LAUSR

Abstract Evolution of C4 photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C4 species, but several C3‐C4 intermediates, mainly in the genus Moricandia. Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C3‐C4 intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C3 relative M. moricandioides enabled us to unravel the specific C3‐C4 characteristics in these Moricandia lines. Reduced CO2 compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C3 to C3‐C4 intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P‐protein, followed by dominant activity of a bundle sheath‐specific element in its promoter. In contrast to recent models, installation of the C3‐C4 pathway was not accompanied by enhanced activity of the C4 cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C4 in Moricandia.