Ancestral light and chloroplast regulation form the foundations for C₄ gene expression

C₄ photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C₄ pathway is found in more than 60 plant lineages 1 but the molecular enablers of this evolution are poorly understood. In particular, it is unclear how non-photosynthetic proteins in the ancestral C₃ system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C₄ leaves. Here, we provide clear evidence that in C₃ leaves, genes encoding key enzymes of the C₄ pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C₄ leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C₄ cycle genes by light and the chloroplast in the ancestral C₃ state has facilitated the repeated evolution of the complex and convergent C₄ trait.