C4-like metabolism and HCO3- use in submerged leaves of Ottelia cordata lacking Kranz anatomy at both low and high CO2 concentrations.

To date, only a few submerged plants have been reported to perform C4 and CAM. Ottelia cordata is a heteroblastic aquatic plant developing both submerged and floating leaves throughout its life cycle. Previous research found that, besides HCO3- use, the submerged leaves of O. cordata can also perform C4 metabolism. However, it remains unclear how the HCO3- use or the C4 pathway, and the anatomical structure, respond to varying CO2 environments. Therefore, we examined the anatomical structures and carbon-concentrating mechanisms in O. cordata submerged leaves under high (HC) and low CO2 (LC) conditions. Our results revealed the leaf consists of an upper and lower layer of epidermal cells, separated by large air spaces and two layers of mesophyll cells, without the presence of Kranz anatomy. Both epidermal and mesophyll cells contained chloroplasts, but starch grains were larger in the mesophyll chloroplasts than in the epidermal cells. Additionally, the area of the upper epidermal cells, mesophyll cells, air spaces, chloroplast, and starch grains significantly increased under HC. Moreover, the ability to utilize HCO3- was stronger under LC. The activity of photosynthetic enzymes, kinetics of O2 evolution, and δ13C confirmed the C4 pathway under both HC and LC. However, the organic acid results indicated that CAM was not operational in either HC or LC. In summary, our findings suggested that the ability to use HCO3- and a C4-like metabolism may occur in the submerged leaves of O. cordata, despite the absence of Kranz anatomy, across both HC and LC environments.