Neofunctionalisation of basic helix-loop-helix proteins occurred when embryophytes colonised the land

ROOT HAIR DEFECTIVE SIX-LIKE (RSL) genes control the development of structures from single cells at the surface of embryophytes (land plants) such as rhizoids and root hairs. RSL proteins constitute a subclass (VIIIc) of the basic helix-loop-helix (bHLH) class VIII transcription factor family. The Charophyceae form the only class of streptophyte algae with tissue-like structures and rhizoids. To determine if the function of RSL genes in the control of cell differentiation in embryophytes was inherited from a streptophyte algal ancestor, we identified the single class VIII bHLH gene from the charophyceaen alga Chara braunii (CbbHLHVIII). CbbHLHVIII is sister to the RSL proteins; they constitute a monophyletic group. Expression of CbbHLHVIII does not compensate for loss of RSL functions in Marchantia polymorpha or Arabidopsis thaliana. In C. braunii CbbHLHVIII is expressed at sites of morphogenesis but not in rhizoids. This finding indicates that C. braunii class VIII protein is functionally different from land plant RSL proteins. This result suggests that the function of RSL proteins in cell differentiation at the plant surface evolved by neofunctionalisation in the land plants lineage after its divergence from its last common ancestor with C. braunii, at or before the colonisation of the land by embryophytes.