EVOLUTION OF THE C-4 PHOSPHOENOLPYRUVATE CARBOXYLASE PROMOTER OF THE C-4 DICOT FLAVERIA-TRINERVIA - AN EXPRESSION ANALYSIS IN THE C-3 PLANT TOBACCO

The key enzymes of photosynthetic carbon assimilation in C-4 plants have evolved from C-3 isoforms which were present in the C-3 ancestral species. We are interested in the molecular changes responsible for the novel expression pattern of C-4 genes and are focussing on phosphoenolpyruvate carboxylase (PEPCase) of the genus Flaveria. The C-4 isoform of PEPCase in the C-4 plant F. trinervia is encoded by the ppcA subgroup of the PEPcase gene family and is abundantly expressed in the mesophyll cells of leaves. The orthologous ppcA genes of the C-3 plant F. pringlei are only weakly expressed and their transcripts do not accumulate in a leaf-specific manner but, rather, are present in all plant organs. To answer the question whether the differences in the expression levels of the ppcA genes from F. pringlei and F. trinervia are caused by changes in the 5' upstream regions of the genes or by C-4-specific trans-regulatory factors, varying parts of the 5' flanking region of the ppcA1 genes of both species were fused to the beta-glucuronidase (GUS) gene and inserted in the tobacco genome. GUS expression analysis of transgenic plants revealed that the level of expression of the Flaveria ppcA1 genes are recapitulated in the heterologous C-3 plant tobacco. Hence, the 5' upstream region of the ppcA1 gene of F. trinervia contains regulatory cis-elements that are responsible for the C-4-specific, abundant expression of this gene. These sequences are located upstream of position -500 relative to the transcription initiation site. Interestingly, the ppcA1 promoter (position -2118 to position +66) of F. trinervia is specifically active in palisade parenchyma cells of transgenic tobacco, suggesting that the mesophyll specificity of this promoter is partially maintained in tobacco.