Cytological and biochemical markers of autophagy in wheat suspension culture cells under abiotic stress

A.B. Mazina a,b*, S.A. Dmitrieva b**, A.G. Renkova b***, J.N. Valitova b****, V.R. Khabibrakhmanova b,c*****, F.V. Minibayeva a,b******,

aKazan Federal University, Kazan, 420008 Russia

bKazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center,

Russian Academy of Sciences, Kazan, 420111 Russia

cKazan National Research Technological University, Kazan, 420015 Russia

E-mail: *abmazina@gmail.com, **s_dmitrieva@list.ru, ***renkova@kibb.knc.ru, ****yulavalitova@mail.ru, *****venerakhabibrakhmanova@gmail.com, ******minibayeva@kibb.knc.ru

Received March 10, 2020

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DOI: 10.26907/2542-064X.2020.4.541-556

For citation: Mazina A.B., Dmitrieva  S.A., Renkova А.Г., Valitova J.N., Khabibrakhma­nova V.R., Minibayeva F.V. Cytological and biochemical markers of autophagy in wheat suspension culture cells under abiotic stress. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2020, vol. 162, no. 4, pp. 541–556. doi: 10.26907/2542-064X.2020.4.541-556. (In Russian)

Abstract

Autophagy is a highly conserved process of intracellular degradation of damaged, oxidized or excess macromolecules and organelles in eukaryotic cells. Under optimal conditions, the baseline level of autophagy is low. However, the activity of autophagic processes greatly increases in response to stress. In this paper, we analyzed the biochemical characteristics of autophagosomes that are formed in the wheat suspension culture cells in response to starvation and as a result of treatment with polyamine spermine. High activity of acid phosphatase and protease, the marker enzymes of lytic vesicles, and changes in the lipid composition were observed in the autophagosomes isolated from the sucrose-deficient cells. It was revealed that polyamine spermine can induce аutophagy, which was accompanied with the changes in the cellular redox status and the energy status of mitochondria. The spermine-induced autophagosomes were characterized by the high enzymatic activity of acid phosphatase and protease. The results obtained suggest the functional universality of the main biochemical markers of autophagosomes in plant cells.

Keywords: suspension culture of wheat, autophagosomes, starvation, spermine, acid phosphatase, protease

Acknowledgments. The study is performed in part within the state assignment to FRC Kazan Scientific Center, Russian Academy of Sciences (description of autophagic activity) and according to the Russian Government Program of Competitive Growth of Kazan Federal University (isolation of autophagosomes).

Autophagic processes in the wheat suspension culture cells were studied using the Triticum timopheevii (Zhuk.) Zhuk.  strain from the All-Russian Collection of Cell Cultures of Higher Plants, K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences (Moscow).

Figure Captions

Fig. 1. Visualization of autophagosomes in the wheat suspension culture cells under starvation (a–b) and in the subcellular fractions with high activity of acid phosphatase (c–f): a – control; b – starvation; c – fraction no. 6, control; d – fraction no. 6, starvation; e – fraction no. 7, control; f – fraction no. 7, starvation.

Fig. 2. Distribution of the activities of acid phosphatase (a) and protease (b) in the subcellular fractions after separation in the Percoll gradient. Gray color – activities in the control variant, white color – activities in the cells under starvation. Fraction numbers are given sequentially, from the top to the bottom of the centrifugal tube.

Fig. 3. Visualization of autophagosomes in the wheat suspension culture cells after 3 h of spermine action: a – control; b – 1 μM of spermine; c – 10 μM of spermine; d – 100 μM of spermine. Viability of the wheat suspension culture cells after 12 h of spermine action: e – control; f – 1 μM of spermine; g – 10 μM of spermine; h – 100 μM of spermine. Mitochondrial membrane potential in the wheat suspension culture cells after 3 hours of spermine action: i – control; j – 1 μM of spermine; k – 10 μM of spermine; l – 100 μM of spermine. Scale bar – 50 μM. LT – LysoTracker Red, PI – Propidium Iodide, TMRM – Tetramethylrhodamine Methyl Ester.

Fig. 4. Visualization of autophagosomes in the subcellular fractions with increased activity of acid phosphatase: a – fraction no. 4, control; b – fraction no. 4, 10 μM of spermine; c – fraction no. 7, control; d – fraction no. 7, 10 μM of spermine.

Fig. 5. Distribution of the activities of acid phosphatase (a) and protease (b) in the subcellular fractions after separation in the Percoll gradient. Gray color – activities in the control variant, white color – activities in the cells treated with spermine. Fraction numbers are given sequentially, from the top to the bottom of the centrifugal tube.

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