Re-creation of a Key Step in the Evolutionary Switch from C₃ to C₄ Leaf Anatomy

The C₄ photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C₄ plants are higher yielding than C₃ plants, efforts are underway to introduce the C₄ pathway into the C₃ crop rice. This is an ambitious endeavor; however, the C₄ pathway evolved from C₃ on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C₄ rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C₃ to so-called "proto-Kranz" anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in "proto-Kranz" species, and, as such, a key step toward engineering C₄ rice has been achieved.