Active carbon uptake in Laminaria digitata and L-saccharina (Phaeophyta) is driven by a proton pump in the plasma membrane

Mechanisms for inorganic carbon acquisition in Laminaria digitata (Hudson) Lamour and L. saccharina (L.) Lamour from the Swedish west coast were studied in pH-drift experiments, using several inhibitors for different types of carbon uptake across the cell membrane. Throughout the experiments total carbon decreased in concert with a pH increase while alkalinity stayed relatively stable. Addition of acetazolamide had a strong inhibitory effect on the carbon uptake rate in L. digitata and the anion exchange protein inhibitor DIDS had a small inhibitory effect above pH 9.5. These results indicate that carbon uptake in L. digitata depends on the presence of an external carbonic anhydrase while direct bicarbonate uptake may contribute at high pH. These two mechanisms have previously been shown to occur in L. saccharina. We show that two inhibitors of H+-ATPases, vanadate and erythrosin B, also decreased carbon uptake rates in both Laminaria species. The effect of erythrosin B was immediate and it probably acts on the outside of the cell membrane. Contrarily, vanadate needs to be transported into the cell, where it competes with the phosphate from ATP for the aspartic acid phosphorylation site on the plasma membrane P-type H+-ATPase. Therefore, 1-2 h of pH drift were usually required before an inhibitory effect became apparent. Additional experiments with P-enriched and P-starved L. saccharina corroborated this process. Based on these results we suggest that the investigated Laminaria species, besides external carbonic anhydrase and DIDS-sensitive anion exchange, also possess a mechanism for the active uptake of carbon, which is dependent on plasma membrane P-type H+-ATPase activity. This paper also reports on the buffering capacity of the inhibitors when used in natural seawater, an aspect that has been neglected in previous studies.