450 million years ago, the transition of plants from aquatic to terrestrial life marked a pivotal moment in evolution. As plants adapted to life on land, they developed protection mechanisms against drying. On the other hands, land plant lost their ability to survive underwater.

Some plants, known as aquatic plants, returned to aquatic environments. These plants underwent significant changes, developing soft, tender bodies and small, narrow leaves to withstand water currents. They are exclusively aquatic and cannot survive on land.

In contrast, amphibious plants, which can thrive in both terrestrial and submerged conditions, have evolved unique adaptations to cope with fluctuating environments. These plants, often found in riparian zones, exhibit heterophylly, a phenomenon where they develop leaves with different morphological and physiological characteristics in response to water levels. This adaptation allows them to form hard leaves in terrestrial conditions, similar to typical land plants, and soft, dissected leaves underwater, akin to aquatic plants. Heterophylly has independently evolved in various plant taxa and serves as a typical example of convergent evolution.

Rorippa aquatica, an amphibious plant native to North America, shows remarkable heterophylly in response to environmental cues such as temperature, light, and submergence. Its leaves become more dissected and narrow when submerged than when on land.

 Being a member of the Brassicaceae family and closely related to Arabidopsis thaliana, R. aquatica is an ideal model for studying heterophylly. However, the genome information, which is needed for molecular analysis, of R. aquatica had been lacking.

In our study, the genome of R. aquatica was fully assembled and annotated using next-generation sequencing technology. The genome consists of 15 chromosomes with a total length of approximately 450 Mbase, containing 46,197 genes, significantly larger than related diploid species. Comparative genomics revealed that R. aquatica underwent tetraploidization, with chromosomes clustered into two subgenome groups. One subgenome (subgenome A) is closely related to R. islandica, diverging 4.2 million years ago, while the other (subgenome B) separated from an ancestor of subgenome A species 7.2 million years ago, suggesting allopolyploidization.

Transcriptome analysis identified differentially expressed genes between aerial and submerged conditions related to pathways such as shade avoidance, leaf polarity, and response to plant hormones. Ethylene, a plant hormone, was found to induce submerged-type leaves even in terrestrial conditions, indicating its role as a signal for heterophylly in response to submergence. Because ethylene accumulates underwater, it likely plays a role in the shared submergence response among amphibious plants.

The assembly of the R. aquatica reference genome provides valuable insights into the molecular mechanisms and evolution of heterophylly. While there is much more to uncover about the molecular mechanisms underlying heterophylly, the establishment of genomic information for R. aquatica paves the way for future research in this area.