Expression of maize PEPC in rice reveals a strategy for establishing a functional C4 CO2-concentrating mechanism

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first irreversible reaction of the CO2 concentrating mechanism (CCM) in C4 photosynthesis. Engineering C4 photosynthesis into the C3 plant rice (Oryza sativa) to increase photosynthetic efficiency requires sufficient activity and proper regulation of PEPC. In previous studies, in vivo PEPC activity remains low despite substantial in vitro activity. Here, we tested the activity and regulation of PEPC expressed in rice using either the coding (cDNA) or genomic (gDNA) sequences of the C4 photosynthetic isoform of PEPC from maize (Zea mays) driven by the ZmPEPC promoter, termed cDNAlines and gDNAlines, respectively. We quantified PEPC enzyme characteristics in vitro, estimated in planta PEPC activity using 13CO2 labeling, and assessed diel phosphorylation status. Both cDNAlines and gDNAlines showed higher PEPC activities than wild-type rice, but in planta activity was