A. A. Ponomareva

Oxidative stress-induced autophagy in plants: the role of mitochondria.

The strictly regulated removal of oxidized structures is a universal stress response of eukaryotic cells that targets damaged or toxic components for vacuolar or lysosomal degradation. Autophagy stands at the crossroad between cell survival and death. It promotes survival by degrading proteins and organelles damaged during oxidative stress, but it is also activated as a part of death programs, when the damage cannot be overcome. Evidence is accumulating that the cellular sites of ROS production and signaling may be primary targets of autophagy. Therefore, autophagosomal targeting of mitochondria (mitophagy) is of particular importance. Mitophagy is a selective process that can specifically target dysfunctional mitochondria, but also mitophagy may play a role in controlling the number and quality of mitochondria during stress. Here we review the mechanisms of both non-specific autophagy and mitochondrial targeting in plants, drawing analogies and emphasizing differences with yeast and mammalian systems.

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis.

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.

Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

Intracellular origin and ultrastructure of platelet-derived microparticles

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell‐activating stimulus. Thrombin‐treated platelets fall apart and form microparticles that contain cellular organelles.

Phytopathogenicity of avian mycoplasma Mycoplasma gallisepticum S6: Morphologic and ultracytostructural changes in plants infected with the vegetative forms and the viable but nonculturable forms of the bacterium

The data obtained in this study proved that Mycoplasma gallisepticum S6 known as avian pathogen had a phytopathogenic potential. The vegetative forms and the viable but nonculturable (VBNC) forms of this mycoplasma could infect the plants via an assemblage of rootlets, invade different tissues, persist there and cause destructive events characteristic to phytomycoplasmoses. In comparison with the vegetative forms, the VBNC forms induced more prominent destructive changes. This phenomenon might be connected to increasing expression of proteins responsible for virulence in the bacterial cells. The fact that M. gallisepticum S6 could demonstrate virulent features (infectivity, invasiveness, persistence and toxigenicity) in regard to plants seems to require a development of new ways for controlling phytomycoplasmoses taking into account the probable presence of asymptomatic carriers of this bacterium.

Sterol binding by methyl‐β‐cyclodextrin and nystatin – comparative analysis of biochemical and physiological consequences for plants

The dependence of membrane function on its sterol component has been intensively studied with model lipids and isolated animal membranes, but to a much lesser extent with plant membranes. Depleting membrane sterols could be predicted to have a strong effect on membrane activity and have harmful physiological consequences. In this study, we characterized membrane lipid composition, membrane permeability for ions, some physiological parameters, such as H2O2 accumulation, formation of autophagosomal vacuoles, and expression of peroxidase and autophagic genes, and cell viability in the roots of wheat (Triticum aestivum L.) seedlings in the presence of two agents that specifically bind to endogenous sterols. The polyene antibiotic nystatin binds to endogenous sterols, forming so‐called ‘nystatin pores’ or ‘channels’ in the membrane, and methyl‐β‐cyclodextrin has the capacity to sequester sterols in its hydrophobic core. Unexpectedly, although application of both methyl‐β‐cyclodextrin and nystatin reduced the sterol content, their effects on membrane permeability, oxidative status and autophagosome formation in roots differed dramatically. For comparison, we also tested the effects of the antibiotic gramicidin S, which does not bind to sterols but forms nonspecific channels in the membrane. Gramicidin S considerably increased membrane permeability, caused oxidative stress, and reduced cell viability. Our results suggest that a decrease in the sterol content is, in itself, not sufficient to have deleterious effects on a cell. The disturbance of membrane integrity, rather than the decrease in the sterol content, is responsible for the toxicity of sterol‐binding compounds.

Stromule-like protrusions of plastid membrane envelope in root cells

The work presents the results of the electron-microscopy visualization of stromule-like protrusions of plastid membrane envelope in root cells. Cases of the appearance of a long, narrow protrusion of the outer membrane, in which the shorter protrusion of the plastid envelope inner membrane was located, are discussed. The possible role of cytoskeleton and plastoskeleton in formation of outer and inner protrusions, respectively, is considered. It is concluded that items of the structure and functions of stromules in plant cells are to be considered to be the same as the structure and functions of the intracavity space of endoplasmic reticulum.

Protonophores as inducers of energy-dependent changes of ultrastructure of mitochondria in wheat root cells.

The membrane potential of plasmalemma, the release of K+ ions into incubation medium, respiratory gas exchange, the ATP content, and changes in the ultrastructure in cells of excised roots of wheat seedlings have been studied under the effect of protonophores 2,4-DNP (2,4-dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenylhydrazone). After 1–4 h, a drop occurred in the plasmalemma membrane potential, as well as the release of K+ ions into incubation solution and the suppression of the intensity of oxygen absorption by cells. Mitochondria were of ovoid shape and had numerous and clearly outlined, slightly swollen cristae, which corresponds to the condensed type of the organelles. Additionally, a peculiar spatial arrangement of cristae in mitochondria has been revealed (as piles parallel to each other, as well as in the form of fans and of propellers) under the effects of protonophores. After 5 h of the action of protonophores against a background of the significant stimulation of oxygen consumption, low membrane potential, and a decrease in the functional activity of mitochondria, the destruction of the cell ultrastructure began. It is suggested that the revealed conformational transitions of mitochondria reflect gradual changes in their functional activity and the functional state of the cells under the long action of protonophores.

Structural characterization of platelets and platelet microvesicles

Platelets are blood cells without nuclei, which, in conjunction with fibrin, cause bleeding to stop (hemostasis). Cellular microvesicles are microscopic particles released into extracellular space under activation and/or apoptosis of cells of different types. Platelet microvesicles form the main population of blood circulating through microvesicles and play an important role in the reactions of hemostasis, thrombosis, and many other (patho)physiological processes. Despite the large number of studies that have been devoted to the function of platelet microvesicles, the mechanisms of their formation and structural details remain poorly understood. The ultrastructure of the initial platelets and microvesicles formed in vitro from resting cells and platelets activated by arachidonic acid, ADP, thrombin, and calcium ionophore A23187 is investigated in this study. The intracellular origin, stages of formation, structural diversity, and size of microvesicles were analyzed according to the results of transmission electron microscopy of human platelets and isolated microvesicles. It was shown that thrombin, unlike other activators, not only stimulates microvesiculation of the plasma membrane, but also causes decomposition of cells with the formation of subcellular particles that have sizes comparable with the size of the microvesicles from the outer membrane of the cells. Some of these microparticles are cellular organelles surrounded by a thin membrane. The size of isolated microvesicles ranges from 30 to 500 nm, but their size distribution depends on the nature of the activating stimulus. The obtained results contain new data on the formation of platelet microvesicles and their structural diversity, which are important for understanding of their multiple functions in health and disease.

Changes in mitochondrial shape in wheat root cells exposed to mitochondrial poisons

It is found that mitochondrial poisons not only modify the functional activity of the organelles but also change the shape of some mitochondria. The ring-shaped organelles were found to appear in root cells of 5- to 6-day-old seedlings of spring wheat (Triticum aestivum L., cvs. Moskovskaya 35 and Lyuba). Using a technique of serial sections, we have shown that the circular profiles of mitochondria within the cell correspond in most cases to bowl-shaped organelles. Spatial reorganization of mitochondria did not depend directly on the respiration rate, duration of exposure to inhibitors, and inhibitor specificity. These observations indicate the reversibility of spatial rearrangements and general nonspecific nature of the detected morphological changes.