Single-cell transcriptomics unveils photosynthetic adaptation in submersed leaves of heterophyllous Nuphar pumila.

C4 photosynthesis has emerged in various plant lineages as an adaptive response to abiotic stress, as it allows for higher photosynthetic efficiency. C4 photosynthesis has been uncovered in both terrestrial and aquatic plants. However, the extent to which C4 photosynthesis serves as an adaptation to submerged environments in aquatic plants remains elusive. Here, we studied the heterophyllous aquatic plant Nuphar pumila, which has phenotypically distinct leaves depending on the level of submergence. By applying 13C labeling experiments, submersed leaves of N. pumila were shown to exhibit both C4 and C3 photosynthesis, whereas floating leaves are limited to C3 photosynthesis. Leaf-specific single-cell trajectories of epidermal and bundle sheath cells show that, in contrast to terrestrial C4 plants, CO2 fixation in C4 photosynthesis in submersed leaves primarily occurs in the epidermal cells rather than in the mesophyll cells, allowing the leaves to cope with limited inorganic carbon in water. The combined presence of C3 and C4 photosynthesis, along with tissue-specific adaptations in the heterophyllous aquatic plant N. pumila, demonstrates how C4 photosynthesis in aquatic environments likely evolved as an adaptive trait from C3 photosynthesis.