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).