The bHLH transcription factor OsPRI1 activates the Setaria viridis PEPC1 promoter in rice

Photosynthetic efficiency is reduced by the dual role of Rubisco, which acts either as a carboxylase or as an oxygenase, the latter leading to photorespiration. C₄ photosynthesis evolved as a carbon-concentrating mechanism to reduce photorespiration. To engineer C₄ into a C₃ plant, it is essential to understand how C₄ genes, such as phosphoenolpyruvate carboxylase (PEPC1), are regulated to be expressed at high levels and in a cell-specific manner. Yeast one-hybrid screening was used to show that OsPRI1, a rice bHLH transcription factor involved in iron homeostasis, binds to the Setaria viridis PEPC1 promoter. This promoter drives mesophyll-specific gene expression in rice. The role of OsPRI1 in planta was characterized using a rice line harbouring SvPEPC1_pro ::GUS. We show that OsPRI1 activates the S. viridis PEPC1 promoter by binding to an N-box in the proximal promoter, and that GUS activity is highly reduced in SvPEPC1_pro ::GUS lines when OsPRI1 is mutated. Cross-species comparisons showed that the SvPRI1 homolog binds to the SvPEPC1 promoter but the maize ZmPRI1 does not bind to the ZmPEPC1 promoter. Our results suggest that elements of the iron homeostasis pathway were co-opted to regulate PEPC1 gene expression during the evolution of some but not all C₄ species.