The mitochondria of major taxonomic groups possess the ability to import DNA from their environment; however, the cellular role of this process remains not fully understood. Collectively, all mitochondria within a cell form the chondriome, whose structure is crucial for organelle interactions and maintenance of cellular homeostasis. No data are currently available regarding the impact of altered mitochondrial fusion and fission dynamics-which leads to the structural disruption of the chondriome-on DNA import into mitochondria. This study aimed to examine the relationship between chondriome structure and the import of foreign DNA into mitochondria using Arabidopsis knockout lines characterized by impaired fusion and fission processes (drp3, friendly). Inactivation of the mitochondrial fission protein isoform DRP3B, which leads to the formation of highly elongated mitochondria, reduced the import activity of a 2.7 kb fragment. Mitochondrial clustering, characteristic of the friendly knockout mutant, also led to a decrease in the import level of this fragment, while the import activity of a short DNA fragment (0.27 kb) remained unchanged. Thus, the normal functioning of the mechanism responsible for importing larger DNA fragments requires maintaining a balance between mitochondrial fusion and fission. The observed correlation between DNA import activity and changes in chondriome structure provides new insights into the role of DNA import and its contribution to cellular stability.

Yellowing is the first visually observable sign of plant leaf senescence. We found that Arabidopsis double knockout mutant gdh1gdh2 for genes of NAD(H)-dependent glutamate dehydrogenase retains green color of the leaves (stay-green phenotype) during a dark-induced senescence, in contrast to wild-type plants, whose leaves turn yellow. When the gdh1gdh2 plants are exposed to the dark more than four days, they demonstrate slower chlorophyll degradation than in the wild-type plants under the same conditions, as well as dysregulation of chlorophyll breakdown genes encoding chlorophyll b reductase, Mg-dechelatase, pheophytinase and pheophorbide a oxygenase. The slowed degradation of chlorophyll b in gdh1gdh2 plants significantly alters the chlorophyll a/b ratio. Ion leakage in the mutant plants increases significantly from four to eight days in the darkness, correlating with their premature death during this period. The discovered facts suggest a functional connection between activity of NAD(H)-dependent glutamate dehydrogenase and dark-induced senescence progress in Arabidopsis.

The transcription of Arabidopsis organellar genes is performed by three nuclear-encoded RNA polymerases: RPOTm, RPOTmp, and RPOTp. The RPOTmp protein possesses ambiguous transit peptides, allowing participation in gene expression control in both mitochondria and chloroplasts, although its function in plastids is still under discussion. Here, we show that the overexpression of RPOTmp in Arabidopsis, targeted either to mitochondria or chloroplasts, disturbs the dormant seed state, and it causes the following effects: earlier germination, decreased ABA sensitivity, faster seedling growth, and earlier flowering. The germination of RPOTmp overexpressors is less sensitive to NaCl, while rpotmp knockout is highly vulnerable to salt stress. We found that mitochondrial dysfunction in the rpotmp mutant induces an unknown retrograde response pathway that bypasses AOX and ANAC017. Here, we show that RPOTmp transcribes the accD, clpP, and rpoB genes in plastids and up to 22 genes in mitochondria.

Genetic transformation of higher eukaryote mitochondria in vivo is an unresolved and important problem. For efficient expression of foreign genetic material in mitochondria, it is necessary to select regulatory elements that provide a high level of transcription and transcript stability. This work is aimed at studying the effectiveness of regulatory elements of mitochondrial genes flanking exogenous DNA using the phenomenon of natural competence of plant mitochondria. For this purpose, genetic constructs carrying the GFP gene under the control of the promoter regions of the RRN26 or COX1 genes and one of the two 3'-untranslated regions (3'-UTR) of mitochondrial genes were imported into isolated Arabidopsis mitochondria, followed by transcription in organello . It was shown that the level of GFP expression under the control of promoters of the RRN26 or COX1 genes in organello correlates with the level of transcription of these genes observed in vivo. At the same time, the presence of the tRNA Trp sequence in the 3'-UTR leads to a higher level of the GFP transcript than the presence in this region of the 3'-UTR of the NAD4 gene containing the binding site of the MTSF1 protein. The results we obtained open prospects for creating a system for efficient transformation of the mitochondrial genome.

