Gymnosperm and fern multifunctional bHLH transcription factors control stomatal development

Abstract

Stomatal development is driven by a conserved family of basic Helix-loop-helix (bHLH) transcription factors. In the angiosperm model Arabidopsis thaliana, SPEECHLESS (SPCH), MUTE and FAMA show pronounced yet incompletely separated stage-specific functions. How the functional specialization observed within angiosperms evolved has remained unclear. Here, we perform the first functional analysis of stomatal regulators from the fern Ceratopteris richardii and the gymnosperm Picea abies (Norway spruce). We show that both species possess a single SPCH/MUTE-like gene (CrSM and PaSM) together with a single FAMA-like gene (CrFAMA and PaFAMA). Cross-species complementation in Arabidopsis revealed broad but biased developmental capacities: all four bHLHs could promote stomatal-lineage entry, whereas SM more effectively supported early-to-intermediate transitions and FAMA showed stronger capacities at later stages. PaSM and PaFAMA showed overlapping but distinct expression patterns within spruce stomatal lineage, with PaSM enriched at early to middle developmental stages and PaFAMA present across a broader developmental window. Dominant-negative perturbation of either gene in spruce was associated with altered stomatal-file patterning while producing partially distinct effects on mature stomatal-complex morphology. Together, these findings show that PaSM and PaFAMA display broad and in-part overlapping developmental roles within spruce stomatal lineage. Our data supports the model in which angiosperm stage specialization emerged through progressive partitioning of broader, overlapping developmental capacities.