Plant Molecular Biology 26: 1775-1783, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1775 The C 3 plant Flaveria pringlei contains a plastidic NADP-malic enzyme which is orthologous to the C 4 isoform of the C 4 plant F. trinervia B~bel Lipka, Klaus Steinm/aller, Elke Rosche, Dagmar Btrsch and Peter Westhoff* Institut fiir Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universitiit, D-40225 Diisseldorf, Germany (* author for correspondence) Received 11 March 1994; accepted in revised form 7 September 1994 Key words: C 4 photosynthesis, molecular evolution, nadp-malic enzyme, flaveria Abstract 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 ho- mologous 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~o 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. Introduction NADP-dependent malic enzyme (NADP-ME; oxaloacetate-decarboxylating, EC 1.1.1.40) is an abundant protein in malate-transporting C4 plants and serves as a diagnostic criterion for the classification of C4 plants [ 10, 12]. The enzyme is located in bundle-sheath chloroplasts and ca- talyses the oxidative decarboxylation of malate [13]. NADP-malic enzyme plays also a central role in crassulacean acid metabolism [35] and, moreover, NADP-ME activity has been detected in non-photosynthetic tissues of Ca and C4 plants. It has been proposed that this non-photosynthetic NADP-ME is involved in an alternative branch of the glycolytic pathway and that it functions in The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X78069.