Regulation and localization of key enzymes during the induction of Kranz-less, C4-type photosynthesis in Hydrilla verticillata

Kranz-less, C-4-type photosynthesis was induced in the submersed monocot Hydrilla verticillata (L.f.) Royle. During a 12-d induction period the CO2 compensation point and O-2 inhibition of photosynthesis declined linearly. Phosphoenolpyruvate carboxylase (PEPC) activity increased 16-fold, with the major increase occurring within 3 d. Asparagine and alanine aminotransferases were also induced rapidly. Pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (ME) activities increased 10-fold but slowly over 15 d. Total ribulose-1,5-bisphosphate carboxylase/oxygenase activity did not increase, and its activation declined from 82 to 50%. Western blots for PEPC, PPDK, and NADP-ME indicated that increased protein levels were involved in their induction. The H. verticillata NADP-ME polypeptide was larger (90 kD) than the maize C-4 enzyme (62 kD). PEPC and PPDK exhibited up-regulation in the light. Subcellular fractionation of C-4-type leaves showed that PEPC was cytosolic, whereas PPDK and NADP-ME were located in the chloroplasts. The O-2 inhibition of photosynthesis was doubled when C-4-type but not C-3-type leaves were exposed to diethyl oxalacetate, a PEPC inhibitor. The data are consistent with a C-4-cycle concentrating CO2 in H. verticillata chloroplasts and indicate that Kranz anatomy is not obligatory for C-4-type photosynthesis. H. verticillata predates modern terrestrial C-4 monocots; therefore, this inducible CO2-concentrating mechanism may represent an ancient form of C-4 photosynthesis.