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bioRxiv · 10.1101/2025.11.10.687565

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

Iida, M.·Elhazzime, K.·Vaten, A.
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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 showed that all the four bHLHs displayed broad developmental capacities and promoted asymmetric stomatal-lineage entry divisions, a function associated with angiosperm SPCH. Moreover, PaSM promoted pore-like structure formation in the fama background, indicating a capacity extending into a developmental transition controlled by angiosperm FAMA. To examine the functions of PaSM and PaFAMA in the native context, we generated transgenic spruce lines expressing dominant-negative variants of these genes. Perturbation of either bHLH was associated with fewer epidermal cells between adjacent stomata, whereas the two constructs produced partially distinct effects on mature stomatal-complex morphology. Together, our findings show that the fern and gymnosperm stomatal bHLHs examined here possess broad and overlapping developmental capacities. These results support a model in which the stage-specific functions of angiosperm stomatal regulators evolved through the progressive refinement and partitioning of broader, partially overlapping developmental capacities.

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Iida, M., Elhazzime, K., Vaten, A.. 2025-11-11. Fern and gymnosperm SPCH/MUTE and FAMA can regulate multiple cell fate transitions during stomatal development. https://doi.org/10.1101/2025.11.10.687565

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