Evolutionary-conserved RLF, a cytochrome b₅-like heme-binding protein, regulates organ development in Marchantia...

In Arabidopsis thaliana, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome b₅-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root (LR) 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 two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in A. thaliana. In addition, we show that MpRLF, a RLF ortholog in the bryophyte Marchantia polymorpha, also binds to heme in vitro and that MpRLF rescues the LR formation phenotype of the A. thaliana rlf mutant. Mprlf^ge, the loss-of-function mutation in MpRLF, resulted in delayed thallus growth and inhibited both gemma cup and reproductive organ formation. Our findings indicate that MpRLF is essential for proper vegetative and reproductive development in M. polymorpha. This suggests that RLF-dependent redox reaction systems are conserved across diverse plant species and were independently co-opted for organ development in bryophyte and seed plant evolution.