Epigenetic Regulation Links Bacterial Signaling to Growth and Reproductive Development in the Green Macroalga Ulva compressa (Chlorophyta)

DNA (deoxyribonucleic acid) methylation is a key epigenetic mechanism that regulates gene expression and developmental transitions in eukaryotes. Interestingly, algal growth- and morphogenesis-promoting bacteria can reduce global DNA methylation levels in the green seaweed Ulva (Chlorophyta), highlighting DNA methylation as a dynamic and environmentally responsive epigenetic mechanism in marine macroalgae. We hypothesized that DNA methylation serves as a rapid and essential epigenetic regulatory mechanism during gametogenesis in the model organism Ulva compressa (cultivar U. mutabilis slender). We further propose that this process is controlled by sporulation inhibitors - extracellular compounds long known to regulate reproduction in Ulva, yet whose molecular mode of action has remained elusive. Using ultra-high-performance liquid chromatography-mass spectrometry (HPLC–HRMS), we quantified global levels of 5-methylcytosine (5mC) and N6-methyladenine (6mA) across defined life-cycle stages of Ulva, including gametogenesis induction and gamete release. We observed a rapid and pronounced increase in 5mC immediately following removal of sporulation inhibitors, consistent with active epigenetic reprogramming. In contrast, 6mA was detected only in mature thallus (vegetative tissue of macroalgae) and gametes, and not throughout gametogenesis, indicating a distinct yet minor role for this DNA modification in Ulva development. Re-addition of sporulation inhibitors prevented both gametogenesis and the characteristic methylation dynamics, indicating that these not-yet-characterized inhibitors act as epigenetic modulators. Together, our findings identify DNA methylation as a key regulatory layer linking bacterial cues, sporulation control, and reproductive development in Ulva, with direct implications for the epigenetic management of growth and reproduction in marine algal biotechnology.