A conserved cytochrome for organ development

Abstract

The Arabidopsis genome encodes 15 cytochrome b5 proteins, most of which have metabolic functions such as nitrate reduction or sphingolipid desaturation. Of these cytochrome b5 proteins, only RLF is reported to be required for organ development. Specifically, rlf mutants display defective lateral root formation as well as reduced primary root growth and smaller leaf size. In these mutants, auxin signalling is unaffected, suggesting that the role of RLF in organ development is independent of auxin signalling. Cytochrome b5 proteins have a haem-binding domain and coordinated haem is required for their electron carrier function. However, it is not clear whether haem binding and thus the canonical electron carrier function are required for RLF’s role in organ development, and whether this role is conserved in plants.

The researchers confirmed in vitro haem binding to purified Arabidopsis RLF spectroscopically by the presence of the typical Soret and α/β bands. Two conserved histidine residues were identified, which are required for in vitro haem binding. The lateral root phenotype of rlf mutants could be complemented with wild-type RLF but not when either of the two histidine residues were mutated to alanine, suggesting that haem binding is also required for the in planta functionality of RLF. Phylogenetic analysis showed that RLF orthologues form a separate clade among cytochrome b5 proteins that is conserved in plants. The wild-type RLF from M. polymorpha was confirmed to bind haem in vitro, and MpRLF could fully complement the lateral root phenotype of Arabidopsis rlf mutants, suggesting functional conservation. In Marchantia, RLF is expressed both in vegetative and reproductive growth phases, and mutants display stunted growth and various defects in the development of organs with both vegetative and reproductive functions. These phenotypes can be partially complemented by the Arabidopsis RLF orthologue, again showing considerable functional conservation. Transcriptomics showed that predominantly genes with metabolic functions were differentially expressed between rlf mutants and the wild type, including genes involved in nitrate metabolism, which is known to affect lateral root formation. This might suggest that RLF affects organ development through a metabolic function; it would be interesting to confirm such a hypothesis by finding direct interactors of RLF in future.