The phenomenon of DNA import into mitochondria has been shown for all major groups of eukaryotes. In plants and animals, DNA import seems to occur in different ways. It has been known that nucleic acids enter plant organelles through alternative channels, depending on the size of the imported molecules. Mitochondrial import of small DNA (up to 300 bp) partially overlaps with the mechanism of tRNA import, at least at the level of the outer membrane. It is noteworthy that, in plants, tRNA import involves components of the protein import apparatus, whose role in DNA transport has not yet been studied. In this work, we studied the role of individual components of the TIM inner membrane translocase in the process of DNA import into isolated Arabidopsis mitochondria and their possible association with the porin VDAC1. Using knockout mutants for the genes encoding Tim17 or Tim23 protein isoforms, we demonstrated for the first time the involvement of these proteins in the import of DNA fragments of different lengths. In addition, inhibition of transport channels with specific antibodies to VDAC1 led to a decrease in the level of DNA import into wild-type mitochondria, which made it possible to establish the specific involvement of this porin isoform in DNA import. In the tim17-1 knockout mutant, there was an additional decrease in the efficiency of DNA import in the presence of antibodies to VDAC1 compared to the wild type line. The results obtained indicate the involvement of the Tim17-1 and Tim23-2 proteins in the mechanism of DNA import into plant mitochondria. At the same time, Tim23-2 may be part of the channel formed with the participation of VDAC1, while Tim17-1, apparently, is involved in an alternative DNA import pathway independent of VDAC1. The identification of membrane carrier proteins involved in various DNA import pathways will make it possible to use the natural ability of mitochondria to import DNA as a convenient biotechnological tool for transforming the mitochondrial genome.

Background. The mitochondrial electron transport chain, in particular the respiratory complex I, is one of the main sources of reactive oxygen species (ROS) in living cells. Suppression of the complex I activity in human cells through chemical inhibition or mutations that disrupt the functionality of this complex leads to a significant increase in the content of ROS. The complex I mutants are also known for plants; however, it is still not clear whether the suppression of the activity of this complex leads to an increase in the level of ROS and the development of oxidative stress. Purpose. To study the level of superoxide, hydrogen peroxide and sensitivity to prooxidants in Arabidopsis ndufs4 cells with complex I inactivated by insertional mutagenesis. Materials and methods. A suspension culture of wild-type Arabidopsis cells and a line with knockout the NDUFS4 complex I subunit was used. The level of hydrogen peroxide in the cells was determined using dichlorofluorescein. The superoxide content was estimated by cell staining in the presence of nitroblue tetrazolium. Results. It has been shown that inactivation of the NADH-dehydrogenase complex in Arabidopsis cells due to the absence of one of its subunits leads to a decrease in the content of superoxide and hydrogen peroxide. It was also shown that the level of ROS in cells treated with the menadione and hydrogen peroxide increased several times in wild-type cells, but remained almost unchanged in ndufs4 cells. Conclusion. Suppression of the activity of the NADH dehydrogenase complex in plant cells, but not in animal cells, leads to a decrease in the content of both hydrogen peroxide and superoxide. Cells of ndufs4 line have an increased ability to detoxify ROS, possibly associated with the constant mobilization of antioxidant defense systems.

Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways.

Expression of Arabidopsis thaliana glutamate dehydrogenase genes is very low in the light and high in the dark. The molecular signals and mechanisms that provide the light-dependent glutamate dehydrogenase genes regulation remain unknown. The aim of this work was to study a role of redox signals which occur during light shifts in the regulation of the glutamate dehydrogenase genes expression. Using photosynthetic electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl benzoquinone (DBMIB) we demonstrate that transcript levels of the GDH1 and GDH2 genes in Arabidopsis leaves change in accordance with a redox state of plastoquinone pool: they are low when it is highly reduced and high when it is oxidized. Hydrogen peroxide or high light treatment did not result in decreasing of GDH1 or GDH2 expression, so reactive oxygen species cannot be the signals that reduce expression of these genes during dark-to-light shifts. There was no significant difference between the glucose content in the leaves of plants treated with DCMU and the plants treated with DBMIB, so glucose is not the only or the main factor that regulates expression of the studied genes. We presume that expression of Arabidopsis GDH1 and GDH2 genes depends on the plastoquinone pool redox state. Expression of Arabidopsis glutamate dehydrogenase genes during light-to-dark and dark-to-light shiftsdepends on chloroplast electron transport chain redox state rather than reactive oxygen species or glucose content.

