Evolution of C4 Phosphoenolpyruvate Carboxykinase in Grasses, from Genotype to Phenotype

C-4 photosynthesis is an adaptation over the classical C-3 pathway that has evolved multiple times independently. These convergences are accompanied by strong variations among the independent C-4 lineages. The decarboxylating enzyme used to release CO2 around Rubisco particularly differs between C-4 species, a criterion used to distinguish three distinct biochemical C-4 subtypes. The phosphoenolpyruvate carboxykinase (PCK) serves as a primary decarboxylase in a minority of C-4 species. This enzyme is also present in C-3 plants, where it is responsible for nonphotosynthetic functions. The genetic changes responsible for the evolution of C-4-specific PCK are still unidentified. Using phylogenetic analyses on PCK sequences isolated from C-3 and C-4 grasses, this study aimed at resolving the evolutionary history of C-4-specific PCK enzymes. Four independent evolutions of C-4-PCK were shown to be driven by positive selection, and nine C-4-adaptive sites underwent parallel genetic changes in different C-4 lineages. C-4-adaptive residues were also observed in C-4 species from the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) subtype and particularly in all taxa where a PCK shuttle was previously suggested to complement the NADP-ME pathway. Acquisitions of C-4-specific PCKs were mapped on a species tree, which revealed that the PCK subtype probably appeared at the base of the Chloridoideae subfamily and was then recurrently lost and secondarily reacquired at least three times. Linking the genotype to subtype phenotype shed new lights on the evolutionary transitions between the different C-4 subtypes.