Wetlands are strongly affected by seasonal hydrological changes and extreme drought events that can cause low water levels. In response, some freshwater plants can produce emergent growth forms that experience a drastic change in environmental conditions. Potamogeton wrightii is a freshwater plant, which grows primarily underwater but can also produce terrestrial shoots when emerged. The objective of this study was to investigate the anatomical and physiological responses of P. wrightii to these two distinct environments, as well as the genetic responses behind them. Aerial leaves of P. wrightii were thicker, and had larger amounts of cutin and wax, developed stomata, had a greater tolerance to strong light, and a greater photochemical efficiency. In contrast, submerged leaves had a greater ability to use HCO3- and to synthesize photosynthetic pigments. The differentially expressed genes including cutin and wax biosynthesis, photosynthesis-antenna proteins and photosynthesis pathways, clarified the molecular adaptive mechanisms in P. wrightii to aquatic and terrestrial environments. The capacity of P. wrightii to survive fluctuating water level can be attributed to its genotype that resulted from its evolution from land plants and its phenotypic plasticity. Further work is needed to assess the possibility and costs of aerial leaves to survive when re-submerged.

Acclimation to variable CO2 was studied in floating leaves of the freshwater monocot Ottelia cordata grown in either low or high CO2. The most striking anatomical variations responding to high CO2 included the enlarged upper epidermal cells and the decreased area of epidermal chloroplasts. Stomata that distributed on the upper surface, and the stomatic chamber area, showed no significant response to high CO2. pH-drift experiments indicated that floating leaves of O. cordata were able to use bicarbonate regardless of CO2 concentrations. Photosynthetic enzyme activities and patterns of organic acids fluctuation confirmed that floating leaves of O. cordata can operate CAM only at low CO2, and perform C4-like metabolism at both high and low CO2. Overall, the present results imply that the floating leaves of O. cordata does not just rely on the atmospheric CO2 for its inorganic carbon, but is also dependent on CO2 and bicarbonate in the water. By showing these effects of CO2 variation, we highlight the need for further experimental studies on the regulatory mechanisms in O. cordata floating leaves, that prevent futile cycling among the three CO2 concentrating mechanisms (bicarbonate use, C4, and CAM metabolism) and the strategy for exploiting atmospheric CO2, as well as studies on the detailed biochemical pathway for C4 and CAM metabolism in this species.

Ottelia alismoides (Hydrocharitaceae) is a heterophyllous freshwater macrophyte which shows heteroblastic heterophylly during plant development, and differs in carbon dioxide-concentrating mechanisms (CCMs) at different stage. This study investigated the anatomy and CCMs in the first stage of leaf during O. alismoides development, linear juvenile leaves. It comprised three layers of cell and enriched air spaces, but not significantly affected by CO2 concentration ([CO2]), while leaf thickness and chloroplast ultrastructure were significantly affected by [CO2]. The linear juvenile leaves could perform C-4 photosynthesis at low [CO2]; the two types of chloroplasts distributed in epidermal and mesophyll cells respectively, with different shape and starch content, might provide the structural basis for C-4 operation. The pH-drift data showed that the linear juvenile leaves could use HCO3- regardless of [CO2]. Whereas, CAM and C-4 operation could only be induced at low [CO2]. When compared the CCMs among all the heteromorphic leaves in O. alismoides, it is shown that CCMs in the O. alismoides heteromorphic leaves become more diversified during the plants development: HCO3- use was present in all leaf-types regardless of the [CO2]; CAM and C-4 were only induced at low [CO2] and became more efficient in mature leaves. The anatomy was also regulated to adapt to the functions of CCMs, and ultimately, the ovate mature leaves can operate C-4 constitutively.

BACKGROUND AND AIMS: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.METHODS: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.KEY RESULTS: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.CONCLUSIONS: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.