MOLECULAR EVOLUTION OF C-4 PHOSPHOENOLPYRUVATE CARBOXYLASE IN THE GENUS FLAVERIA

C-4 plants are known to be of polyphyletic origin and have evolved ind ependently several times during the evolution of angiosperms. We are i nterested in understanding the molecular changes that the C-4 genes ha ve undergone as they were adapted to their new functions in C-4 photos ynthesis and are using the C-4 phosphoenolpyruvate carboxylase (PEPC) of the genus Flaveria as a model. The PEPCs of F. trinervia (C-4) and F. pringlei (C-3) are encoded by a gene family that is composed of at least three different gene classes named ppcA, ppcB and ppcC. The C-4 PEPC of F. trinervia is encoded by the ppcA gene class and is expresse d at high levels only in the mesophyll cells of the leaves. The neares t neighbour to the ppcA gene class of F. trinervia is found in F. prin glei. Comparisons of this pair of orthologous gene classes are used to identify the C-4-specific differences between the enzymatic propertie s of the ppcA PEPCs and the activities of the ppcA promoters. The two ppcA PEPCs are 96% identical, but differ in the K-m for phosphoeno/pyr uvate (PEP) and their inhibition by malate. Chimerical PEPCs are prese ntly constructed to map the differences in the enzymatic properties of the C-4 and C-3 PEPC isoforms. To investigate determinants for the C- 4 specific expression pattern, the 5' flanking regions of the ppcA1 ge nes of F. trinervia and F. pringlei were fused to the uidA reporter ge ne encoding beta-glucuronidase and transformed into the C-4 plant F. b identis and the C-3 species tobacco. In F. bidentis, the C-4 ppcA1 pro moter drives a high level of expression of the transgene only in the m esophyll cells, while the C-3 ppcA1 promoter leads to low levels of ex pression in leaves, stems and roots. Determinants for the C-4 specific expression of the ppcA1 gene of F. trinervia must therefore be locate d in the 5'-flanking region of this gene. Further analyses showed that two regions, a proximal and a distal segment, are sufficient to gener ate the C-4 specific expression pattern. In tobacco, the C-4 ppcA1 pro moter is preferentially expressed in the palisade parenchyma cells of the leaves. These results indicate that the major events during the ev olution of the C-4 ppcA promoter occurred at the promoter level.