Brassicaceae display diverse photorespiratory carbon recapturing mechanisms

Carbon concentrating mechanisms enhance the carboxylase efficiency of the central photosynthetic enzyme rubisco by providing supra-atmospheric concentrations of CO 2 in its surrounding. In the C 4 photosynthesis pathway, this is achieved by combinatory changes to leaf biochemistry and anatomy. Carbon concentration by the photorespiratory glycine shuttle requires fewer and less complex modifications. It could represent an early step during evolution from C 3 to C 4 photosynthesis and an inspiration for engineering approaches. Plants displaying CO 2 compensation points between 10 to 40 ppm are therefore often termed ‘C 3 –C 4 intermediates’. In the present study, we perform a physiological, biochemical and anatomical survey of a large number of Brassicaceae species to better understand the C 3 -C 4 intermediate phenotype. Our phylogenetic analysis suggested that C 3 -C 4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathways showed considerable variation between the species but also within species. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C 3 -C 4 classified accessions indicating a crucial role of anatomical features for CO 2 concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual plant accessions, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of PEPC activities suggests that C 4 -like shuttles have not evolve in the investigated Brassicaceae. Highlight Our physiological, biochemical and anatomical survey of Brassicaceae revels multiple evolution of C 3 -C 4 intermediacy connected to variation in photorespiratory carbon recapturing efficiency and a distinct C 3 -C 4 bundle sheath anatomy.