SPEECHLESS duplication in grasses expands potential for environmental regulation of stomatal development

Plants acquire atmospheric carbon dioxide for photosynthesis while minimizing water loss and do so by regulating stomatal function and development. The ancestral basic helix-loop-helix transcription factor (TF) gene that drove stomata production in early land plants diversified in sequence and function to become paralogs SPEECHLESS (SPCH), MUTE, and FAMA. Extant angiosperms use these three TFs and their heterodimer partners to regulate stomatal cell identities. Grasses exhibit a particularly interesting set of duplications and losses of SPCH. Using phylogenetic methods, we tracked the duplication of SPCH to the Poaceae-specific rho whole genome duplication and demonstrated that both paralogs remain under selection. By following responses to environmental change in B. distachyon plants bearing mutations in either BdSPCH1 or BdSPCH2 , we reveal paralog-specific divergence in response to light or temperature shifts, and further show this behavior is conserved O. sativa SPCH paralogs. Plausible molecular mechanisms underpinning paralog divergence, and cellular mechanisms driving the stomatal phenotypes are supported by analyses of RNA and protein expression in B. distachyon and sequence variation among grasses. These studies suggest ways in which a duplication of a key stomatal regulator enables adaptation and could inform genetic strategies to mitigate anticipated stressors in agronomically important plants.