Could one of only three known DNA markers—N4-methylcytosine (4mC)—be absent in all but microbes? Until recently, 4mC had only been documented in bacteria, where it serves as a defense mechanism. Now, researchers led by Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) have discovered that this epigenetic marker is essential for sperm development and maturation in the liverwort Marchantia polymorpha, a key organism in plant evolution. Their findings, published in Cell, represent a significant milestone in plant molecular biology.

A relative of mosses, Marchantia is among the oldest land plants and offers unique insights into early plant evolution. It retains a primitive form of reproduction reliant on water—sperm are released into rain droplets and swim to fertilize nearby female plants. Despite its evolutionary importance, the molecular basis of its sperm development has remained largely unknown.

Feng’s team uses Marchantia as a model to uncover hidden mechanisms behind plant reproduction. Their study reveals that 4mC, long believed to be exclusive to microbes, plays a pivotal role in the development of viable, motile sperm in this plant. Our study provides conclusive evidence for a new DNA marker in plants or animals with a crucial function—it’s essential for sexual reproduction in Marchantia. These insights also open possibilities for biotechnological applications, where gene expression could be modulated without altering DNA sequences.

In bacteria, 4mC acts as an immune shield, masking DNA from restriction enzymes. It is one of three known epigenetic DNA markers, along with 5mC (5-methylcytosine) and 6mA (6-methyladenine). While 5mC and 6mA have been observed in eukaryotes, 4mC had not been conclusively detected—until now.

In studying sperm development in Marchantia, Feng’s group identified two distinct waves of DNA methylation. The first, involving 5mC, suppressed transposable elements. The second wave targeted CG dinucleotides within coding regions—something 5mC alone could not account for. At the same time, genes resembling bacterial N4-methyltransferases were highly expressed. This led the team to investigate the presence of 4mC using a suite of molecular techniques. 

Their results were striking. They found that 4mC accounted for about 15percentage  of methylated cytosines in mature sperm—far higher than typical microbial levels. These levels are extraordinary. No bacterium carries this much methylation. This helped validate our conclusion.

Functionally, 4mC proved indispensable. Without it, sperm motility dropped significantly, fertility was impaired, and early embryo development was compromised. These effects underscore its role in reproductive success.

Interestingly, 4mC has only been confirmed in Marchantia so far. However, Feng suggests it may exist in other organisms but only during specific developmental stages. In mammals, for example, early embryogenesis includes large-scale epigenetic reprogramming that might temporarily involve 4mC. We may simply have missed the right window in other species, she notes.

Caution is warranted in the field of DNA methylation. A prior claim that 6mA was widespread in eukaryotes was later refuted, traced back to bacterial contamination. Aware of this, Feng’s group used rigorous, independent techniques to rule out similar artifacts. We were meticulous, Feng emphasizes. “But the sheer volume of 4mC also helped—it couldn’t have come from bacteria.

As for how Marchantia acquired the ability to methylate 4mC, the researchers believe horizontal gene transfer (HGT) from bacteria is the most likely explanation. HGT is known to have played a role in plant adaptation to land, and this epigenetic trait may have been a useful byproduct of such an event. Nature is full of such evolutionary shortcuts, Feng says. The acquisition of 4mC methylation may have been one that stuck—and proved highly beneficial.

The implications of this work go beyond plant biology. By unraveling the function of 4mC in a living system, researchers can begin to explore its use in epigenetic genome editing—a technology that modifies gene expression without changing the DNA code. This could have transformative potential in medicine, agriculture, and biotechnology.

This study not only revises what we know about DNA chemistry in plants but also points to how ancient lineages can reveal innovations that modern systems may obscure. With 4mC now confirmed in plants, the hunt is on to see where else it might be hiding.

Source: https://ist.ac.at/en/news/from-bacterial-immunity-to-plant-sex/