The initial formation of the photosynthetic apparatus in plants occurs during photomorphogenesis. The red/far-red (phytochromes) and blue (cryptochrome) light protein-photoreceptors play the most important role in photomorphogenesis initiation and regulation. The exited phytochrome and cryptochrome molecules can interact with transcription factors, changing the expression of nuclear genes, which encode the proteins of the plant photosynthetic apparatus. Since light is a variable factor, plants have developed appropriate adaptation mechanisms, including their photosynthetic apparatus protection. The mechanism of state transitions ensures a rapid adaptation of the photosynthetic apparatus. This adaptation mechanism increases the adsorption efficiency under current light conditions and prevents intensive generation of active forms of oxygen in chloroplasts, which leads to photo-oxidation and even cell death. This work aims to determine the role of photoreceptors - phytochromes A and B, as well as cryptochrome 1 and 2 - in regulating the process of state transitions in the Arabidopsis thaliana model plant. Arabidopsis mutants with the defects on A and B phytochromes and cryptochrome 1 and 2 genes were used as the research objects. The blue native electrophoresis in polyacrylamide gel was used to visualise state transitions. It was found that these photoreceptors had no direct effect on the redox-regulation of the state transitions mechanism in Arabidopsis. Presumably, these photoreceptors protect the photosynthetic apparatus from excessive light not by regulating the state transitions but indirectly, through regulating the chlorophyll, carotenoid and antioxidant components content.

It is known that mitochondrial population of living organisms is heterogeneous. Using the density gradient of Percoll or sucrose, we obtained and characterized two distinct mitochondrial subfractions (named “light” and “heavy” according to their sedimentation) from three plant species (Brassica rapa, Zea mays, and Arabidopsis thaliana). Electron microscopy showed that mitochondria of a light subfraction have a poorly developed cristae structure. The respiratory control of these mitochondria was reduced; however the degree of their outer membrane integrity remained high. These data suggest that mitochondrial subfractions purified in the density gradient differ in their maturity. We carried out DNA import assays to study the ability of these two subfractions for DNA transport. We have shown that the mitochondria of light subfraction in all three plant species uptakes DNA more efficiently than the mitochondria of heavy subfraction. In addition, import of DNA with different sizes into the light and heavy mitochondrial subfractions has different sensitivity to specific inhibitors of the voltage-dependent anion channel and adenine nucleotide translocase, proteins playing a central role in DNA transport across the mitochondrial membrane. The obtained results suggest that DNA import depends on organization of the transport system in the double mitochondrial membrane influenced by the maturity of the mitochondria.

The influence of low‐intensity laser radiation (LILR) on the changes in the content of anthocyanins, kaempferol, quercetin and their glycosides in the leaves of 5‐week‐old plants of Arabidopsis thaliana L. was studied by means of methods of high‐performance liquid chromatography and gas chromatography mass spectrometry (GC‐MS). It was found that in the leaves subjected to a stimulating He‐Ne laser radiation dose (3.6 J cm−2, continuous wave radiation, wavelength—632.8 nm, exposure time—5 min), the radiation induced an increase in the content of such compounds, the most significant one being in the case of anthocyanins (9 times). The present study also revealed an increase in the antioxidant potential of kaempferol, quercetin and their glycosides as a result of laser exposure. This increase was due to the preferential synthesis of compounds with a larger number of OH‐groups on the phenyl ring. Thus, the content of quercetin, which has five OH‐groups in its structure, increased almost by three times as compared to the control.

