The molecular evolution of phosphoenolpyruvate carboxylase in the genus Flaveria

Abstract

To unravel the key changes which accompanied the transition from C3 to C4 photosynthesis the genus Flaveria with its wide range of C3-C4 intermediate species was selected as a model system. The C4 phosphoenolpyruvate carboxylase (PEPC) gene (ppcA) of the C4 species F. trinervia and its orthologous counterpart of the C3 plant F. pringlei were chosen to identify the C4-specific changes both at the level of enzyme properties and gene expression determinants. Two regions were identified in the C4 and C3 ppcA PEPCs which are responsible for most of the kinetic differences with respect to the substrate PEP. The region between amino acids 296 and 437 is essential for activation by glucose-6-phosphate. The carboxyterminal segment between amino acids 645 and 966 contains a serine at position 774 which is conserved in all C4 PEPC isoforms and is a key determinant for Km-PEP. To understand the sequence of events taken during the evolution of the C4 isoform the ppcA PEPCs of the C3-C4 intermediate species F. pubescens and F. brownii were analyzed. The cis-regulatory signals which are required for the mesophyll-specific expression of the C4 PEPC gene were studied by comparing the expression profiles of the ppcA promoters of F. trinervia and F. pringlei in transgenic F. bidentis. It was found that the C4 ppcA promoter contains an enhancer-like 5' distal region that is necessary for mesophyll-specific expression. When inserted into the promotor of the orthologous ppcA gene of F. pringlei this 5' distal region adds a mesophyll-specific expression component to the C3 promotor.