Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development

Stomatal development driving bHLHs are conserved -- it is likely that the genome of the first plant with stomata already carried a single gene ancestral to these genes. It has been proposed that the ancestral bHLH gene may have duplicated and gained novel functions which lead to the stage-specific functions characterizing three angiosperm stomatal master regulators, SPEECHLESS (SPCH), MUTE, and FAMA. However, it has remained unexplored when and how these stage-specific functions evolved. Here, we perform the first functional analysis of stomatal regulators from non-angiosperm vascular plants by using fern Ceratopteris richardii and gymnosperm Picea abies as model species. We show that the genomes of these species contain a single gene encoding SPCH/MUTE (SM), and another gene encoding FAMA. These factors are likely to share a common ancestral gene with angiosperm SPCH and MUTE, and angiosperm FAMA, respectively. Our work shows that C.fern and spruce SM and FAMA can promote gene functions resembling angiosperm SPCH and MUTE, and FAMA functions, respectively. However, all the four proteins display ability to drive asymmetric entry divisions in the stomatal lineage characteristic previously associated specifically to angiosperm SPCH. Further, PaSM is sufficient to promote stomatal pore formation, suggesting that it can control subset of FAMA target genes. Our work demonstrates that SM and FAMA in early evolved vascular plants already show functional divergence thus resembling angiosperm stomatal regulators, however, they also display multifunctional properties.