COI Notice: One of the authors of this paper is my Principal Investigator (P.I.). I, however, had no involvement in this paper or the experiments done in it.

When it comes to understanding plant genetics and the potential for improvements in growth and development, it should be argued that the humble mitochondria deserves the bulk of attention over the nucleus. That’s because, somewhat unlike though similar to  animal cells, the mitochondrial genome of plants is markedly more influential for the specific growth and energy development traits desired by all who grow our green cousins. Normally, one would think the size comparison between the two, with animal mitochondria having around 16,000 nucleotides, while plant mitochondrial vary in size from 190,000 nucleotide bases long to over 11 million for some of the largest found thus far, would be a relevant example for this importance. However, despite that, there isn’t a notable difference in the number of actual coding genes between these extremes, with around 40-50 genes in general within the entire mitochondrial plant genome.

Breaking The Master Circle

The comparative gene numbers is complicated further by the mysteries of how plant mitochondrial genomes are structured, something that has plagued plant biologists for decades. When mapping the genomes for sequencing, our computational machinery routinely pops out a circle containing the entire sequence, commonly termed the master circle. Most people think this would imply that plant mitochondrial genomes are circular molecules just like those in bacteria. This is consistent with the fact that mitochondria and chloroplasts in eukaryotic cells originated from bacteria. But things are nowhere near that simple.

If more discerning techniques are utilized, the master circle hypothesis falls apart, because of a lack of evidence for its existence. It appears to be a result of how our sequencing machines naturally produce genome maps, but also the real structure of the mitochondrial genome in plants plays a role in how this misconception came about. Research into this true identity was initially slow to develop due to inherent usage of these circular sequence results from plant model species. Once labs began probing more directly, however, the alternative physical nature of plant mitochondrial genomes was laid bare front and center. 

Even so, it is due to the massive size of plant mitochondrial genomes and the similarly large number of repeat nucleotide sequences and even inverted repeats that there have been experiments showing a structure of many linear strands that can be overlapped into a circle. Multiple overlapping strands can, when laid piece by piece, be fitted into the puzzle as a circle even though they don’t exist in reality in that form. The inverted repeat sequences, it should be noted, likely played a role in why expansion of the overall genome has occurred over time thanks to their potential for sequence duplication. The true shape of the plant mitochondrial genome can feature multi-branched linear structures or perhaps even a few small circles as well, with those subcircles only making up a portion of the total genome sequence. Just from those comparisons one can see how this is a tricky puzzle of knotted string. 

Finding The Lettuce Solution

As a hopeful way to resolve all of the past scientific investigation on the topic into a singular conclusion and statement, a collaborative team of researchers in the field of plant mitochondrial genomes from the University of California – Davis, a visiting member from University of Nebraska – Lincoln, and two members from Wageningen University in the Netherlands all came together to push understanding forward. And they did so with the help of the simple and unassuming lettuce. 

The team started with a sequence of the lettuce mitochondrial genome, alongside similar sequences of its wild relatives, prickly lettuce and willowleaf lettuce, as a comparison. They made sure to alter the assembly parameters to allow for a genome that can have multiple forms and arrangements so that the machinery would not try to assume a circular genome, nor would it assume the sequence to be entirely linear. Different types of segments were used to confirm accuracy between them and to give a high level of quality to the final determined sequence structure. 

It was quickly discovered that the mitochondrial genomes of lettuce and prickly lettuce are identical, forcing their results to be combined and only the differences from willowleaf lettuce were able to be compared. A major inversion around one of the repeat sequences and a plasmid that was integrated into the genome itself were the main distinct features of willowleaf lettuce, along with many minor changes and an overall rearrangement of the genome. 

Microscopy and fluorescent fluorophores allowed the researchers to confirm that there are multiple forms the mitochondrial genome can take, including linear, branched, and circular forms that were smaller than the genome. Within each species, there were different arrangements of the segments of sequence (referred to as isoforms). Their finding that most of the DNA remained in the wells when running a size separation gel, along with a long smear of small material, indicated that most of the mitochondrial genomes are present as small linear segments and larger branched pieces. The action of recombination can explain the presence of subcircles, branched molecules and isoforms.

The Circle Is Not Unbroken

With this, the group was able to rather definitively conclude the nature of the mitochondrial genomes of lettuce and its two cousins. And while it has been well known for some time within the small field of researchers studying plant mitochondrial genomes that they are not circular, the publication of such data in the literature has often defaulted to showing the mitochondrial genomes as circular merely because that’s the output format of the sequencing and assembly machinery. Now, with this new methodology and programming, accurate sequence maps can be made that truly reflect what each of these plant mitochondrial genomes look like and how they are structured. 

A better understanding of the structure, and therefore function, of the plant mitochondrial genome can potentially open up new capabilities to produce beneficial cultivars and otherwise determine individual gene function and interactions within the overall genome. In time, we may be able to fully unlock the potential for improving the plants we grow in ways we can only dream about right now. 

Press Article

Study Article

Photo CCs: Starr-170627-0207-Lactuca sativa-variety of salad greens in rows-Hydroponics Greenhouse Sand Island-Midway Atoll (36319966221) from Wikimedia Commons

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