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

C₄ photosynthesis has evolved repeatedly fromthe ancestral C₃ state to generate a carbon concentratingmechanism that increases photosynthetic efficiency. This specialized formof photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world's most productive crops. The C₄ cycle is accomplished through cell-typespecific accumulation of enzymes but cis-elements and transcription factors controlling C₄ photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C₄ plants evolved from ancestral components found in C₃ species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C₄ 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 C₃ and C₄ Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C₃ state. We conclude that C₄ photosynthesis has co-opted an ancient C₃ 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 C₄ photosynthesis.