Microtubules (MTs) play an important role in the regulation of autophagy development in yeast and animal as well as in plant cells. MTs participate in maturation and traffic of autophagosomes through their dynamic state changes and post-translational modifications of tubulin, namely acetylation. We subjected Arabidopsis thaliana seedlings to metabolic-, salt-, osmotic stresses as well as irradiation of ultraviolet B and investigated the involvement of plant MTs in the development of stress-induced autophagy via tubulin acetylation. For this purpose Arabidopsis thaliana line expressing autophagy-related protein 8 h (atg8h)-GFP was generated to investigate autophagy, applying the level of free GFP as an indicator of autophagy development. Using autophagosome confocal imaging and Western blot analysis of Atg8 post-translational lipidation and synchronous GFP release it was shown that all examined stressful stimuli led to pronounced development of autophagy, particularly in different root tissues. Moreover, autophagy development was accompanied by α-tubulin acetylation under all stressful conditions. Presented data indicate the possible role of the post-translational acetylation of α-tubulin in the mediation of plant stress-induced autophagy.

Проведено транскрипційний аналіз основних генів, залучених до реалізації аутофагії, індукованої опроміненням ультрафіолетом В (УФ-В) у клітинах Arabidopsis thaliana. Зміни рівня експресії більшості ізотипів α-ту буліну та atg8 мали чітко визначений дозо-та часозалежний характер. Істотне підвищення рівня експресії генів tub4 і atg8a через 3 та 24 год після опромінення в обох дозах опосередковано свідчить про наявність взаємозв'язку між структурними одиницями аутофагосом і мікротрубочками. Отримані профілі експресії генів elp3/деацетилаз та гексокіназ вказують на роль ацетилювання α-тубуліну як регуляторної модифікації, необхідної для здійснення аутофагічної відповіді-етапу, що передує розвитку програмованої клітинної загибелі.

Aim. The aim of this work was to investigate changes in expression profiles of key genes involved in the development of autophagy by means of microtubules under the influence of sucrose starvation, osmotic and salt stresses. Methods. Arabidopsis thaliana seeds were sown aseptically on Murashige and Skoog solid medium. Salt and osmotic stresses were simulated by seed germination and seedlings cultivation on the media containing 150 mM NaCl and 10 mM mannitol, respectively. For investigation of starvation-induced autophagy plants were germinated and grown on sucrose-free medium. Results. Changes in expression of α-tubulin and atg8 genes had clearly defined stressdependent nature. Overexpression of tua1 and atg8e under starvation; tua3 and atg8f under osmotic stress; tua3 and atg8f, atg8e during salt stress indirectly testifies interaction between the structural units of autophagosomes and microtubules. It was shown that influence of investigated abiotic stimuli results in overexpression of elp3 and hda6 genes. Small increase in expression levels of hexokinase 2 and 3 was demonstrated. Conclusions. Transcriptome analysis of key genes involved in realization of autophagy induced by sucrose starvation, osmotic and salt stresses in Arabidopsis thaliana cells was conducted. Received data indirectly testifies interaction between the structural units of autophagosomes and microtubules and enables to point α-tubulin and atg8 genes, which are specific for the realization of autophagy induced by a certain abiotic stimuli. Expression profiles of elp3/deacetylases as well as hexokinases indicate the critical role of α-tubulin acetylation for autophagic response, that is involved in the development of programmed cell death.Keywords: autophagy, sucrose starvation, osmotic stress, salt stress, transcriptome analysis, α-tubulin, atg8.

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