Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 dicot Flaveria trinervia: an expression analysis in the C3 plant tobacco

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Abstract
The key enzymes of photosynthetic carbon assimilation in C4 plants have evolved from C3 isoforms which were present in the C3 ancestral species. We are interested in the molecular changes responsible for the novel expression pattern of C4 genes and are focussing on phosphoenolpyruvate carboxylase (PEPCase) of the genus Flaveria. The C4 isoform of PEPCase in the C4 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 C3 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 C4-specific trans-regulatory factors, varying parts of the 5′ flanking region of the ppcA1 genes of both species were fused to the β-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 C3 plant tobacco. Hence, the 5′ upstream region of the ppcA1 gene of F. trinervia contains regulatory cis-elements that are responsible for the C4-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.