[Differential Expression of a Foreign Gene in Arabidopsis Mitochondria In Organelle].

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.

Inactivation of the TIM complex components leads to a decrease in the level of DNA import into Arabidopsis mitochondria.

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.

Somatic embryogenesis in Larix: the state of art and perspectives.

Clonal propagation of conifers using somatic embryogenesis is essential for the selection of tree species, and for the implementation of afforestation and reforestation. In combination with cryopreservation, somatic embryogenesis creates the basis for the development of economically valuable lines of clones and elite genotypes. The industrial use of such genetically verified clone lines in forestry can significantly increase forest productivity compared to any conventional methods for improving tree crops that are available. Larch is considered as one of the main conifer candidates for large-scale reforestation, not only due to the vastness of its habitat, but also due to the unique quality of its wood, rapid growth and high ecological plasticity. However, the vast majority of larch species are characterized by uneven yields and extremely low seed quality. In this regard, obtaining planting material for reforestation from larch seeds on seed plantations is not advisable, but can be successfully implemented in afforestation programs using somatic embryogenesis technologies. Research on the somatic embryogenesis of larch has been conducted for over 30 years, which allowed considerable experience in this field to be accumulated. To date, the conditions for the initiation and maintenance of embryogenic cultures, as well as for the formation and development of somatic embryos have been determined. Significant progress has been made in the study of both the factors affecting these processes and the molecular mechanisms that underlie the various stages of embryogenesis. Nevertheless, despite the successes achieved, knowledge available today on the somatic embryogenesis of representatives of the genus Larix is still not enough to develop technologies for producing valuable plant-breeding material in vitro. This review analyzes the current state of research on the problem of somatic embryogenesis of representatives of the genus Larix. Particular attention is paid to the choice of explants for somatic embryogenesis, the composition of the media for cultivation, the dependence of the potential of somatic embryogenesis on the duration of cultivation, and the genetic control of somatic embryogenesis.

DNA Import into Plant Mitochondria: Complex Approach for in organello and in vivo Studies.

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.

Import of Proteins and Nucleic Acids into Mitochondria.

Many mitochondrial genes have been transferred to the nucleus in course of evolution. The products of expression of these genes, being still necessary for organelle function, are imported there from the cytosol. Molecular mechanisms of protein import are studied much deeper than those of nucleic acids. The latter, it seems to us, retards the development of mitochondrial genome editing technologies. In this review, we describe mechanisms of DNA, RNA, and protein import into mitochondria of different eukaryotes. The description is given for the natural processes, as well as for artificial targeting of macromolecules into mitochondria for therapy. Also, we discuss different approaches to introduce changes into the mitochondrial DNA sequence.

Extracellular Actin in Health and Disease.

This review considers the functions of extracellular actin - cell surface bound, associated with extracellular matrix, or freely circulating. The role of this protein in different pathological processes is analyzed: its toxic effects and involvement in autoimmune diseases as an autoantigen. The extracellular actin clearance system and its role in protection against the negative effects of actin are characterized. Levels of free-circulating actin, anti-actin immunoglobulins, and components of the actin clearance system as prognostic biomarkers for different diseases are reviewed. Experimental approaches to protection against excessive amounts of free-circulating F-actin are discussed.

DNA Import into Mitochondria.

In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.

Glutathione Reductase Gene Expression Depends on Chloroplast Signals in Arabidopsis thaliana.

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H2O2 level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.

Level of Blood Cell-Free Circulating Mitochondrial DNA as a Novel Biomarker of Acute Myocardial Ischemia.

Changes in the level of blood cell-free circulating mitochondrial DNA were examined during experimental adrenaline-induced myocardial injury in rats. The amount of mitochondrial DNA in the blood was significantly elevated at 48 and 72 h after subcutaneous injection of adrenaline solution, and it was accompanied by development of multiple small-focal myocardial ischemia. This suggests that the measured level of blood cell-free circulating mitochondrial DNA might be used as a biomarker of acute myocardial ischemia.

Induction of Arabidopsis gdh2 gene expression during changes in redox state of the mitochondrial respiratory chain.

Expression of the gdh2 gene encoding the alpha-subunit of mitochondrial glutamate dehydrogenase depends on redox state of the mitochondrial electron transport chain. Treatment of Arabidopsis thaliana cell suspension with antimycin A, a respiratory chain complex III inhibitor, resulted in an increase in gdh2 transcripts within 2 h. Inhibition of complex I by rotenone did not influence the transcript level, but treatment with potassium cyanide, a complex IV inhibitor, also increased the transcript content. Thus, gdh2 gene expression obviously responds to changes in the respiratory chain segment localized between complexes I and III. Lack of activation of gene expression after treatment of a cell suspension with hydrogen peroxide and the prooxidant paraquat and results of experiments with antioxidants suggest that gdh2 gene expression is not associated with increased content of reactive oxygen species generated during inhibition of the electron transport chain. Protein phosphorylation by serine/threonine protein kinases is the essential step required for signal transduction into nucleus resulting in the induction of gdh2 expression.