From bacterial immunity to plant sex

It sometimes takes a living fossil to make some of the most interesting findings about life and evolution. A relative of mosses, the liverwort Marchantia is one of the oldest existing plant forms and probably the oldest lineage of plants to colonize land. In addition to its central function in the plants' transition from an aquatic to a terrestrial ecosystem, Marchantia still uses an ancestral form of reproduction that is no longer found in most plants. Whereas other plants have evolved sexual reproduction independently of water, Marchantia sperm is released into rainwater drops splashing on the plants' surface and swims to fertilize the nearby female plants. However, many molecular aspects of Marchantia sperm function have remained in the dark to this day.

The group of Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) uses this unique plant as a model to uncover yet unknown molecular principles that shed new light on the evolution of sexual reproduction. And their latest findings are no less than groundbreaking. "Our study provides conclusive evidence for a new DNA marker in plants or animals with a very important function: It is essential for sexual reproduction in Marchantia, specifically affecting sperm development in male plants," says Feng. The findings could have applications in biotechnology, opening up perspectives for regulating gene expression without altering the underlying DNA sequence.

A long search beyond the realm of microbes

N4-methylcytosine (4mC) is a form of immunity in bacteria, camouflaging their genome from enzymes that degrade foreign sequences by adding a methyl chemical group to target locations. It is one of the three known DNA markers: 4mC, 5mC, and 6mA. Since these chemical markers only 'mask' the underlying genetic code without mutating it, they are called "epigenetic markers," based on the Greek prefix epi, meaning 'over,' 'outside of,' or 'around.' While 4mC and 5mC are chemical modifications of the cytosine nucleotide -- one of the building blocks of DNA -- 6mA is the only one to target the adenine nucleotide. Since it was never conclusively documented in plants or animals, 4mC has interested the scientific community for a long time.

The levels are "crazy" -- but needed for agile sperm

In their quest to decipher the molecular mechanisms of sperm function in Marchantia, Feng and her team distinguished two waves of extensive DNA methylation during sperm development. These two waves literally cover the sperm's genome. They showed that the first wave corresponded to 5mC, a DNA modification already known in animals and plants that targets and silences so-called "jumping genes." However, 5mC alone could not explain the second wave of extensive methylation, which targets short sequences of two nucleotides scattered all over coding genes, the so-called CG (cytosine-guanine) dinucleotides. In addition, the team showed that genes with a sequence similarity to N4-cytosine methyltransferases, the enzymes that catalyze 4mC methylation in bacteria, were expressed in the same window of the plant's sperm development. Could the team just have stumbled upon the enzymes for 4mC outside of microbes?

Motivated by this discovery, they used myriad quantitative techniques to show that 4mC was indeed the culprit. They found that 4mC alone was responsible for a whopping fifteen percent of methylated cytosines in the mature Marchantia sperm as opposed to less than one percent in bacteria. "Having used a battery of conclusive experimental approaches, we can safely say we are sure of our results," says Feng. "The levels of 4mC we detected in Marchantia sperm are crazy to think of." Thus, the team demonstrated that the extensive 5mC and 4mC methylation waves were orchestrated during the plant's sperm development. In addition, they showed that these "crazy" levels of 4mC had key functions in the Marchantia sperm. Without 4mC, the plant's sperm can no longer compete for fertilization: its swimming becomes slower and less directional, its fertility is drastically decreased, and even in the rare cases of successful fertilization, the plant embryo's early development is affected.

Horizontal gene transfer

Another form of DNA methylation, 6mA, was erroneously reported by other researchers in several organisms before its detection was traced to bacterial contamination. This has left a bad taste in scientists' mouths and led to increased scrutiny in the DNA methylation field. "It is very important to use multiple independent methods for the detection of DNA methylation, and we wanted to make absolutely sure that our claims were sound. But in a way, the high level of 4mC was helpful for our confidence. After all, no bacterium carries this much methylation," says Feng. But how did 4mC arise in Marchantia in the first place? The answer lies in transferring genetic material between different species outside of sexual reproduction, an evolutionary phenomenon called horizontal gene transfer, or HGT.

HGT has been reported to happen frequently -- on an evolutionary scale -- between bacteria and plants. This is most probably how 4mC reached Marchantia. HGT events from soil bacteria to plants have contributed to the plant's transition from an aquatic to a terrestrial environment. "This might be one of nature's ad-hoc events that boosted evolution. The horizontal transfer of genetic material from bacteria to Marchantia turned out quite useful for the plants, so the trait was kept and selected for," explains Feng.

More 4mC waiting to be discovered?

The liverwort is the only plant group where 4mC has been conclusively documented to date. "However, our findings are probably not a coincidence," says Feng. In fact, early developmental stages in mammals usually include extensive methylation reprogramming, completely erasing and re-establishing the DNA epigenetic modifications. "So, we might need to look at a specific window of development in other plants and animals. 4mC might also be there, waiting to be discovered."

The present study sheds light on a fundamental function of sexual reproduction that evolved in nature and could find applications in biotechnology. By dissecting the precise function of the 4mC epigenetic modification in Marchantia, this molecular mechanism could potentially be used as a tool for epigenetic genome editing, i.e., methods that target gene expression without altering the DNA sequence.

The present study was started 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.