Evidence that inducible C4-type photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submersed monocot

Hydrilla verticillata (L.f.) Royle exhibits an inducible C₄-type photosynthetic cycle, but lacks Kranz anatomy. Leaves in the C₄-type state (but not C₃-type) contained up to 5-fold higher internal dissolved inorganic carbon (DIC) concentrations than the medium, indicating that they possessed a CO₂-concentrating mechanism (CCM). Several lines of evidence indicated that the chloroplast was the likely site of CO₂ generation. From C₄-type leaf [DIC] measurements, the estimated chloroplastic free [CO₂] was 400 mmol m^?3. This gave a calculated 2% O₂ inhibition of photosynthesis, which was identical to the measured value, and provided independent evidence that the estimated [CO₂] was close to the true value. A homogeneous distribution of DIC in the C₄-type leaf could not account for such a high [CO₂], or the resultant low O₂ inhibition. For C₃-type leaves the estimated chloroplastic [CO₂] was only 7 mmol m^?3, which gave high, and similar, calculated and measured O₂ inhibition values of 22 and 26%, respectively. The CCM did not appear to be located at the plasma membrane, as it operated at low and high pH, indicating that it was independent of use of HCO₃^? from the medium. Also, both C₃^? and C₄-type Hydrilla leaves showed pH polarity in the light, with abaxial and adaxial boundary layer values of about pH 4·0 and 10·5, respectively. Thus, pH polarity was not a direct component of the CCM, though it probably improved access to HCO₃. Additionally, iodoacetamide and methyl viologen greatly reduced abaxial acidification, but not the steady-state CCM. Inhibitor studies suggested that the CCM required photosynthetically generated ATP, but Calvin cycle activity was not essential. Both leaf types accumulated DIC in the dark by an ATP-requiring process, possibly respiration, and C₄-type leaves fixed CO₂ at 11·8% of the light rate. The operation of a CCM to minimize photorespiration, and the ability to recapture respiratory CO₂ at night, would conserve DIC in a densely vegetated lake environment where daytime [CO₂] is severely limiting, while [O₂] and temperatures are high.