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 <2.1% of in vitro activity. PEPC from gDNAlines had similar affinity to HCO3- but decreased affinity to phosphoenolpyruvate (PEP) in the absence of the positive effector glucose-6-phosphate. In contrast to maize, PEPC in rice lines was primarily phosphorylated in the dark. Estimated PEP concentrations in rice were higher than those required for half-maximal activity of maize PEPC, suggesting that PEP availability was not limiting. The estimated malate pool was higher than required to inhibit phosphorylated and especially dephosphorylated maize PEPC, consistent with malate inhibiting activity in vivo and compounded by a mismatch in light activation. These results indicate that relieving inhibitory and regulatory constraints, rather than further increasing extractable PEPC capacity, is likely required to increase PEPC-dependent CO2 capture in engineered rice.