Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C4 system of Hydrilla verticillata

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C-4 plant. It typically exhibits C-3 photosynthetic characteristics, but exposure to low [CO2] induces a C-4 system in which the C-4 and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C-4 induction. This and enzyme kinetic data were consistent with it being the C-4 photosynthesis isoform. However, the C-4 signature serine of terrestrial plant C-4 isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C sequences, was present. Western analyses of C and C-4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C-3, non-graminaceous C-4, and Crassulacean acid metabolism PEPCs but not with the graminaceous C-4, and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C-4 angiosperms.