In recent years, it has become clear that for mitochondria in photosynthetic plant cells, the main role of buffer capacity is becoming, allowing to keep under control the balance of ATP production, regulation of the level of pyridine nucleotide reduction, generation of ROS and RNS, as well as optimize the main metabolic flows, which is especially important under stress. Non-conjugated respiration is an important mechanism for achieving stable operation and maximum efficiency of photosynthetic cells. It is actively used in the light and becomes even more important under stress in lighting conditions. The most important part of these non-canonical mitochondrial functions is provided by alternative mitochondrial respiratory enzymes.

Low temperature is an important factor limiting plant viability and productivity. Along with other stresses, low temperatures increase the generation of reactive oxygen species, which are signaling molecules that can damage cell components. As well as representing one of the main targets of oxidative damage during stress, mitochondria represent a significant source of reactive oxygen species. Plant mitochondria have a large number of enzymes providing alternative electron transport pathways, many of which are activated under stress. Our aim was to assess the effect of low positive temperatures and increased expression of the heterologous gene NDB2 (alternative external NADH dehydrogenase of mitochondria) on the generation of reactive oxygen species, which involve an alternative respiratory chain in mitochondria and the expression of stress proteins under lighting conditions in Nicotiana tabacum tobacco leaves. In the leaves of tobacco plants with increased expression of the Arabidopsis thaliana NDB2 (AtNDB2) gene, a decrease in reactive oxygen species production was observed under normal and low temperature conditions. The results indicate that the heterologous Arabidopsis thaliana NDB2 gene is involved in increasing the activity of the alternative electron transport chain in mitochondria, which reduces the level of reactive oxygen species generation and affects the content of stress proteins under normal and low-temperature exposure.

Numerous biochemical and structural studies into the native organization of oxidative phosphorylation in the mitochondria of various eukaryotic organisms have convincingly shown that respiratory complexes can associate with one another to form higher-order structures referred to as supercomplexes. Plant mitochondria are distinguished by a more complicated organization of the respiratory chain due to the presence of a number of alternative oxidoreductases. It is considered that these enzymes do not physically interact with those of the cytochrome pathway. However, the available literature data obtained on yeast mitochondria suggests the possibility of such an association. In this regard, we aimed to study the native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in plant mitochondria. The work was performed on six-day etiolated pea seedlings. The 2D BN/SDS-PAGE in combination with immunochemistry found that, in pea organelles, the main part of the populations of NDA and NDB alternative NAD(P)H dehydrogenases were included in superstructures with masses of 700, 780, and 900 kDa. Additionally, NDA was detected in the region of 1480 and 1600 kDa, and NDB was registered at values of 1330, 340, and 100-120 kDa. An analysis of subunit profiles of the observed associations and a colorimetric detection of ATPase activity in 1D BN-gel suggested that the major part of the NDA and NDB populations identified by the available antibodies was associated with ATP synthase and represented a heterogeneous population of ATP-synthasomes, assumably, with a NDA2/NDB2Va/b1-2 composition. The rest of the enzymes were likely to be part of the NDA2/NDB2III2IV and NDA2IV1Va2 supercomplexes. The physiological significance of the association of alternative NAD(P)H dehydrogenases with ATP synthase requires further study.

The o-nitrobenzyl ester group is known for more than 50 years as a convenient protecting group and fragment for the design of photosensitive systems, including drug delivery agents. Polymeric amines with these groups are promising systems for medical applications, but the lack of basic knowledge about the possible degradation of these compounds under physiological conditions restrains the development of real preparations. We synthesized five new polyamines with the insertion of the o-nitrobenzyl ester group into the chain, including an oligomeric mixture with a weight average number of amine groups equal to 21. The ester group in the polyamine chain was found to be sensitive to hydrolysis at neutral and alkaline pH values. The presence of an amine group near the ester group facilitates hydrolysis through coordination with a hydroxyl ion or water molecule. The catalytic action of the amine moieties is prevented by protonation of the amine at low pH values or by interaction with polymeric acid. The study of photodegradation of new polyamines by HPLC-MS showed the formation of a number of products, most of which were not considered in previous works with o-nitrobenzyl ester derivatives. The o-nitrobenzyl ester group is used in the design of gene delivery systems and other biomedical preparations as a fragment that can be degraded by light. Our results indicate that photodegradation of o-nitrobenzyl esters can produce a number of compounds whose structure and safety for living organisms must be evaluated before use. On the other hand, the discovered hydrolyzability of o-nitrobenzyl esters catalyzed by a neighboring amine group may find application in the development of drug delivery systems. We have shown that the lifetime of o-nitrobenzyl ester containing polyamines is sufficient for gene delivery. Self-degradation of the delivery agent after penetration into a living cell is a desirable case for the release of the delivered agent.

