Shadrina R. The role of autophagy in the response of Arabidopsis thaliana to microgravity conditions and the participation of the microtubules in mediating this process

The research is dedicated to the analysis of autophagy processes in Arabidopsis thaliana plants under the influence of microgravity, with a particular focus on the role of microtubules in this process. Autophagy, as an intracellular mechanism of degradation and recycling of macromolecules and organelles, is a key factor in the adaptation of plants to various stress conditions. Currently, studying the physiological role of autophagy under stress is one of the actual problems of biology, as depending on the degree of cell damage, this process can either contribute to survival or cell death. Therefore, the scientific work focuses on the induction of autophagy processes under conditions of altered gravity. Studying plant adaptation to microgravity is key for developing life support systems in space. Recent experiments have shown that Arabidopsis thaliana and other species can develop and yield seeds in space conditions, however, they undergo molecular changes. Researching plant adaptation involving autophagy in simulated space conditions is extremely important, as it allows creating a foundation for further space research in plant cultivation, and subsequently food production in altered gravity conditions. A. thaliana plants of the Columbia Col-0 ecotype and two transgenic lines of A. thaliana were used as research material: GFP-ATG8a and GFP-MAP4. Stress conditions were modeled using a clinostat on which the plants were grown (4 rpm rotation mode). The use of a clinostat allows to simulate microgravity conditions, which is important for the study of plant adaptation mechanisms. Autophagosomes were visualized using laser confocal microscopy. This method allows to obtain detailed images of intracellular structures and processes, which is key for the analysis of autophagy. Molecular genetic methods were used to evaluate the gene expression of ATG, α- and β-tubulin proteins in the studied plants. These methods allow us to quantify gene expression levels and identify changes that occur under the influence of microgravity. It was analyzed the effect of microgravity on the growth and elongation of A. thaliana seedlings. As a result of our studies, we found that wedge-statement did not affect seed germination and did not cause significant deviations in the morphology of seedling shoots. Delayed shoot development was observed only on the 6th and 9th day of wedging, and on the 12th day the shoots were practically morphologically indistinguishable from control plants. Also, 12-day-old seedlings had a regular leaf rosette consisting of 4-6 oval green leaves. At the same time, seedlings growing under experimental conditions, unlike the control, had disoriented root growth, which was a consequence of a constant change in the gravity vector. The analysis of the morphology and development of the main roots of seedlings revealed differences in their growth zones. In particular, the tensile and transitional zones were shorter under wedging conditions compared to control plants. Quantitative analysis of the average area of epidermal cells in the transition zone showed insignificant differences (29 % less) in the size of the treated plants compared to the control. The gravitropic slope angle (GSA) was also analyzed. The results showed a significant increase in GSA in A. thaliana seedlings under wedge conditions, indicating a deviation from the normal vertical root orientation, as in the control. The development of stress-induced autophagy at the morphological level was studied by fluorescence and confocal laser scanning microscopy. The characteristic signs of autophagy development under the influence of microgravity were revealed, in particular, an increase in the number of autophagosomes in root cells. Using the fluorescent dye MDC, the appearance of structures ranging in size from 3 to 20 μm was detected, indicating the active development of autophagy under the influence of stressful microgravity conditions. The results obtained using the LTR dye and the transgenic line of A. thaliana (GFP-ATG8a) in both cases showed the induction of autophagy and the maximum level of autophagosome formation in the epidermal cells of the root transition zone of A. thaliana plants after 6 days of cultivation. The number of autophagosomes was counted using the transgenic line of A. thaliana GFP-ATG8a. The results showed a significant increase in the number of autophagosomes in cells exposed to microgravity, indicating the activation of autophagy as a defense mechanism. On the 6th day of cultivation, the most significant change in the number of autophagosomes was observed (more than 2-fold) compared to the control, although on the 9th day the number of autophagosomes decreased in the cells of the root transition zone, they were still more than in the control plants. On the 12th day of cultivation, the number of autophagosomes in the cells of plant roots under conditions of clonostatization was similar to that of control plants.