: Sugars play an important role in plant metabolism. They are not only a source of energy, but also an integral part of the intracellular signalling network. Signals involving sugars regulate a variety of metabolic reactions, e.g. carbon and nitrogen metabolism, and produce plant response to biotic and abiotic stresses. This paper provides a brief review of modern publications about sugars acting as metabolites and signalling molecules. The well-known and recently discovered pathways for the transmission of sugar-mediated signals are described, along with their role in the regulation of plant metabolism and expression of nuclear genes. Hexokinase 1 (an enzyme for glucose synthesis), which was one of the first studied participants in the sugar-mediated signalling network, regulates the expression of many nuclear genes in response to changes in the glucose level. Hexokinase 1 should be considered as a broad-spectrum regulator due to its participation in the transmission of mitochondrial-nuclear signals and regulation of aging processes in plants, which functions lay beyond the regulation of metabolic reactions. Of particular interest is the participation of sugar-mediated signals in the switching of metabolic reactions in response to changes in carbohydrate concentra-tions. Sugar deficiency deactivates most energy-intensive processes, at the same time as stimulating photo-synthesis or (in its absence) enhancing catabolic reactions due to activation of the SnRK1 kinase. Under suf-ficient sugar levels, the SnRK1 kinase is suppressed and the TOR kinase is activated, which stimulates ana-bolic reactions and growth. The role of competing SnRK1 and TOR kinases as the main regulators of such a process is considered, along with the function of signals mediated by trehalose-6-phosphate highly similar to the TOR signalling pathway. All these regulatory mechanisms enable plants to adapt to environmental changes and maintain homeostasis. It is possible that additional information will be obtained in the nearest future on the interaction of energy and stress-dependent signals in plants through the antagonism of TOR and SnRK1.

Natural competence of mitochondria for DNA uptake has been known for the last 20 years. Until the present time, all studies of this process have been conducted exclusively in isolated mitochondria, as no system for investigation of the DNA transport into the mitochondria in intact cells has been available. The objective of this work was to improve and standardize the existing approaches for investigating DNA import into plant mitochondria in an in organello system. A method for detecting the import of fluorescently labeled DNA substrates has been developed. Based on the features of DNA import into the mitochondria, we suggested an efficient method for the evaluation of the DNA import efficiency by quantitative PCR. We also developed and characterized the in vivo system that allows to detect DNA transport from the cytoplasm to the mitochondrial matrix in Arabidopsis thaliana protoplasts. A combination of the proposed techniques for studying the DNA uptake by plant mitochondria might be useful for elucidating whether the properties of the mitochondrial DNA import established in the in organello system are preserved in vivo.

The Agrobacterium-mediated transformation of a duckweed plant (Lemna minor L.) with the use of the organogenic callus and nucleotide sequence of a gene for hirudin-1 optimized for expression in plants has been performed. Eight transgenic plant lines transformed by hirudin and seven lines transformed by the gene for β-glucuronidase were obtained. The expression of the glucuronidase gene was proven by histochemical staining and Western blot. ELISA of the transgenic plants showed that the content of β-glucuronidase in them varied from 0.28 to 1.43% of the total soluble protein. The expression of the hirudin-1 gene was confirmed by RT-PCR, with the maximum hirudin accumulation being equal to 0.02% of the total soluble protein. The results of the study can be used in the development of an expression system using the duckweed plant to obtain hirudin and other recombinant proteins for pharmaceutical use.

«Genome uncoupled» (gun) is a molecular phenotype manifesting in a high expression of a number of nuclear photosynthesis associated genes in plants with arrested chloroplast biogenesis. The chloroplast biogenesis arrest could be achieved by growing of plants in presence of lincomycin (plastid translation inhibitor) or norflurazon (carotenoid biosynthesis inhibitor). We have found that Arabidopsis thaliana gun1-1gun5-1 double mutant seedlings with gun phenotype associated with both lincomycin and norflurazon demonstrate a repressed root growth when germinated on lincomycin but not norflurazon-containing media. Statistical analysis has demonstrated a high negative correlation between root length and gun phenotype evaluated on both LHCB1.2 and HEMA1 genes expression in three gun mutants of Arabidopsis: gun1-1, cch1-1 and the double mutant gun1-1gun5-1. Our results demonstrate that regulatory signals of plastid origin might participate in root development.

Аннотация.Изолированные митохондрии растений обладают способностью импортировать молекулы ДНК.В данной работе импорт ДНК проводили в реконструированной системе, включающей изолированные митохондрии клубней картофеля и

2 Федеральное государственное бюджетное образовательное учреждение высшего образования «Иркутский государственный университет»