A CO2-flux mechanism operating via pH-polarity in Hydrilla verticillata leaves with C-3 and C-4 photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C-4-based, CO2 concentrating mechanism (CCM) that concentrates CO2 in the chloroplasts. Both C-3 and C-4 Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m(-)3), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO2 flux mechanism (CFM), making HCO3- available for photosynthesis by conversion to CO2. DIC at 10 mol m(-3) completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m(-3) DIC. At low irradiance (50 mu mol photons m(-2) s(-1)), where CCM activity in C-4 leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C-3 leaves decreased. In C-4 leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO2 fixation in Hydrilla, also suppressed acidification and alkalization, especially in C-4 leaves. IA does not inhibit the C-4 CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H+-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP(+) ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation.