Molecular Evolution of C4 Phosphoenolpyruvate Carboxylase in the Genus Flaveria

C4 plants are known to be of polyphyletic origin and have evolved independently several times during the evolution of angiosperms. We are interested in understanding the molecular changes that the C4 genes have undergone as they were adapted to their new functions in C4 photosynthesis and are using the C4 phosphoenolpyruvate carboxylase (PEPC) of the genus Flaveria as a model. The PEPCs of F. trinervia (C4) and F. pringlei (C3) are encoded by a gene family that is composed of at least three different gene classes named ppcA, ppcB and ppcC. The C4 PEPC of F. trinervia is encoded by the ppcA gene class and is expressed at high levels only in the mesophyll cells of the leaves. The nearest neighbour to the ppcA gene class of F. trinervia is found in F. pringlei. Comparisons of this pair of orthologous gene classes are used to identify the C4 -specific differences between the enzymatic properties of the ppcA PEPCs and the activities of the ppcA promoters. The two ppcA PEPCs are 96% identical, but differ in the Km for phosphoenolpyruvate (PEP) and their inhibition by malate. Chimerical PEPCs are presently constructed to map the differences in the enzymatic properties of the C4 and C3 PEPC isoforms. To investigate determinants for the C4 specific expression pattern, the 5´ flanking regions of the ppcA1 genes of F. trinervia and F. pringlei were fused to the uidA reporter gene encoding ß-glucuronidase and transformed into the C4 plant F. bidentis and the C3 species tobacco. In F. bidentis, the C4ppcA1 promoter drives a high level of expression of the transgene only in the mesophyll cells, while the C3ppcA1 promoter leads to low levels of expression in leaves, stems and roots. Determinants for the C4 specific expression of the ppcA1 gene of F. trinervia must therefore be located in the 5´-flanking region of this gene. Further analyses showed that two regions, a proximal and a distal segment, are sufficient to generate the C4 specific expression pattern. In tobacco, the C4ppcA1 promoter is preferentially expressed in the palisade parenchyma cells of the leaves. These results indicate that the major events during the evolution of the C4ppcA promoter occurred at the promoter level.