NADP-dependent malic enzyme gene family in a facultative single-cell C4 photosynthesis system

The facultative single-cell C4 plant Hydrilla verticillata switches from C3 to C4 photosynthesis during growth at low CO2 concentrations. In C4 leaves, intracellular segregation of the C4 and C3 cycles between cell compartments enables CO2 to be concentrated in the chloroplasts. The objective of this work was to identify and characterize the specific NADP+-dependent malic enzyme (NADP-ME) isoform that provides CO2 for Rubisco in C4 leaves. Three genes encoding NADP-ME isoforms were identified in Hydrilla. Expression studies showed light-dependent increases in hvme1 transcripts during induction of the C4 cycle in Hydrilla leaves, but not for the other two isoforms, hmve2 and hvme3. The specific activity of NADP-ME was higher in C4 than in C3 leaves but it was not light regulated. An NADP-ME protein band of ∼66 kD was present in C3 and C4 leaves with a similar pl to that predicted for the hvme1 polypeptide (5.5 to 6.0). HVME1 was the only isoform with a predicted transit peptide, and was found only in chloroplast fractions where it occurred as a tetramer. A phylogenetic analysis suggested that at least four duplication events gave rise to chloroplastic NADPME isoforms, and that the Hydrilla hvme1 may be derived from the cytosolic hvme3. The molecular and kinetic properties for HVME1m and HVME3 fusion proteins were compared. The mature form of hvme1, HVME1m, showed similar molecular mass (64 kD) and pl (6.0) values to the band detected in C4 leaves. Moreover, kinetic analyses showed that HVME1m had characteristics of a chloroplastic isoform, unlike HVME3. HVME1m had an optimum pH of 7.8, lower Km values for malate (0.68 mM) and NADP + (0.010 mM), and up to a three-fold higher kcat value (47.8 s-1) than HVME3. This is the first molecular and biochemical characterization of the NADP-ME gene family in a single-cell C 4 plant. Hydrilla is a model for attempts to enhance productivity in C3 crop species, such as rice, by introducing a C4 cycle. The results of this study suggest that isoforms with specific regulatory features, such as found in HVME1m, may be needed to successfully engineer a C4 cycle in a C3 crop plant.