Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants

The evolution of C₄ photosynthesis from C₃ ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C₃, C₃–C₄ intermediate and C₄ species from 12 lineages of C₄ evolution. All C₃ species had more chloroplasts per M cell area than their C₄ relatives in high‐light growth conditions. C₃ species also had higher chloroplast coverage of the M cell periphery than C₄ species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C₄ lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C₄ species than their C₃ relatives. C₃–C₄ intermediate species generally exhibited similar patterns as their C₃ relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C₄ function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C₄ M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C₄ photosynthesis.