Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1 , NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1 . The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.

Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential.

The mechanism of yeast cell death induced by heat shock was found to be dependent on the intensity of heat exposure. Moderate (45°C) heat shock strongly increased the generation of reactive oxygen species (ROS) and cell death. Pretreatment with cycloheximide (at 30°C) suppressed cell death, but produced no effect on ROS production. The protective effect was absent if cycloheximide was added immediately before heat exposure and the cells were incubated with the drug during the heat treatment and recovery period. The rate of ROS production and protective effect of cycloheximide on viability were significantly decreased in the case of severe (50°C) heat shock. Treatment with cycloheximide at 39°C inhibited the induction of Hsp104 synthesis and suppressed the development of induced thermotolerance to severe shock (50°C), but it had no effect on induced thermotolerance to moderate (45°C) heat shock. At the same time, Hsp104 effectively protected cells from death independently of the intensity of heat exposure. These data indicate that moderate heat shock induced programmed cell death in the yeast cells, and cycloheximide suppressed this process by inhibiting general synthesis of proteins.

Повышение содержания Hsp104 (heat shock protein) при тепловом стрессе играет ключевую роль в развитии термотолерантности в клетках дрожжей Saccharomyces cerevisiae. Синтез Hsp104 S. cerevisiae повышается при тепловом стрессе и при переходе дрожжей в стационарную фазу роста. Показано, что утрата митохондриальной ДНК (мутация petite) подавляла индукцию синтеза Hsp104 при тепловом стрессе (39°С) и при переходе в стационарную фазу роста. Одновременно мутация petite подавляла повышение активности антиоксидантных ферментов в стационарной фазе, что сопровождалось снижением термотолерантности. В то же время мутация petite ингибировала продукцию активных форм кислорода и защищала клетки от гибели при действии теплового шока в логарифмической фазе роста. Полученные результаты указывают, что нарушение функционального состояния митохондрий подавляет экспрессию ядерных генов при переходе клеток дрожжей в стационарную фазу роста.

Тепловой стресс вызывает у растений повышение потенциала на внутренней митохондриальной мембране (мт ), что сопровождается активацией экспрессии белков теплового шока (БТШ, или HSP). Обработка амиодароном (АМД) клеток Saccharomyces cerevisiae приводит к параллельному повышению уровня Ca2+ в цитозоле ([Ca2+]цит) и мт , а также к индукции синтеза Hsp104. Предположили, что причиной гиперполяризации является повышение [Ca2+]цит. В настоящей работе изучали эффект АМД (0–100 мкМ) на жизнеспособность, экспрессию БТШ, мт и [Ca2+]цит, используя культуру клеток Arabidopsis thaliana (L.) Heynh. Обработка АМД приводила к увеличению [Ca2+]цит, что сопровождалось повышением мт и активацией экспрессии гена HSP101. Повышение [Ca2+]цит и экспрессия HSP101 наблюдались также при обработке протонофором СССР (карбонил-цианид(m-хлорфенил)гидразоном, 4 мкМ), снижающим мт . Полученные данные указывают на то, что митохондрии растительной клетки, изменяя потенциал на внутренней митохондриальной мембране, модулируют содержание Ca2+ в цитозоле и таким образом участвуют в ретроградной регуляции экспрессии HSP101.

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.

Heat stress in plants elevates the potential across the inner mitochondrial membrane (mtΔψ) and activates the expression of heat shock proteins (HSPs). The treatment of Saccharomyces cerevisiae cells with amiodarone (AMD) elevated the cytosolic Ca2+ level ([Ca2+]cyt) in parallel with (mtΔψ) increase and led to the induction of Hsp104 synthesis. The hyperpolarization was presumably due to the increase in [Ca2+]cyt. In the present study the effects of AMD (0–100 μM) on cell viability, HSP expression, mtΔψ, and [Ca2+]cyt were investigated using the cell culture of Arabidopsis thaliana (L.) Heynh. The treatment of cultured cells with AMD led to the elevation of [Ca2+]cyt, which was accompanied by the increase in mtΔψ and by activation of HSP101 expression. The increase in [Ca2+]cyt and expression of HSP101 were also observed upon the treatment with the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone, 4 μM) known to diminish mtΔψ. The results suggest that plant cell mitochondria modulate the cytosolic Ca2+ level by changing the potential at the inner mitochondrial membrane and, thereby, participate in the retrograde regulation of HSP101 expression.

Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca2+ level in cells of the yeast Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca2+ level. It is supposed that the change in intracellular Ca2+ concentration plays an important role in activation of the HSP104 gene expression and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium homeostasis is discussed.

Many biotic and abiotic stresses cause an increase of cytosolic Ca level in cells. Calcium is one of the most important second messengers, regulating many various activities in the cell and was known to affect expression of stress activated genes. Mild heat shock induces the expression of heat shock proteins (Hsps) which protect cell from drastic heat shock exposure. There are some literature data permitting to suggest that transient elevation of cytosolic Ca level in plant cells is important for activation of Hsps expression. On the other hand mitochondria are known to regulate the intracellular calcium and reactive oxygen species signaling. It has been shown recently that mild heat shock induces hyperpolarization of inner mitochondrial membrane in plant and yeast cells and this event is critically important for activation of Hsps expression. To reveal the relationship between mitochondrial activity, intracellular calcium homeostasis and Hsps expression an antiarrhythmic drug amiodarone (AMD) have been used. AMD is known to cause transient increase of cytosolic Ca level in Saccharomyces cerevisiae. Obtained results have showed that AMD treatment induced the synthesis of Hsp104p in S. cerevisiae cells and Hsp101p in A. thaliana cell culture. Induction of Hsp104p synthesis leads to enhanced yeast capability to survive lethal heat shock exposure. Development of S. cerevisiae thermotolerance depended significantly on the presence of Hsp104p. Elevation of Hsp104p level in the result of AMD treatment was shown to be governed by activity of Msn2p and Msn4p transcription factors. Deletion of the MSN2 and MSN4 genes abrogated the AMD ability to induce Hsp104p synthesis. Mild heat shock and AMD treatment induced the hyperpolarization of the inner mitochondrial membrane in yeast and Arabidopsis cells which accompanied by HSP synthesis and development of thermotolerance. It was suggested that increase of cytosolic Ca level after AMD treatment directly or indirectly causes the activation of mitochondrial activity which leads to hyperpolarization of the inner mitochondrial membrane and production of reactive oxygen species (ROS). Modulation of cellular Ca 2+ and ROS signals by mitochondria is assumed to play a prominent role in activation of Hsps expression in yeast and plant cells.

Fluorine is a component of atmospheric emissions in industrial areas. It negatively affects plant development and weakens the defense systems, thus making plants vulnerable to extreme environmental conditions. The heat shock proteins (HSP) are known to promote the plant resistance to various biotic and abiotic stresses. We studied the action of sodium fluoride (NaF) on growth, viability, respiration, transmembrane electric potential at the inner mitochondrial membrane (mtΔΨ), the development of induced thermotolerance, and HSP synthesis in the cell culture of Arabidopsis thaliana (L) Heynh (accession Columbia). The treatment with 20 mM NaF (for 120 min) had no negative influence on viability of the cell culture but inhibited the development of induced thermotolerance and suppressed the induction of HSP (Hsp101 and Hsp17.6) synthesis during mild heat stress (37°C). At the same time, the treatment with NaF inhibited respiration and suppressed the increase in mtΔΨ induced by mild heat stress. Hence, the negative impact of NaF on plants might arise from its ability to inhibit synthesis of stress proteins indispensible for plant adaptation to changing environmental conditions.

Effect of calcium ions on heat tolerance of Saccharomyces cerevisiae and on the induction of Hsp104 synthesis by this microorganism was studied. Short-term (30 min) treatment with CaCl 2 at 30°C enhanced the heat tolerance to the lethal heat shock (50°C); the synthesis of Hsp104 was induced as well. The effect of Ca 2+ on the heat tolerance and Hsp104 synthesis was shown to be ion-specific and was inhibited by LaCl 3 , which is known to block calcium ion channels on the cytoplasmic membrane. The effect of Ca 2+ depended on the potential of the inner mitochondrial membrane. When the cells were treated with sodium azide, which reduced the electrochemical potential, the effect of calcium both on heat tolerance and Hsp104 synthesis was suppressed. Depending on the concentration of exogenous Ca 2+ and the ambient conditions, calcium ions may either induce or inhibit the expression of the stress genes and cell viability.