Drought stress usually causes huge economic losses for tobacco industries. Drought stress exhibits multifaceted impacts on tobacco systems through inducing changes at different levels, such as physiological and chemical changes, changes of gene transcription and metabolic changes. Understanding how plants respond and adapt to drought stress helps generate engineered plants with enhanced drought resistance. In this study, we conducted multiple time point-related physiological, biochemical,transcriptomic and metabolic assays using K326 and its derived mutant 28 (M28) with contrasting drought tolerance. Through integrative analyses of transcriptome and metabolome,we observed dramatic changes of gene expression and metabolic profiles between M28 and K326 before and after drought treatment. we found that some of DEGs function as key enzymes responsible for ABA biosynthesis and metabolic pathway, thereby mitigating impairment of drought stress through ABA signaling dependent pathways. Four DEGs were involved in nitrogen metabolism, leading to synthesis of glutamate (Glu) starting from NO-3 /NO-2 that serves as an indicator for stress responses. Importantly, through regulatory network analyses, we detected several drought induced TFs that regulate expression of genes responsible for ABA biosynthesis through network, indicating direct and indirect involvement of TFs in drought responses in tobacco. Thus, our study sheds some mechanistic insights into how plant responding to drought stress through transcriptomic and metabolic changes in tobacco. It also provides some key TF or non-TF gene candidates for engineering manipulation for breeding new tobacco varieties with enhanced drought tolerance.

The study aims to confirm the functional activity and localization of the At_NDB2 transgenic protein of Arabidopsis in tobacco cells and to evaluate the effect produced by the permanent expression of external alternative NADH dehydrogenase on the resistance of a heat-loving plant to low temperatures. Proteins and mitochondria were isolated from the leaves of tobacco plants grown at 25 °С (day/night) at the 7-leaf stage. At_NDB2 protein localization in mitochondria was determined via electrophoresis and immunoblotting. The functional activity of At_NDB2 was confirmed through the polarography of isolated mitochondria and the specific mitochondrial complex I inhibitor (rotenone). It was also found that the cyanide-resistant respiration rate and the activity of an alternative oxidase enzyme were significantly higher in transgenic plants than in wild-type plants. In order to determine the resistance to low temperatures, the parent and transgenic tobacco plants were grown in soil until the 2–3 and 6–7 leaf stages, after which they were kept at 3 to -3 °С for a day in the dark and left to regrow at 25 °С for seven days. Although it was previously shown that oxidative stress is reduced in transgenic plants at low temperatures as compared to wild-type plants, it was established that the tolerance of transgenic and nontransformed plants does not differ. Thus, alternative NADH dehydrogenase activity was found to reduce oxidative stress and increase alternative oxidase activity, without enhancing the resistance of Nicotiana tabacum to negative temperatures.

Novel lines of tobacco (Nicotiana tabacum L.), highly expressing the AtNDB2 gene (NDB2 from Arabidopsis thaliana (L.) Heynh.), were produced with the help of agrobacterial transformation followed by a selection. The transgenic 13s line, possessing typical exterior and the AtNDB2 expression level, was compared with the initial wild type of N. tabacum regarding the parameters of growth and respiratory activity under optimal and suboptimal temperatures. It was found that the total and alternative respiration increased and the superoxide generation decreased in the 13s plants under the suboptimal temperature. The growth rate was decreased in the plants highly expressing the AtNDB2 gene in comparison with the control wild type plants, especially at the temperature below the optimum. Possible causes of the found changes are discussed.

Based on the readily available 3-organyl-5-(chloromethyl)isoxazoles, a number of previously unknown watersoluble conjugates of isoxazoles with thiourea, amino acids, some secondary and tertiary amines, and thioglycolic acid were synthesized. The bacteriostatic activity of aforementioned compounds has been studied against Enterococcus durans B-603, Bacillus subtilis B-407, Rhodococcus qingshengii Ac-2784D, and Escherichia coli B-1238 microorganisms (provided by All-Russian Collection of Microorganisms, VKM). The influence of the nature of the substituents in positions 3 and 5 of the isoxazole ring on the antimicrobial activity of the obtained compounds has been determined. It is found that the highest bacteriostatic effect is observed for compounds containing 4-methoxyphenyl or 5-nitrofuran-2-yl substituents in position 3 of the isoxazole ring as well as methylene group in position 5 bearing residues of L-proline or N-Ac-L-cysteine (5a-d, MIC 0.06-2.5 & mu;g/ml). The leading compounds showed low cytotoxicity on normal human skin fibroblast cells (NAF1nor) and low acute toxicity on mice in comparison with the well-known isoxazole-containing antibiotic oxacillin.

