Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

C 4 photosynthesis has evolved repeatedly from the ancestral C 3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C 4 cycle is accomplished through cell-type specific accumulation of enzymes but cis -elements and transcription factors controlling C 4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme ( NADP-ME ) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C 4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C 4 NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans -activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis -elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis -elements is found in both C 3 and C 4 species of the PACMAD clade. Our analysis is consistent with this cis -element pair originating from a single motif present in the ancestral C 3 state. We conclude that C 4 photosynthesis has co-opted an ancient C 3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C 4 photosynthesis.