Ottelia alismoides is a constitutive C₄ plant and bicarbonate user, and has facultative crassulacean acid metabolism (CAM) at low CO₂. Acclimation to a factorial combination of light and CO₂ showed that the ratio of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (>5) is in the range of that of C₄ plants. This and short-term response experiments showed that the activity of PEPC and pyruvate phosphate dikinase (PPDK) was high even at the end of the night, consistent with night-time acid accumulation and daytime carbon fixation. The diel acidity change was maximal at high light and low CO₂ at 17–25 µequiv g⁻¹ FW. Decarboxylation proceeded at ~2–3 µequiv g⁻¹ FW h⁻¹, starting at the beginning of the photoperiod, but did not occur at high CO₂; the rate was greater at high, compared with low light. There was an inverse relationship between starch formation and acidity loss. Acidity changes account for up to 21% of starch production and stimulate early morning photosynthesis, but night-time accumulation of acid traps <6% of respiratory carbon release. Ottelia alismoides is the only known species to operate CAM and C₄ in the same tissue, and one of only two known aquatic species to operate CAM and bicarbonate use.

Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO₂-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO₂ concentration and how they may underlie C₄ photosynthesis. Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO₂ concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C₄ photosynthesis were present. When O. alismoides was grown at low CO₂, the leaves performed both C₄ and CAM photosynthesis whereas at high CO₂ leaves used C₄ photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO₂, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO₂, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO₂, for example via C₄ metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO₂. The structural similarity of leaves at low and high CO₂ is consistent with the constitutive nature of bicarbonate and C₄ photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C₄ photosynthesis even though Kranz anatomy is absent.