The study of the supramolecular organization of the mitochondrial oxidative phosphorylation system (OXPHOS) in various eukaryotes has led to the accumulation of a considerable amount of data on the composition, stoichiometry, and architecture of its constituent superstructures. However, the link between the features of system arrangement and the biological characteristics of the studied organisms has been poorly explored. Here, we report a comparative investigation into supramolecular and functional OXPHOS organization in the mitochondria of etiolated shoots of winter wheat (Triticum aestivum L.), maize (Zea mays L.), and pea (Pisum sativum L.). Investigations based on BN-PAGE, in-gel activity assays, and densitometric analysis revealed both similarities and specific OXPHOS features apparently related to the life strategies of each species. Frost-resistant winter wheat was distinguished by highly stable basic I1III2IVa/b respirasomes and V2 dimers, highly active complex I, and labile complex IV, which were probably essential for effective OXPHOS adaptation during hypothermia. Maize, a C4 plant, had the highly stable dimers IV2 and V2, less active complex I, and active alternative NAD(P)H dehydrogenases. The latter fact could contribute to successful chloroplast–mitochondrial cooperation, which is essential for highly efficient photosynthesis in this species. The pea OXPHOS contained detergent-resistant high-molecular respirasomes I1–2III2IVn, highly active complexes IV and V, and stable succinate dehydrogenase, suggesting an active energy metabolism in organelles of this plant. The results and conclusions are in good agreement with the literature data on the respiratory activity of mitochondria from these species and are summarized in a proposed scheme of organization of OXPHOS fragments.

Tertiary phosphine oxides, phosphine sulfides, and phosphine selenides containing pyridine, imidazole, and pyrazole groups have been synthesized via the reaction of elemental phosphorus or secondary phosphine oxides with functional pyridines, imidazoles, and pyrazoles. Alkyl tris(2-pyridylethyl)phosphonium iodide and bromide are also obtained by quaternization of the corresponding phosphine. Antimicrobial activity of the synthesized compounds, including nitrogen-containing heterocycles, phosphorus, selenium, and sulfur, with respect to Enterococcus durans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa microorganisms is evaluated. It is found that phosphine chalcogenides bearing imidazole (14, 19), pyrazole (13), and pyridine fragments (5, 9) and phosphonium salts (11, 12) can be considered as new promising antibacterial agents. For some synthesized compounds, LC50 is determined. Phosphine oxide with methylpyrazole fragments (13) and phosphonium salts (11, 12) show strong profile of antimicrobial activity, and cytotoxic effect of phosphonium bromide having a long chain radical (12) is by order of magnitude higher than that of cisplatin. We believe that the results obtained may contribute to the development of highly effective agents for the treatment and prevention of bacterial infections and cancers.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance.

The antitumor properties of aqueous extracts of Inonotus rheades basidiomycete mushrooms were evaluated when the fungi were cultured on wood discs and wood shavings (birch) at 25 ± 1°C in the dark and under constant illumination with blue light (12.8 W/m2). The aqueous fraction containing water-soluble polysaccharides was isolated and analyzed: WS-5 fraction cultured on wood discs under blue light illumination, WS-8 fraction grown under similar conditions on wood shavings; WS-6 fraction grown in the dark on wood discs; and WS-7 fraction grown under similar conditions on wood shavings. The antitumor effects of the extracts were studied on the model of Ehrlich ascites carcinoma in mice. It was found that the extracts isolated from the basidial fungus Inonotus rheades exhibit antitumor properties and that their accumulation during growth is determined by different cultivation conditions.

