The alternative reality of plant mitochondrial DNA: One ring does not rule them all

Abstract

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms.

The Tempo and Mode of Angiosperm Mitochondrial Genome Divergence Inferred from Intraspecific Variation in Arabidopsis thaliana

“…Other analyses of intraspecific mitogenome variation in systems such as A. thaliana (Davila et al 2011), Beta vulgaris (Darracq et al 2011), and Zea mays (Allen et al 2007;Darracq et al 2010) have generally focused on structural rearrangements resulting from repeat-mediated recombination. Indeed, at an even finer level, angiosperm mitogenomes are really a population of alternative structures that interconvert via recombination and coexist within cells and tissues in a single individual (Palmer and Shields 1984;Gualberto and Newton 2017;Kozik et al 2019). As such, these structural rearrangements are arguably the most dynamic element of plant mtDNA evolution, and rapid shifts in the predominant structure (referred to as substoichiometric shifting) are often observed on very short generational timescales (Abdelnoor et al 2003; Arrieta-Montiel and Mackenzie 2011).…”

“…Angiosperm mitogenome sequencing projects typically report genome assemblies represented as a single circular structure, but it is widely accepted that this is an oversimplification resulting from mapping and that the physical form of angiosperm mtDNA involves complex branching structures (Bendich 1993;Sloan 2013;Kozik et al 2019). These branching structures likely reflect the activity of DNA replication, which is thought to be initiated by recombination-dependent mechanisms and not depend on a single origin of replication (Cupp and Nielsen 2014).…”

Discordant evolution of organellar genomes in peas (PisumL.)

“…Evidently, there are various manners to resolve the overlapping circular DNAs into a master circle since each join/repeat has two entrances and two exits that implies four possible configurations of the DNA pass, in a manner of the '8' figure or as two circles. This fact probably reflects the nature of plant mitochondrial DNA which exists as a set of interconverting sub-genomic molecules due to homologous recombination between repeated regions (Gualberto et al, 2014;Kozik et al, 2019). In fact, each of the alternative configurations was represented by some contigs of the starting set.…”

“…It is well known that plant mitochondrial genomes exist as several structural isoforms (circular, linear or branched) permanently undergoing rearrangements at their repeated segments (Gualberto et al, 2014;Kozik et al, 2019). The here assembled mitogenomes represent isoforms, where possible, consisting of one master circle,.…”

Structure–Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System

“…Finally, recombination at small repeats drives the apparition of new open reading frames associated with traits like cytoplasmic male sterility [71,72]. The notion that recombination is dependent on the length of the repeat is challenged by comparing new mitochondrial DNA sequences between domesticated and wild-type cultivars and by following the evolutionary history between species [73,74].…”