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 (e.g. rhizoids, root hairs, gemmae, mucilage papillae) that develop from single cells at the surface of diverse groups of land plants. RSL proteins constitute a subclass (VIIIc) of the basic helix loop helix (bHLH) class VIII transcription factor family. We set out to determine if the function of RSL genes in the control of cell differentiation in land plants was inherited from streptophyte algal ancestor. The Charophyceae are a monophyletic class of streptophyte algae with tissue-like structures and rhizoids. We identified the single class VIII bHLH gene from the charophyceaen alga Chara braunii (CbbHLHVIII). Phylogenetic analysis suggests that this protein is sister to the RSL (bHLH subclass VIIIc) proteins and together they constitute a monophyletic group. Expression of CbbHLHVIII does not compensate for loss of the RSL function in either Marchantia polymorpha or Arabidopsis thaliana. Furthermore, CbbHLHVIII is expressed at sites of morphogenesis in C. braunii (e.g. the apices, nodes and gametangia) but not in rhizoids. This indicates that C. braunii class VIII protein is functionally different from land plant RSL proteins; they control rhizoid development in land plants but not in the charophycean algae. These data are consistent with the hypothesis that RSL proteins and their function in the differentiation of cells at the plant surface evolved in the lineage leading to land plants after the divergence of the land plants and C. braunii from their last common ancestor. This may have occurred by neofunctionalisation at or before the colonisation of the land by streptophytes.