Scots pine (Pinus sylvestris L.) and Siberian spruce (Picea obovata Ledeb.), which are common for the south of Eastern Siberia, differ from each other in their ecophysiological characteristics. Scots pine is more thermophilic and drought-resistant, while Siberian spruce is more hydrophilic and tolerates cooling better. In different seasons, both species accumulate water-soluble sugars (WSS) in their needles, which are one of the factors of cold and drought resistance of needles. The level of WSS accumulation in needles can be influenced by both factors determined by the tree species and territorial ones, associated with the climate of the growing region. For the first time, this study introduces a comparison of changes in WSS content in Scots pine and Siberian spruce growing in the south of Eastern Siberia during two annual cycles. During the observation period, winter 2015-2016 was colder than winter 2016-2017, and the growth period 2015 was warmer and wetter than the same period in 2016. WSS content in Scots pine needles was found to be less subject to fluctuations and, according to the results of most of the observations made, to be higher than in Siberian spruce needles both in more favorable and less favorable conditions for the physiological activity. The exception was the period of November-February 2015-2016 (when WSS content in the needles of both species was the same) and March-April 2016 (when it was higher in Siberian spruce needles). The ability of Scots pine to maintain higher WSS reserve in its needles is possibly one of the mechanisms that allow trees of this species to successfully compete with Siberian spruce and occupy larger territories under the extreme conditions of the south of Eastern Siberia.

The functional parameters of mitochondria (intactness, oxidation rates of succinate and NAD·H, respiratory control coefficient, ADP/O ratio, and protein content) were studied in freshly harvested and cold-stored tubers of potato Solanum tuberosum L. cv. Skarb, transformed with a gene gox encoding the glucose oxidase from Penicillium funiculosum . Using untransformed potatoes and potatoes transformed with a vector without the target gene and vector constructs carrying the native and modified gox gene, it was shown that its expression causes changes in the rates of oxidation of the studied substrates and the degree of coupling of oxidation and phosphorylation, reduces the potential activity of the alternative oxidase, and induces the synthesis of a number of protective mitochondrial proteins involved in the regulation of the formation of ROS during storage in the cold. The role of hydrogen peroxide in the mechanisms of growth and stress resistance of plants, and its participation in the respiratory metabolism of plant cells is discussed.

Plants can minimise the damaging effects of high temperatures through numerous protective mechanisms; however, it is largely unknown how these mechanisms respond to extreme temperatures associated with wildfire. We investigated the effect of experimental burning (EB) on the accumulation of stress heat shock proteins (Hsps), which are one of the factors of thermotolerance in plants, in the needles of Scots pine (Pinus sylvestris L.). Previous fire exposure led not only to short- and long-term changes in the content of stress proteins in needles but also to changes in the accumulation of these proteins in response to reheating. The content of Hsp 101, Hsp 70 and Hsp 17.6 in the needles increased on the second day after EB (short-term effect of fire). Three years after EB, the content of Hsps in the fire-exposed needles was lower compared with the control needles. When these needles were subjected to the heat stress test at 45°C, the content of Hsps increased, whereas the content of Hsps in control needles decreased. Our results suggest that Scots pine needles retain a fairly long-term ‘stress memory’, expressed through proteomic defence mechanisms, to wildfire heat-induced damage.

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

The aim was to determine whether the antioxidant, cytotoxic and virucidal properties of aqueous extracts isolated from the Inonotus rheades basidiomycete depend on the illumination of the mycelium during cultivation. Effects of blue light illumination on the mycelium of I. rheades, which was cultivated on birch wood at 25 +/- 1 degrees C in the dark and under a constant illumination of 12.8 W/m(2) were studied. In the course of the work, two fractions of water-soluble polysaccharides were obtained: BP-5 - isolated from the mycelium grown under blue light; BP-6 - isolated from mycelium grown in the dark. Two fractions of water-soluble polysaccharides were obtained during the study: BP-5 - water-soluble polysaccharides isolated from the mycelium grown under blue light; BP-6 - water-soluble polysaccharides isolated from the mycelium grown in the dark. The extract from the mycelium grown under blue light showed a greater antioxidant activity than that from the mycelium grown in the dark. An analysis of the effect of the extracts under study on a test culture of tumour cells showed that the extracts cause the death of some amount of the cells on the 6th day of coincubation. The cytostatic effect of the extracts was also manifested following 6 days. In comparison with the control, the density of the culture at maximum concentrations decreased to 60% and 20% for BP-6 and BP5, respectively. The results of measuring the antiviral activity of the extracts showed that BP-5 and BP-6 completely destroy tick-borne encephalitis viruses. It was experimentally shown that, after normalisation of the pH values, both extracts contain components exhibiting a significant antiviral effect. The inhibition index for BP-5 and BP-6 comprised 3 and 2 Ig PFU/ ml, respectively. This suggests that the concentration of virucidal components in the extract from mycelium grown under blue light is approximately 10 times higher than that in the extract from the mycelium grown in the dark. Thus, the extracts from the mycelium of I. rheades grown on birch discs contain substances exhibiting antioxidant, cytostatic and virucidal properties. The accumulation of these properties can be stimulated by blue light illumination.

