2.3. Respiration measurement and determination of AOX capacity
Total respiration, and cytochrome and alternative respiratory
activities of S. minima leaves were measured polarographically
with the Clark electrode (Yellow Springs Instrument Co. USA) and
recorded in a Gilson oxygraph (Gilson Medical Electronics, Inc.USA), at 28 ◦C in a thermostatted cell. Floating and submerged
leaves from both Cr-untreated (control) and Cr-treated plants
were harvested in the sixth day and sliced with a razor blade
at approximately 1 mm strips to yield 70 and 100 mg (wet plant
biomass) of floating and submerged leaves, respectively. Strips
were submerged in 2 mL of an air saturated solution (initial concentration
of O2 was considered to be 240 M) containing 50 mM
NaH2PO4/Na2HPO4 buffer, pH 7.0. This pH value was selected
because it is similar to pH of the artificial pond. The oxygen
consumption was measured using 1 mM KCN as inhibitor of the
cytochrome pathway and 3 mM SHAM (salicylhydroxamic acid) as
inhibitor of the alternative oxidase. These concentrations of KCN
and SHAM had no apparent side effect in our system and produced
maximal inhibition, as indicated by titration curves using different
concentrations of each inhibitor in the presence and absence
of the other. Steady rates of respiratory O2 consumption were
determined after about 20 min under assay conditions. Alternative
oxidase capacity (AOX capacity) defined as the SHAM-sensitive O2
uptake in presence of KCN [37], was determined by subtracting the
rate of O2 uptake in presence of both KCN and SHAM from the rate
of O2 uptake in presence of only KCN. The O2 uptake in presence of
SHAM and KCN is called residual respiration. Although the role of
residual respiration is unclear it has been suggested that it is apparently
related to the activity of supplementary terminal oxidases,
including Cu-containing oxidases and/or other non-mitochondrial
oxidases [38,39]. Respiration data were expressed as nmol O2 mg−1
FW min−1.