2.1 Plant material and growth conditions
O. alismoides seeds were sown in soil from Donghu Lake, adjacent to the laboratory in Wuhan, and covered with sterile tap water with an alkalinity of about 2.2 mequiv L-1 as described (Huang et al., 2018). After a month, seedlings were placed in a 400-L tank (64 cm deep) receiving natural daylight in a glasshouse on the flat roof of the laboratory. The tap water in the tank was changed weekly and snails were removed daily. After nearly two months, the plants in the tank had produced many mature leaves. pH and temperature were measured every day with a combination pH electrode (E-201F, Shanghai Electronics Science Instrument Co., China) connected to a Thermo Orion Dual Star Benchtop pH/ISE Meter. The alkalinity was measured by Gran titration with a standard solution of HCl. CO2 concentrations were calculated from pH, alkalinity, and temperature using the equations in Maberly (1996). Because of their high biomass the plants generated high pH values (8.3-9.7) and low concentrations of CO2 (0.11-6.15 μM) in the tank. Information of the conditions in the tank is shown in Supplementary Data Table S1.
To examine whether HCO3- acquisition was affected by carbon limitation, in a separate experiment O. alismoides was incubated at high and low CO2concentration for 40 days in plastic containers within one of the tanks in the glasshouse as described previously (Zhang et al., 2014). The pH in the low CO2 treatment (LC) ranged from 8.0 to over 9.8 and the CO2 concentration ranged from 0.1 to 13 μM with a mean of 2.4 μM. For the high CO2 treatment (HC), CO2-saturated tap water was added to the buckets twice each day in order to keep the pH between 6.7-6.8, producing CO2 concentrations between 481-1110 μM with a mean of 720 μM (Supplementary Data Table S1). These different CO2 acclimated leaves were used to detect the effect of AZ and DIDS on Ci uptake rate and external CA activity.