Evolution of C(4) Photosynthesis in Flaveria Species

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C(4) photosynthesis, provides CO(2) 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.