Microgravity Induces Interplay Between Atg8, Tua and Tub Gene Expression in Arabidopsis Thaliana

Abstract

Autophagy plays a direct or indirect roles in plant growth and development, pathogen invasion and immunity. It is involved in the control of cell homeostasis and modulates plant response and adaptation to various abiotic stress stimuli. The formation and motility of autophagosomes require microtubule dynamics. The manner in which autophagy can be induced by microgravity in plants has not been previously studied. Investigating how plants can cope with the absence of gravity is a critical task in space biology, since growing plants as a food source on the space station is in particular demand for the future. The present study demonstrates and confirms that microgravity affects the growth and development of plants. Using different GFP-expressing Arabidopsis thaliana lines (GFP-ATG8 and GFP-MAP4), laser scanning confocal microscopy and gene expression analysis of 6 α-tubulin (tua), 9 β-tubulin (tub) and 9 atg8 genes, we can conclude that main microgravity-induced stress response in plants is observed during the first 6-9 days of the microgravity experiments. It is shown that the morphological changes found in plants are directly related to changes in the organization and dynamics of microtubules. It was found that in root cells microtubules become disorganized after 6 days of growing plants under microgravity conditions, which leads to visible morphological changes in the plants. However, after 9-12 days of the experiment no dramatic disturbances in the orientation and organization of MTs were identified. The results obtained using LysoTracker™ Red and A. thaliana transgenic line (GFP-ATG8a) in both cases showed induction of autophagy and maximum level of autophagosome formation in plant root cells after 6 days of plant growth, and then a gradual decrease of autophagosomes in the same cells after 9 and 12 days under microgravity. The results of transcriptome analysis of tubulin and atg8 genes reveal connection between development of autophagy and functioning of microtubules. In particular, the most pronounced increase of expression in response to microgravity exposure was observed of tua2, tua3, tub2, tub3 and atg8i genes. It should be noted that the main responses to stress were established after 6-9 days of exposure to microgravitation, while after 12 days of the experiment, an adaptive response of plants to microgravity-induced stress was observed. The findings provide a basis for further studies of cellular mechanisms of autophagy and their interaction with cytoskeletal structures, in particular microtubules, to enhance plant adaptation to microgravity conditions

Elucidation of Cellular Mechanisms of Autophagy Involvement in Plant Adaptation to Microgravity Conditions

“…As a result of the analysis of the expression of 6 αand 9 β-tubulin genes, an increase in the expression levels of tua2, tua3, tua4, tua6 genes and a decrease in expression of tua4, tua5 genes on the 6 th and 9 th days of cultivation were found (Table 1). On the 12 th day of plant growing under clinostating conditions, none of the six studied genes showed an increase in the level of expression (Table 1) [25]. The tua1 gene is probably not involved in autophagy, as its expression level was consistently low during long-term cultivation.…”

“…Analysis of root apex tissues using two autophagosome markers -monodansyl cadaverine and Lyso-Tracker™ Red -showed that the density of autophagosomes in the root apices of clinostated plants was significantly higher than in the control [24,25,28]. The dynamic of the formation of autophagosomes in the root tissues of control and experimental plants was different.…”

“…The influence of simulated microgravity on the transcriptional activity of αand β-tubulin genes. The transcriptional activity of genes involved in the implementation of stress-induced autophagy, in particular the αand β-tubulin genes of A. thaliana under microgravity conditions was present in [25]. As a result of the analysis of the expression of 6 αand 9 β-tubulin genes, an increase in the expression levels of tua2, tua3, tua4, tua6 genes and a decrease in expression of tua4, tua5 genes on the 6 th and 9 th days of cultivation were found (Table 1).…”

“…Joint expression (coexpressions) of studied genes are highlighted in gray bules in the development of stress-induced autophagy, and provide an opportunity to identify genes specific to stress induced by simulated microgravity. In particular, it was established that certain tua, tub and atg8 genes can be considered as key elements of plant adaptation to prolonged exposure to microgravity [25]. Thus, data on the expression of plant tubulin genes under clinostating were obtained for the first time, that provides a basis for the development of approaches to increase the adaptation of plants during their growing under the influence of such stress.…”