varieties were used in the study: Yanzhen and Miben. When studying the eco-physiological interactions that occur between plants when grafting technology is used, pumpkin plants that were subjected to salt stress were grown, followed by their use as rootstocks for watermelon. The study of the interactions formed when using salt-resistant pumpkin rootstocks was supplemented by an assessment of the impact of arbuscular mycorrhiza on plant metabolism and resistance. According to the results of the study of the effect of salt stress caused by different concentrations of NaCl, the nature of the reaction of morphological features of pumpkin to salt stress was determined. It is shown that against the background of increased salt concentration, the size, and vitality of plants decrease, and a number of negative quality signs appear in them: yellowing of leaves, their twisting, etc. It has been established that changes in the dimensional parameters of plants against the background of salt stress can be an informative indicator in the study of the mechanism of salt resistance and the breeding of salt-resistant varieties of pumpkin. It is shown that plants respond to an increase in the concentration of a salt solution with a statistically significant increase in the salt damage index and the salt damage rate. The results of the research proved that the salt damage index, as a morphological index for assessing plant resistance to salt, is a simple, easy, accurate and reliable indicator for directly detecting the degree of resistance of Cucurbita genus representatives to salt stress. It has been proven that salt stress affects indicators and signs related to the course of photosynthesis and water exchange of pumpkin plants, which leads to a decrease in the following values: photosynthesis rate, stomatal size, stomatal conductance, transpiration rate, as well as changes in chlorophyll content, mainly in the direction of its increase. At the same time, the reduction of indicators that determine the amount of water loss are factors in increasing the resistance of plants to salt stress, and the indicators that determine the intensity of photosynthesis are factors in slowing down the synthesis of carbohydrates and inhibiting plant growth. The degree of variation in chlorophyll content in leaves under salt stress was related to plant variety and salt concentration. An increase in the content of MDA (malondialdehyde) and the permeability of cell membrane structures due to their peroxidation was recorded in pumpkin under salt stress. The relative permeability of the plasma membrane also increased against the background of increasing salt concentration. One of the reasons why NaCl damages the structure of the plasma membrane is the excessive accumulation of Na+ in cells: it replaces Ca2+, which can stabilize and protect the plasma membrane. It was shown that the transformations related to the metabolism of carbohydrates and amino acids in pumpkin play an important role in the complex of processes of response and adaptation to salt stress: with increasing salt concentration, the content of proline and soluble sugars increases. Soluble sugar and proline are essential substances that regulate the osmotic potential and water potential of plants under salt stress conditions. After 7-day salt stress caused by exposure to NaCl, the proline content in the leaves of pumpkin seedlings was significantly higher than in the control. It was established that salt stress affects the absolute and relative indicators of the accumulation of ions of mineral substances, as well as their distribution in plant organs. According to these characteristics, Na+, K+, Ca2+ and Mg2+ ions showed high individuality, which is also a result and evidence of their specific role both in the aspect of ensuring the reaction and adaptation of pumpkin plants to salt stress. It has been found out that in response and adaptation to salt stress in pumpkins at all levels of the organization against the background of the stated general trends, varietal features are clearly manifested. In particular, it was established that the Yanzhen mainly accumulates Na+ in the root system, while the Miben mainly accumulates in the stem. This results in the formation of differences in the varieties and in the accumulation of K+, Ca2+ and Mg2+, as well as differences in the value of the K+/Na+ ratio. In general, according to a complex of physiological and morphometric characteristics, Yanzhen shows a higher resistance to salt. Identification and analysis of the expression of the WRKY gene family in Cucurbita genus representatives under salt stress conditions was carried out. Screened 12 WRKY family transcription factors in pumpkin in response to salt stress, and analysed their phylogenetic relationships, spatio-temporal expression patterns, tissue-specific expression characteristics, and transcriptional activities under salt stress in detail. For to improve and expand research aimed at revealing the genetic aspects of the formation of salt resistance, have developed an efficient transient transformation system for the study of gene function and protein subcellular localization. Using this method, the salt tolerance gene StNHX1 was transiently overexpressed in pumpkin roots and cotyledons. The interactions that occur between the scion and the salt-resistant pumpkin rootstock when using the technology of grafting in the cultivation of gourds were studied. The influence of salt-resistant rootstocks on the metabolism of rootstocks has been clarified and proved, the feasibility of using grafting technology to increase the resistance of plants to salt stress, increase the yield of crops and obtain high-quality products has been shown. It is shown that the use of arbuscular mycorrhizal fungi in the cultivation of melon crops based on grafting technology leads to a change in plant metabolism, affects the quality of the obtained products and contributes to increasing the resistance of plants to adverse environmental factors. Scientific novelty is inherent in the research results obtained during the dissertation. For the first time, based on the results of observations during the entire growing season, the influence of salt stress on the complex of quantitative and qualitative morphological features of pumpkin plants was investigated. The features and regularities of the accumulation of ions of mineral substances in the vegetative and generative organs of pumpkins under conditions of salt stress have been established. The physiological and biochemical transformations that occur in pumpkins against the background of salt stress have been comprehensively investigated and clarified. The methodological aspects of assessing the impact of salt stress during the pumpkin ontogenesis were improved, and the method of vitality analysis was used for the first time to assess the vitality of pumpkin plants. An efficient system of temporary transformation was developed to study the function of genes and subcellular localization of protein in pumpkin. The theoretical and practical principles of using salt-resistant pumpkin rootstocks for the cultivation of melon crops have gained further development. The materials of the dissertation work are used in the educational process of the Department of Ecology and Botany of Sumy National Agrarian University. The research results were reviewed and discussed at 6 conferences of various ranks. 12 scientific works have been published based on the dissertation materials: 3 articles in journals included in the Scopus, Web of Science database, 3 articles in specialized scientific publications of Ukraine, 6 publications in conference materials.