The Mitochondrial Genome: Comparison
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Please note this is a comparison between Version 1 by Serge Rozov and Version 2 by Camila Xu.

Mitochondria are powerhouses of the cell and implement oxidative phosphorylation processes. Similar to plastids, mitochondria are endosymbionts of a pro-eukaryotic cell, have their own genome, and their own transcriptional and translational machinery.

  • transgene plants
  • transplastomic plants
  • mitochondrial genome

1. Introduction

Mitochondria are powerhouses of the cell and implement oxidative phosphorylation processes. Similar to plastids, mitochondria are endosymbionts of a pro-eukaryotic cell, have their own genome, and their own transcriptional and translational machinery. Unlike the plastid genome (represented by approximately 100 plastome copies), the genome of mitochondria (mitogenome) is not so over-replicated in most plant species. Animal mitochondrial genomes are rather small (~15 kbp); on the contrary, the plant mitogenome is considerably larger and varies in size by two orders of magnitude depending on the species: from 66 kbp in the hemiparasitic mistletoe (Viscum scurruloideum, Santalaceae) [1] to 11,300 kbp in the sand catchfly (Silene conica, Caryophyllaceae) [2]. On average, mitogenome size in terrestrial plants (300–400 kbp) is two- to threefold longer as compared with the plastid genome (100–200 kbp) [3]. In contrast to the plastid genome, coding genes in the mitogenome are much less densely arranged, and their total number is only 3–66 [4].

2. Specific Features of the Organization and Function of the Plant Mitochondrial Genome

As compared with the conserved plastid genome and compact animal mitochondrial genome, the plant mitochondrial genome possesses several unique specific features. Cases of extensive homologous recombination of plant mitochondrial DNA (mtDNA) are frequent, underlying its propensity for reorganization. The structure and size of plant mtDNA considerably changes because of intensive proliferation of mobile genetic elements, an increase in introns, and insertions of foreign DNA (nuclear, plastid, viral, or bacterial) [2]. Along with a single circular chromosome, as in A. thaliana [5], many plant species contain several circular or even linear chromosomes in their mitochondrial genome. For instance, cucumber (Cucumis sativus) has three mitochondrial chromosomes with lengths of 1156, 84, and 45 kbp [6]. Another example is S. conica, the 11,300 kbp genome which is composed of 128 circular molecules replicating independently of one another [2]. A key feature of the organization of plant mitochondrial genomes is that the noncoding rather than coding sequences constitute its main body. The copy number of the plant mitogenome is considerably lower as compared with animals and pronouncedly varies depending on the developmental stage or analyzed tissue. In particular, the maximum observed copy number of the A. thaliana ATP1 gene is approximately 280 copies per cell, being lower than the average number of mitochondria per cell (approximately 500). This observation suggests that plant mitochondria can carry a partial mitochondrial genome or even be devoid of it [7]. The plant mitochondrial genome contains direct and inverted repeats, which are involved at a high rate in the recombination and rearrangement of mtDNA (e.g., 6.5 and 4.2 kbp repeats in Arabidopsis mtDNA) [8]. Recombination also involves short repeats but at a considerably lower rate. In general, plant mtDNA in vivo is most likely represented by a set of recombination intermediates, concatemers, subgenomic circular molecules, and plasmids. A certain part of plant mtDNA exists in a single-stranded form (as observed in Chenopodium album) [9].
The plant mitochondrial genome has the form of a nucleoid, i.e., nucleoprotein particles anchored to the inner mitochondrial membrane. The role of the nucleoid is mtDNA compaction and regulation of its metabolism and transcriptional activity, while its external components are involved in signaling systems. Note that nucleoids of plant mitochondria contain many proteins binding single-stranded rather than double-stranded DNA. Some proteins of the nucleoid are double-targeted and function in plastids as well. All nucleoid proteins of A. thaliana mitochondria are encoded in the cell nucleus [10]. Unlike plastids, which carry two types of RNA polymerases (PEPs (encoded in the plastid genome) and NEPs (encoded in the nucleus)), mitochondria of almost all eukaryotes have lost bacterial-type polymerases (PEPs), and hence NEPs exclusively participate in transcription (some of them function in plastids, too) [11].

