Elucidation of cellular mechanisms of autophagy involvement in plant adaptation to microgravity conditions

Heading: 
Space Life Sciences
1Yemets, AI, 1Plokhovska, SH, 1Shadrina, RYu., 1Kravets, OA, 1Blume, Ya.B
1Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine, Kyiv, Ukraine
Space Sci. & Technol. 2023, 29 ;(2):22-31
https://doi.org/10.15407/knit2023.02.022
Publication Language: English
Abstract: 
It was shown that clinostating conditions induce autophagy without increasing of programmed cell death (PCD) index in the epidermal cells of the root apex of A. thaliana seedlings. After the phase of activation of autophagy, its regulatory weakening occurs, which probably indicates adaptive changes to the conditions of clinostating. The induction of autophagy correlates with an increase in the expression levels of atg8 genes, some of which (atg8e and atg8i) may be involved in the implementation of autophagy under the microgravity condition. The transcriptional activity of cytoskeleton genes involved in the implementation of stress-induced autophagy, in particular α- and β-tubulin genes, was analyzed. Joint coexpression of α- and β-tubulin genes and atg8 under microgravity conditions was revealed. These results illustrate the role of the cytoskeleton in the development of microgravity-induced autophagy and make it possible to identify genes specific to this type of stress.
            The induction of autophagy and PСD was studied under the action of gamma- irradiation as a concomitant microgravity factor of space flights, as well as under the combined action of acute irradiation and clinostating. Gamma-irradiation in doses equivalent to those in the spacecraft cabin (1 - 6 Gy) induced dose-dependent changes in the topology and cytogenetic state of the root apical meristem, as well as slightly inhibited of the early plant development. In the meristem, heterogeneity increased, PCD indexes, mainly proliferative death and autophagy, increased. With the combined action of gamma irradiation (2 Gy) and clinostating, the density of autophagosomes in the epidermal cell root apices of 6-day-old seedlings increased (24 hours after irradiation), and after 4 days it decreased, compared to the non-irradiated control.
            Treatment of seeds of A. thaliana with a NO donor had a stimulating effect on plant development, increased the content of endogenous NO in root tissues and the resistance of plants to clinostating. Under clinostating conditions, compared to the control, the optimum concentration of NO decreased, possibly due to the contribution of NO to the generation of ROS. The negative effect of NO scavenger on seedling growth was enhanced by clinostating, including increased accumulation of autophagosomes in epidermal cells. These data indicate that endogenous NO content is an important component of intracellular signaling mechanisms involved in the response of plant cells to simulated microgravity, including autophagy induction mechanisms. The obtained data deepen the understanding of the molecular mechanisms of the development of stress-induced autophagy, in particular the involvement of different isotopes of ATG8 proteins and their interaction with α- and β-tubulins, as well as other molecular components involved in the induction of autophagy, and will be the basis for the development of approaches to increase stress resistance and adaptation of plants to the conditions of long space flights. 
Keywords: adaptation, Arabidopsis thaliana, atg8 genes, autophagy, clinostating, gamma-irradiation, gene expression of α- and β-tubulins, nitric oxide
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References: 
1. Aubry-Hivet D., Nziengui H., Rapp K., et al. (2014). Analysis of gene expression during parabolic flights reveals distinct early gravity responses in Arabidopsis roots. Plant Biol., 16, № 1, 129—141.
2. Blume Ya. B., Plokhovska S. H., Shadrina R. Yu., et al. (2022). The role of nitric oxide in Arabidopsis thaliana response to simulated microgravity and the participation of autophagy in the mediation of this process. 44th COSPAR Scientific Assembly (Athens, Greece, July 16—24, 2022): Book of Abstrs.
3. Cao Y., Fan X., Shen Z., et al. (2007). Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity. Cell Biol. Int., 31, № 1, 24—29.
4. Cassia R., Amenta M., Fernández M. B., et al. (2019). The role of nitric oxide in the antioxidant defense of plants exposed to UV-B radiation. Reactive Oxygen, Nitrogen and Sulfur Species in Plants. Eds M. Hasanuzzaman, V. Fotopoulos, K. Nahar, M. Fujita. Wiley-Blackwell, 555—572.
5. Correa Aragunde M. N., Foresi N. P., Lamattina L. (2015). Nitric oxide is a ubiquitous signal for maintaining redox balance in plant cells: regulation of ascorbate peroxidase as a case study. J. Exp. Bot., 66, № 10, 2913—2921.
https://doi.org/10.1093/jxb/erv073. 6. Correll M. J., Pyle T. P., Millar K. D. L., et al. (2013). Transcriptome analyses of Arabidopsis thaliana seedlings grown in space: implications for gravity-responsive genes. Planta, 238, 519—533.
7. Fancy N. N., Bahlmann A-K., Loake G. J. (2017). Nitric oxide function in plant abiotic stress. Plant Cell. Environ., 40, № 4, 462—472.
8. Hu X., Neill S. J., Tang Z., Cai W. (2005). Nitric oxide mediates gravitropic bending in soybean roots. Plant Physiol., 137, № 2, 663—670.
9. Khan M., Al Azawi T. N. I., Pande A., et al. (2021). The role of nitric oxide-induced ATILL6 in growth and disease resistance in Arabidopsis thaliana. Front. Plant Sci., 12, 685156.
10. Kolesnikov Y. S., Kretynin S. V., Volotovsky I. D., et al. (2016). Molecular mechanisms of gravity perception and signal transduction in plants. Protoplasma, 253, № 4, 987—1004.
