Molecular Framework of a Regulatory Circuit Initiating Two-Dimensional Spatial Patterning of Stomatal Lineage

Abstract

Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis.

Author Summary

Generation of self-organized, functional tissue patterns is critical for development and regeneration in multicellular organisms. Small valves on the epidermis of land plants, called stomata, mediate gas-exchange while minimizing water loss. Density and spacing of stomata are regulated by transcription factors that drive differentiation as well as by cell-cell signaling components that regulate entry and spacing of stomatal lineage cells. To unravel how interaction of these components translates into two-dimensional patterning of stomata, we have taken an integrative approach employing molecular genetics, imaging, and mathematical modeling. In this paper we have identified a regulatory circuit controlling the initiation of the stomatal cell lineage. The key elements of the circuit are a positive feedback loop constituting self-activation of the transcription factors SCREAM / SCREAM2 (SCRMs) that requires SPEECHLESS (SPCH), and a negative feedback loop involving the signaling ligand EPF2, the receptor modifier TOO MANY MOUTHS, and the SPCH•SCRMs module. The receptor ERECTA, on the other hand, lies outside of the regulatory loop. Our mathematical modeling recapitulated all known stomatal phenotypes with the addition of two regulatory nodes. This work highlights the molecular framework of a self-organizing patterning system in plants.