[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.

[Heterogeneity of the Mitochondrial Population in Cells of Plants and Other Organisms].

The mitochondrial population is heterogeneous in eukaryotic cells. The heterogeneity of mitochondria can be defined as a variation in certain characteristics of mitochondria within the same or different cells. Differences between mitochondria are possible to classify as nongenetic (structural, morphological, and bioenergetic features) or genetic (differences in mtDNA copy number or sequence). Changes in mtDNA sequence are reflected in the phenomenon of heteroplasmy, which is the presence of several mitochondrial genotypes in a cell or an organism. The review considers the features of the organization and dynamics of the chondriome in plant cells compared with cells of other taxonomic groups of organisms. Particular attention is paid to the factors and mechanisms that lead to mitochondrial heterogeneity, heteroplasmy in plants, possible functional specialization of mitochondria, and the role of these processes in the whole organism. A great number of data indicate that the heterogeneous state of mitochondria in the cell is due, among other factors, to the species-specific features of the mitochondrial dynamics processes that are responsible for the homogeneity of the mitochondrial population.

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.