Phosphoenolpyruvate Carboxylase cDNA phylogeny to investigate the C4 photosynthetic pathway evolution in grasses

Abstract

Convergence for appearance of C4 photosynthetic system has likely occurred in plant evolution. There are evidences for such hypothesis based on phylogenies of specific C4 genes in monocotyledonous and dicotyledonous plants. It was also suggested that C4 photosynthesis evolved independently within grass subfamilies. In C4 plants, the enzyme that allows CO2 fixation is a Phosphoenolpyruvate Carboxylase (PEPCase) isoform called C4 PEPCase. Primers specific of maize - sorghum C4 PEPCase mRNAs and RT-PCR were used to generate partial PEPCase cDNA fragments in 23 C4 grasses. To check the C4 photosynthetic pathway evolution within grasses, fragments of about 1,200 bp were sequenced in 12 species representative of all grass subfamilies displaying C4 species: Panicoideae, Arundinoideae and Chloridoideae. All sequences displayed high homology with known grass C4 PEPCases. We reconstructed a phylogeny based on all known grass PEPCase nucleotide sequences using distances (percent divergence) and the Neighbor Joining algorithm. C4 PEPCases and analogues formed a monophyletic clade in the PEPCase phylogram while three grass C3 PEPCase groups were distinguished. Phylogenetic relationships between species, deduced from C4 PEPCase sequences, were similar to those deduced from other nuclear data: the different subfamilies and tribes were recognized, and the subfamily Chloridoideae displayed a basal position in our C4 PEPCase clade. Our data sustained that C4 PEPCase isoform should have appeared only once in the grass evolution. Thus, we can assume that all grass C4 photosynthetic pathways have derived from a common ancestor and convergent evolution hypothesis for this system inside grass family would be debatable.