Evolutionary conserved RLF, a plant cytochrome b5-like heme-binding protein, is essential for organ development in Marchantia polymorpha

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b₅-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root formation. Whereas the other Cytb5-HBD proteins in A. thaliana regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function, and whether RLF orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme in vitro and that the two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, MpRLF, a RLF ortholog in Marchantia polymorpha, rescues the lateral root formation phenotype of the A. thaliana rlf mutant. Mprlf^ge, the loss-of-function mutation in the MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and antheridiophore formation. Transcriptome analysis using Mprlf^ge revealed that MpRLF affects several metabolic pathways. Our findings indicate that MpRLF is essential for vegetative and reproductive development in M. polymorpha, suggesting that the RLF-dependent redox reaction systems are evolutionarily conserved as crucial mechanisms for organ development across diverse plant species.