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

C-4 photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C-4 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 C-3 system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C-4 leaves. Here, we provide clear evidence that in C-3 leaves, genes encoding key enzymes of the C-4 pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C-4 leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C-4 cycle genes by light and the chloroplast in the ancestral C-3 state has facilitated the repeated evolution of the complex and convergent C-4 trait.