The C3 plant Flaveria pringlei contains a plastidic NADP-malic enzyme which is orthologous to the C4 isoform of the C4 plant F. trinervia.

To study the molecular evolution of NADP-dependent malic enzyme (NADP-ME) in the genus Flaveria a leaf-specific cDNA library of the C3 plant F. pringlei was screened for the presence of sequences homologous to the C4 isoform gene (named modA) of the C4 plant F. trinervia. The cDNAs isolated contained varying numbers of identical restriction fragments suggesting that they were derived from a single gene. This was supported by Southern hybridisation experiments with genomic DNA from F. trinervia and F. pringlei. Nucleotide sequence analysis of a full-size clone identified the presence of a typical plastidic transit peptide and revealed that the mature modA proteins of F. trinervia (C4) and F. pringlei (C3) are 90% similar. These findings indicate that C3 plants, like C4 species, possess a plastidic isoform of NADP-ME and that the modA genes of the two species represent orthologous genes. Northern analyses showed that modA transcripts accumulate to similar levels in leaves, stems and roots of F. pringlei. The expression of this gene in F. pringlei thus appears to be rather constitutive. In contrast, the modA gene of F. trinervia is abundantly expressed in leaves, but maintains its expression in stems and roots. It has to be concluded from these data that the leaf-specific increase in the expression level was a key step which was taken during the evolution of the C4 isoform modA gene starting from a C3 ancestral gene.