Салициловая кислота (СК) может участвовать в развитии устойчивости к абиотическим стрессам, а именно, к тепловому шоку. В нормальных условиях (26°С) обработка экзогенной СК повышала устойчивость гетеротрофной культуры клеток Arabidopsis thaliana (L.) Heynh. к жесткому тепловому шоку (50°С). При мягком тепловом шоке (37°С), который инициирует развитие индуцированной термотолерантности, присутствие СК, напротив, снижало способность клеток арабидопсиса переносить высокую температуру (50°С) и одновременно ингибировало тепловую индукцию синтеза БТШ (Hsp101 и Hsp17.6), важных для развития индуцированной термотолерантности. Поскольку СК подавляла дыхание клеток, активируя альтернативный путь переноса электронов, предполагается, что СК, модулируя функции митохондрий, является эндогенным регулятором экспрессии генов стрессовых белков растений.

Salicylic acid (SA) could be involved in the development of tolerance to abiotic stresses, to heat shock in particular. Under normal conditions (26°C), treatment with SA improved the tolerance of heterotrophic Arabidopsis thaliana (L.) Heynh culture to severe heat shock (50°C). Under mild heat shock (37°C) inducing the development of thermotolerance, the presence of SA, in contrast, reduced the capability of arabidopsis cells to tolerate high temperature (50°C) and simultaneously suppressed induction of HSP synthesis (Hsp101 and Hsp17.6) important for the development of induced thermotolerance. Since SA suppressed cell respiration and activated the alternative pathway of electron transport, SA is supposed, by modulating mitochondria functions, to be an endogenous regulator of plant stress gene expression.

To search the antiseptic agents capable to decontaminate the plants from pathogens the combined effect of moderate heat shock (45oC) and glycolisis inhibitor monoiodoacetate (MIA) on survival of potato pathogen Clavibacter michiganensis ssp. sepedonicus (Cms) and yeast Saccharomyces cerevisiae was studied. Under optimal temperature cultivation (26oC) MIA had no toxic effect on S. cerevisiae but decreased viability of Cms. The lethal effect of MIA significantly increased during heat treatment at 45oC. MIA in the range from 0.1 to 1 mM decreased the thermotolerance of Cms and S. cerevisiae cells in 10-10000 folds in dependence from time of treatment. A minimal concentration of MIA capable to affect the thermotolerance was 0.1 and 0.3 mM for S. cerevisiae and Cms, respectively. The effect of MIA on Cms and yeast survival during heat shock was stronger in logarithmic phase than in stationary ones.

Abstract To search the antiseptic agents capable to decontaminate the plants from pathogens the combined effect of moderate heat shock (45°С) and glycolisis inhibitor monoiodoacetate (MIA) on survival of potato pathogen Clavibacter michiganensis ssp. sepedonicus (Cms) and yeast Saccharomyces cerevisiae was studied. Under optimal temperature cultivation (26°С) MIA had no toxic effect on S. cerevisiae but decreased viability of Cms. The lethal effect of MIA significantly increased during heat treatment at 45°С. MIA in the range from 0.1 to 1 mM decreased the thermotolerance of Cms and S. cerevisiae cells in 10-10000 folds in dependence from time of treatment. A minimal concentration of MIA capable to affect the thermotolerance was 0.1 and 0.3 mM for S. cerevisiae and Cms, respectively. The effect of MIA on Cms and yeast survival during heat shock was stronger in logarithmic phase than in stationary ones.

Mild heat shock induces the synthesis of heat shock proteins (HSP) protecting the cell from damages during subsequent severe heat shock. The nature of the signal inducing transcription of Hsp genes is poorly investigated. We studied the effects of mitochondrial inhibitors, sodium azide and dinitrophenol (DNP) on the development of induced thermotolerance and induction of Hsp101 and Hsp60 syntheses in the suspension culture of Arabidopsis thaliana . The presence of sodium azide and DNP during mild heat shock was shown to suppress heat-induced synthesis of Hsp101 and development of induced thermotolerance in the A. thaliana cultured cells. Severe heat shock (50°C) resulted in programmed cell death as was evident from reduced cell viability and cytochrome c release. The results obtained permit a supposition that the functional state of mitochondria determines Hsp gene expression in A. thaliana subjected to heat shock.

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.