11. Kordyum E. L. (1994). Effects of altered gravity on plant cell processes: results of recent space and clinostatic experiments. Adv. Space Res., 14, № 8, 77—85.
12. Kozeko L. Ye., Buy D. D., Pirko Ya. V., Blume Ya. B., Kordyum E. L. (2018). Clinorotation affects induction of the heat shock response in Arabidopsis thaliana seedlings. Gravit. Space Res., 6, № 1, 2—9.
13. Kravets O. A., Berezhna V. V., Sakada V. I., et al. (2016). Influence clinostating on structure, growth activity and attracting of apical meristem of bean plants. 16th Ukrainian Conference on Space Research (Odesa, Ukraine, August 22—27, 2016): Book of Abstrs, 66.
14. Kravets O. A., Horyunova I. I., Plokhovskaya S. G., et al. (2017). Development of adaptive processes to conditions of modified gravitation at tissue and cellular levels in plants. 17th Ukrainian Conference on Space Research (Odesa, Ukraine, August 21—25, 2017): Book of Abstrs, 60.
15. Kravets O .A., Yemets A. I., Horyunova I. I., Plokhovska S. G., Olenieva V. D., Lytvyn D. І., Spivak S. I., Blume Ya. B. (2018). Investigation of plant cell and tissue adaptive mechanisms to modified microgravity. Space Research in Ukraine. Ed. by O. P. Fedorov. Kyiv: ICD NAS of Ukraine and SCA of Ukraine, 66—72. ISBN 978-966-02-8590-3.
16. Lytvyn D. I., Olenieva V. D., Yemets A. I., Blume Ya. B. (2018). Histochemical analysis of tissue-specific acetylation of α-tubulin as a response for autophagy development in Arabidopsis thaliana induced by different stress factors. Cytol. Genet., 52, № 4, 245—252.
17. Mohd Amnan M. A., Pua T. L., Lau S. E., et al. (2021). Osmotic stress in banana is relieved by exogenousnitric oxide. Peer J., 9, e10879. https://doi.org/10.7717/peerj.10879.
18. Olenieva V. D., Lytvyn D. I., Yemets A. I., Blume Ya. B. (2018). Expression of kinesins involved in the development of autophagy in Arabidopsis thaliana and the contribution of tubulin acetylation to the interaction of ATG8 protein with microtubules. Factors Exp. Evolut. Organisms, 22, 162—168 [in Ukrainian].
19. Olenieva V. D., Lytvyn D. I., Yemets A. I., Blume Ya. B. (2018). Effect of UV-B on the transcriptional profiles of genes of major proteins involved in the development of autophagy with the participation of microtubules. Reports Nat. Acad. Sci. Ukraine, № 1, 100—108.
20. Olenieva V. D., Lytvyn D. I., Yemets A. I., Blume Ya. B. (2018). Influence of sucrose starvation, osmotic and salt stresses on expression profiles of genes involved in the development of autophagy by means of microtubules. Bull. Ukr. Soc. Geneticists and Breeders, 16, № 2, 174—180.
21. Olenieva V., Lytvyn D., Yemets A., Bergounioux C., Blume Ya. (2019). Tubulin acetylation accompanies autophagy development induced by different abiotic stimuli in Arabidopsis thaliana. Cell. Biol. Int., 43, 1056—1064.
22. Paul A. L., Zupanska A. K., Schultz E. R., Ferl R. J. (2013). Organ-specific remodeling of the Arabidopsis transcriptome in response to spaceflight. BMC Plant Biol., 13, 112.
23. Plohovska S. H., Krasylenko Y. A., Yemets A. I. (2019). Nitric oxide modulates actin filament organization in Arabidopsis thaliana primary root cells at low temperatures. Cell. Biol. Int., 43, № 9, 1020—1030.
24. Plokhovska S. H., Shadrina R. Yu., Kravets O. A., Yemets A. I., Blume Ya. B. (2022). The role of nitric oxide in the Arabidopsis thaliana response to simulated microgravity and the involvement of autophagy in this process. Cytol Genet., 56, № 3, 244—252.
25. Shadrina R., Yemets A., Plokhovska S., Blume Ya. (2023). Microgravity induces interplay between atg8, tua and tub gene expression in Arabidopsis thaliana. Preprint available at Research Square.
26. Shadrina R. Yu., Yemets A. I., Blume Ya. B. (2019). Development of autophagy as an adaptive response of Arabidopsis thaliana plants to microgravity conditions. Factors Exp. Evol. Organisms, 25, 327—332.
27. Sun H., Feng F., Liu J, Zhao Q. (2018). Nitric oxide affects rice root growth by regulating auxin transport under nitrate suppl. Front Plant Sci., 9, 659.
28. Yemets A. I., Shadrina R. Yu., Horiunova I. I., et al. (2021). Development of autophagy in plant cells under microgravity: the role of microtubules and atg8 family proteins in autophagosome formation. Space Research in Ukraine. 2018—2020. Ed. O. Fedorov. Kyiv: Akademperiodyka, 79—84.
29. Yun B. W., Skelly M. J., Yin M., et al. (2016). Nitric oxide and S-nitrosoglutathione function additively during plant immunity. New Phytol., 211, № 2, 516—526.
30. Zhan N., Wang C., Chen L., et al. (2018). S-nitrosylation targets GSNO reductase for selective autophagy during hypoxia responses in plants. Mol. Cell., 71, № 1, 142—154.