3. Prospects of Transcriptional and Translational Machinery of Plant Mitochondria for Recombinant Protein Biosynthesis

Long-term efforts to implement genetic transformation of mitochondria have not been fruitful: so far, only mitochondria of the yeasts Saccharomyces cerevisiae, C. reinhardtii [12], and Candida glabrata [13] have been successfully transformed by biolistics. The success of genetic transformation of the mitochondria of these lower eukaryotes is attributable to the specific features of their cell metabolism, which are absent in higher eukaryotes. As for higher plants, the main, albeit unsolved, problem hindering the creation of such a system is the absence of a selection strategy for cells with genetically transformed mitochondria. High antibiotic sensitivity of mitochondrial respiratory chain complexes prevents the use of antibiotics for the selection of transmitochondrial cells or plants.
In the past few years, significant advances have been made in terms of the editing of plant mitogenomes. A system has been developed that can quite effectively modify mitogenomes of land plants with the help of transcription activator-like effector nucleases having mitochondria-targeting localization signals (mitoTALENs) to induce DSBs in targeted mitochondrial genes. By agrobacterial nuclear transformation of rice and rapeseed with this mitoTALEN system, knockouts of cytoplasmic male sterility mitochondrial genes have been obtained in these plants [14]. The floral deep transformation method with mitoTALENs made it possible to obtain a knockout of two genes of ATP synthase [15], and with mitochondria-targeting TALEN-based cytidine deaminase, this approach can replace C:G with T:A pairs in a given region of the mitogenome of A. thaliana [16]. Nevertheless, no events of integration of foreign recombinant genes into the mitogenome have been recorded so far. Be that as it may, this field of gene engineering is developing rapidly, and it is likely that in a few years new tools for editing mitogenomes in the knock-in format based on CRISPR/Cas technologies will be devised.
Thus, plant mitochondria remain the most appealing as the expression system for producing recombinant proteins (and even possibly more attractive than chloroplasts) if we take into account certain fundamental specific features of the genetic system of these organelles. Among the most important of these features is that mitochondria of higher organisms are able to take up (import) DNA, thereby creating favorable conditions for in vivo delivery of genetic constructs to mitochondria during biotechnological manipulations. The mitochondrial plasmids present in the mitogenome of some plant species and their simpler structural–functional organization make them the most suitable for the insertion of target genes. High tolerance of the mitochondrial genome to incorporation of foreign DNA (as evidenced by DNA fragments of nuclear, chloroplast, viral, and unknown origins in the mitogenome) also improves the chances of obtaining an efficient producer. Several years of research aimed at clarifying the functional role of the above-mentioned specific features of the genetic system of plant mitochondria (and at assessing their potential in the relevant biotechnological applications) strongly indicate their promising prospects [17][18][19]. As shown by some authors, it is feasible to insert a reporter gene (a GFP fragment) into the genome of isolated potato, maize, or tobacco mitochondria [20]. Expression of a target gene in mitochondria is undoubtedly preferable to its expression in the nuclear genome, because the production and accumulation of the recombinant protein is confined to the mitochondrial matrix. Consequently, the yield of the target protein is improvable via a decrease in its proteolysis and in its potential cytotoxicity. The use of a polyploid mitochondrial genome to express recombinant proteins offers advantages in terms of both the yield of the target product and lower probability of target gene silencing. As reported in one paper, the activity of mtDNA import considerably increases when a transported linear DNA molecule carries terminal inverted repeats, similarly to linear mitochondrial plasmids and transposons [18]. Recently, the possibility of DNA import into mitochondria was shown in A. thaliana protoplasts [21]. Thus, based on the above results, itwe can be expectedexpect the development of a strategy in the near future that allows transfection of protoplasts with constructs carrying specific sequences that ensure their insertion into the mitogenome [20] and transcription of the target gene. With subsequent regeneration of the cell wall in these protoplasts, it becomes possible to select the transformed cells and either maintain them in cell culture or regenerate whole plants.

References

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