CPOP1 is the key heme synthesis enzyme required for nodule microenvironment control and successful symbiotic nitrogen fixation in Lotus japonicus

Heme is utilized as the cofactor of diverse proteins and plays critical roles in different redox reactions. The fixation active nodules of legumes are abundant with heme-containing leghemoglobin to buffer the oxygen concentration. Symbiotic rhizobia could also synthesize heme, and employ heme for efficient respiration and iron uptake regulation. However, the role and regulation of host-controlled heme production during symbiotic nitrogen fixation have not been directly investigated and experimentally proved. Here, we identified the coproporphyrinogen III oxidase plastid related 1 (CPOP1) as a key regulator of symbiotic heme synthesis enzyme in Lotus japonicus. CPOP1 is specifically highly expressed in nitrogen-fixing nodules, and knocking out CPOP1 alone causes leaf etiolation and dwarfism which could be fully recovered by the exogenous application of nitrogen source, indicating nitrogen fixation defect. The cpop1 mutant shows significantly increased nodule oxygen level and decreased nitrogen fixation activity compared to the wild type (WT). Moreover, cell division and related gene expression of bacteroids are also affected upon CPOP1 knockout. We conclude that CPOP1 is essential for the microaerophilic environment control of infected cells and the activation of rhizobial nitrogenase required for symbiotic nitrogen fixation, through host-regulated nodule heme synthesis. Overall design: In order to reveal core pathways in CPOP1-regulated symbiotic nitrogen fixation, we designed the transcriptome analysis between WT and cpop1 nodules at 10 DPI (young root nodule) and 21 DPI (mature root nodule).