Background. Tick-borne encephalitis virus is dangerous and widespread pathogen that is transmitted to humans through the bites of hard ticks. Wild fungi, such as xylotrophic basidiomycetes, are widely used in traditional medicine to treat the infectious diseases and are promising natural sources of new antiviral agents. It was previously shown that aqueous extracts from the mycelium of the Inonotus rheades (Pers.) P. Karst. (1882) fungus exhibit significant antiviral activity against tick-borne encephalitis virus, however, the mechanisms of this activity remain unclear.Aim. To analyze the relationship between the virucidal properties of I. rheades extract and the substrate on which the cultivation was carried out.Materials and methods. The mycelium was grown either in a standard liquid medium with wort or on wooden disks from birch. Extracts of water-soluble polysaccharides were prepared from both mycelium samples. The concentration of infectious tick-borne encephalitis virus was determined using the method of titration of plaque-forming components (PFU). Approximately 30 000 PFU of tick-borne encephalitis virus was mixed with an equal volume of corresponding I. rheades extract at concentration of 8 mg/mL and incubated for 30 min at 37 °C. Afterwards, the residual infectivity of tick-borne encephalitis virus was determined using the identical virus sample incubated with sterile water as a reference.Results. It was found that treatment of tick-borne encephalitis virus with extracts from I. rheades mycelium resulted in inhibition of the infectivity of the virus in the cell culture. However, the same strain of I. rheades, grown on medium with wort, did not exhibit antiviral properties.Conclusions. Virucidal substances are likely to be not the main metabolites of the mycelium of I. rheades, but are rather metabolized wood polysaccharides. Further research is needed to more accurately identify the active ingredients and assess their antiviral activity.

This study examined effects of the styrylpyrone fraction from mycelium of hymenochaetoid species, Inonotus rheades (Agaricomycetes), on reactive oxygen species inhibition in Thellungiella salsuginea cell culture under oxidative stress. We identified antioxidant effects of styrylpyrones from I. rheades mycelium on oxidative stress in T. salsuginea cell suspension culture using two models of oxidative stress (induced by hyperthermia or hydrogen peroxide). The results showed that the styrylpyrone fraction maintained a high level of cell viability under stress conditions. The effect of pure hispidin on the plant cell culture was insignificant during the development of oxidative damage caused by hydrogen peroxide and was lacking during hyperthermia-induced oxidative stress. Therefore, these results suggest that the protective effect of the styrylpyrone fraction on T. salsuginea cells can be exerted by individual compounds (which are part of the fraction) and their complex.

Mitochondrial ATP synthase is a macromolecular nanomachine, which produces most of the ATP in the cell. This enzyme is located in inner mitochondrial membrane in the form of dimers, which assemble into long rows at the cristae rims. Dimers of ATP synthase are sensitive to detergent treatment in many organisms, as a result of which they mainly dissociate into monomers. Plant enzyme is also very detergent-sensitive, nevertheless, it can be assumed that the detergent-sensitivity or, vice versa, detergent-stability of the enzyme under the treat-ment may vary in different plant species. In this regard, the aim of this work was to study the detergent-stability, as well as the activity of various forms of ATP synthase, solubilized from the mitochondria of different plant species. For this purpose, we used organelles isolated from etiolated pea, winter wheat, and maize shoots. These species belong to different families (Poaceae and Fabaceae), clades (monocots and dicots) and, in addition, differ in their low temperature tolerance. For the solubilization of organelles, a mild non-ionic detergent digitonin was used, which preserved and stabilized the supramolecular associations of membrane proteins. Using 1D BN-PAGE followed by ingel enzyme activity assay, it was shown that ATP synthase in all studied species was solubilized mainly as monomers Va and Vb, dimer Vb2, supercomplex IV1Va2, and minor subcomplex F1. In the course of the study, for the first time, differences in the detergent-stability of dimeric and monomeric forms of the enzyme between the studied species were revealed. It was found that the dimeric form in maize and the monomeric form in winter wheat were the most stable; while, pea ATP synthase had the highest activity. The relationship between the revealed features and the life strategy of the species is assumed and discussed.