Reinfelder et al. reply

Johnston et al. are not convinced that a C4 photosynthetic pathway exists in microalgae. The essential feature of C4 photosynthesis is the fixation of inorganic carbon as a C4 compound that is subsequently decarboxylated to provide CO2 as a substrate for Rubisco in the Calvin cycle. It is known that many microalgae incorporate at least some inorganic carbon directly into C4 compounds such as malate, but the controversy hinges on whether this carbon is released and fixed by Rubisco.

The extrapolation of 14C incorporated into the C4 position of malate to 100% at time zero, which is proposed as a crucial diagnostic test by Johnston et al., is thus not a direct verification of C4 photosynthesis. It would be experimentally difficult to determine and is not mathematically well defined — for example, this approach yields an intercept of only 89% in maize, a well-known C4 plant1. A better demonstration is provided by the transfer of 14C from the C4 compound malate to the C3 compound phosphoglycerate, the first product of Rubisco, as we have shown. Also, we have recently found (unpublished results) that addition of the C4 compound oxaloacetate to compensated cultures of T. weissflogii triggers immediate production of O2 in the light, but no O2 consumption in the dark, which further supports our proposal that this diatom uses C4 photosynthesis.

Johnston et al. also comment on the substrate of PEP carboxykinase and the localization of enzymes. However, oxaloacetate, which is the substrate for PEP carboxykinase, can be produced by oxidation of aspartate or malate. We agree that cellular-fractionation techniques cannot definitively establish how the enzymes of a photosynthetic C4 pathway are compartmentalized — so far, fractionation results have been confirmed by other techniques in the case of carbonic anhydrase, which has been shown to be located in the cytoplasm of T. weissflogii using antibodies.

There is strong evidence that marine diatoms can concentrate inorganic carbon for photosynthesis2,3, but little to indicate how such a mechanism might work. C4 pathways are used to accomplish this in multicellular C4 plants and so may serve the same function in marine diatoms.