Evolution of C4 photosynthesis in Flaveria species -: Isoforms of NADP-malic enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C-4 photosynthesis, provides CO2 to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C-3, C-3-C-4 intermediate, C-4-like, C-4) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C-4 isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C-3 Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C-3 photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C-4 cycle activity. The 62-kD isoform, which is the predominant highly active form in the C-4 species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C-4 metabolism, and a 62-kD form that is necessary for the complete functioning of C-4 photosynthesis.