Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata

Hydrilla verticillata has a facultative single-cell system that changes from C-3 to C-4 photosynthesis. A NADP(+)-dependent malic enzyme (NADP-ME) provides a high [CO2] for Rubisco fixation in the C-4 leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C-4 leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C-4 species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C-3 and C-4 species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The K (m) (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C-4 system in H. verticillata.