Abstract

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 K_m 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.