A transcription factor module mediating C₂ photosynthesis in the Brassicaceae

C₄ photosynthesis has arisen from the ancestral C₃ state in over sixty lineages of angiosperms. It is widely accepted that an early step in C₄ evolution is associated with the appearance of so-called C₂ photosynthesis caused by loss of glycine decarboxylase activity from mesophyll cells followed by activation in the bundle sheath. Although changes in cis to a distal enhancer upstream of the P-subunit of GLYCINE DECARBOXYLASE (GLDP) from C₂ Moricandia enable loss of expression from mesophyll cells, the mechanism then allowing GLDP expression in the bundle sheath is not known. Here we identify a MYC-MYB transcription factor module previously associated with the control of glucosinolate biosynthesis as the basis of this foundational event in the evolution of C₂ photosynthesis. Specifically, we find that in the C₃ state this MYC-MYB module already patterns GLDP expression to bundle sheath cells. As a consequence, when GLDP expression is lost from the mesophyll, the MYC-MYB dependent expression in the bundle sheath is revealed. Evolution of C₂ photosynthesis is thus associated with a MYC-MYB based transcriptional network already present in the C₃ state. This work identifies a molecular genetic mechanism underlying the bundle sheath accumulation of glycine decarboxylase required for C₂ photosynthesis and thus a fundamental step in the evolution of C₄ photosynthesis.