2.2 pH-drift experiments
The pH-drift technique was used to determine the capacity of O. alismoides to utilize HCO3-, and the effects of inhibitors (AZ and DIDS) on photosynthetic Ci uptake (Maberly & Spence, 1983). Measurements were made in a glass and plastic chamber (Maberly, 1990) containing 121 mL of 1 mM HCO3- comprising equimolar concentration of NaHCO3 and KHCO3, a pH electrode (model IP-600-9 Jenco Instruments, USA) and an oxygen electrode (Unisense OX-13298). The chamber was placed in a water bath maintained at 25 ℃ and illuminated from the side by fluorescent tubes that provided 75 μmol photon m-2 s-1 (400-700 nm, Li-Cor sensor connected to a Li-Cor LI-1400 data logger). Prior to the start of the pH drift experiments, the leaves were collected from the tank in the glasshouse in the morning to avoid possible physiological differences caused by a light:dark rhythm of the plant, and then pieces of ~1.1 g fresh weight (FW) of leaf tissue were cut and rinsed in the medium placed in a constant temperature room at 25 ± 2 °C for around 1-4 hours before use. The medium in the incubation chamber was initially bubbled with N2 to reduce O2 concentration ~100 ± 20 μM, which was detected by the oxygen electrode connected to an Unisense microsensor multimeter (Version 2.01) and recorded on a laptop computer. At the start of all drift experiments, the pH of the medium was set to 7.6 with CO2-bubbled medium, and the subsequent changes were measured with the pH electrode connected to a pH meter (model 6311, Jenco Instruments, USA), and recorded on a monitor (TP-LINK, TL-IPC42A-4). The pH-drifts, undertaken at least in triplicate, took 6-23 h to reach an end point value (final pH), which was deemed to be achieved when the pH changed less than 0.01 unit in one hour (Maberly, 1990). After each drift, the dry weight of the plant material and the alkalinity of the medium were measured, allowing Ci concentrations and Ci uptake rates to be calculated (Maberly & Spence, 1983). When photosynthetic Ci uptake rates were plotted against the total carbon concentration (CT) at which the rate occurred, a two-phased response curve was observed. The linear response at higher CT concentration was the consequence of CO2 use, and the extrapolated intercept with the CT axis corresponded to the CO2compensation point (Maberly & Spence, 1983).