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Could it be that one of the few known markers that directly target DNA is exclusively found in microorganisms? Researchers led by Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) have uncovered that N4-methylcytosine (4mC) plays a critical role in the development and maturation of sperm in the liverwort Marchantia polymorpha, a pivotal organism in the evolutionary history of plants. This discovery is elaborated upon in a recent publication in Cell.
Living fossils often provide remarkable insights into evolution and biology. The liverwort Marchantia, which shares a common lineage with mosses, represents one of the most ancient existing plant forms and is believed to be the first group of plants to adapt to terrestrial life. Its significance is heightened by its method of reproduction, which remains tied to aquatic environments—an ancestral approach not commonly seen in most modern plants. In contrast to their more evolved counterparts, the sperm of Marchantia is released into droplets of rainwater and swims to fertilize adjacent female plants. Despite their evolutionary importance, the molecular intricacies of sperm function in these liverworts have remained largely unexplored.
The research group at ISTA aims to delineate molecular mechanisms related to sperm functionality in Marchantia, which may unveil fresh perspectives on the evolution of sexual reproduction. “Our study provides compelling evidence for a novel DNA marker in both plants and animals that serves a crucial role: it is vital for sexual reproduction in Marchantia, particularly influencing the development of sperm in male specimens,” Feng explains. These findings may pave the way for innovative applications in biotechnology, particularly for controlling gene expression without changing the underlying DNA sequence.
A long search beyond the realm of microbes
N4-methylcytosine (4mC) acts as a form of immune defense in bacteria, enabling their genomes to evade degradation by foreign enzymes through the addition of a methyl group to specific sites. It is one of three known DNA markers: 4mC, 5mC, and 6mA. These epigenetic markers operate by ‘masking’ portions of the genetic code without inducing mutations, as suggested by the Greek prefix “epi,” meaning ‘over’ or ‘around.’ While both 4mC and 5mC modify the cytosine nucleotide—one of DNA’s fundamental building blocks—6mA uniquely targets adenine nucleotide. 4mC has long fascinated researchers due to its elusive presence in plants and animals.
The levels are “crazy” — but needed for agile sperm
While investigating the sperm development processes in Marchantia, Feng and her team identified two significant waves of DNA methylation occurring during this reproductive phase. The initial wave was linked to 5mC, an epigenetic marker already established in various animals and plants that silences mobile genetic elements referred to as “jumping genes.” However, the presence of a second wave of extensive methylation, which targets specific sequences of nucleotides throughout coding genes, could not be solely attributed to 5mC. The team also discovered that genes related to N4-cytosine methyltransferases—enzymes responsible for 4mC methylation in bacteria—were active during the critical periods of sperm development in the plant. This raised the question of whether they had identified these enzymes in an organism beyond microbes.
Pursuing this line of inquiry, the researchers employed a series of quantitative techniques to confirm that 4mC was indeed responsible for a significant portion of DNA methylation in mature Marchantia sperm, accounting for approximately 15 percent of methylated cytosines, markedly higher than the less than one percent found in bacteria. “With a comprehensive range of experimental techniques, we are confident in our findings,” Feng remarks. “The levels of 4mC detected in Marchantia sperm are astonishing.” This evidence highlighted the coordination of both 5mC and 4mC methylation waves during sperm development, underscoring that without sufficient 4mC, the sperm’s ability to fertilize is severely hindered—resulting in decreased speed, poor directionality, reduced fertility, and adverse effects on embryo development if fertilization does occur.
Horizontal gene transfer
Previously, claims concerning another form of DNA methylation, 6mA, have been discredited due to contamination issues, which has led to skepticism in the field. “Utilizing various independent methods to verify our findings was crucial. Nevertheless, the elevated levels of 4mC bolstered our confidence; no bacterium exhibits such extensive methylation,” Feng explains. The emergence of 4mC in Marchantia can likely be traced to horizontal gene transfer (HGT), an evolutionary process where genetic material is exchanged between different species outside of reproduction.
HGT is commonly recognized as a significant occurring mechanism between bacteria and plants over evolutionary timescales. This process may be the means by which 4mC was integrated into Marchantia. “This could represent an ad-hoc natural event fueling evolution. The transfer of genetic material from bacteria to Marchantia yielded advantageous traits that were retained and selected in the plant’s evolution,” Feng elaborates.
More 4mC waiting to be discovered?
So far, the liverwort is the only known plant group where 4mC has been conclusively identified. “However, we suspect our findings are not an isolated occurrence,” Feng affirms. In mammals, early development typically involves substantial reprogramming of methylation, suggesting 4mC may be lurking undetected in specific developmental stages of other plants and animals. “Future research should focus on examining these critical developmental windows in various species to identify the potential presence of 4mC,” she notes.
This research not only elucidates a fundamental aspect of the evolutionary process concerning sexual reproduction but also suggests potential extensions into biotechnology. By understanding the specific roles of 4mC in Marchantia, this epigenetic modification may eventually be harnessed for epigenetic genome editing techniques that regulate gene expression without modifying the DNA sequence itself.
This research originated at the Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK, before Xiaoqi Feng joined the Institute of Science and Technology Austria (ISTA) faculty.
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