Diverse mechanisms of adaptive flexibility discovered by multi-species analysis of stomatal development

Stomata are pores in the plant epidermis that control gas exchange between the plant and atmosphere. In Arabidopsis, stomatal development requires the bHLH transcription factor SPEECHLESS (AtSPCH) and perception of signals from adjacent cells, from other parts of the plant and from the environment. SPCH is thought to be a critical target for environmental inputs into development. Despite the power of Arabidopsis as a model for stomatal development, we found crop plants like tomatoes often lean on different cellular and genetic strategies to achieve optimal stomatal distributions. By making genetically encoded reporters of the stomatal lineage, and long-term confocal microscopy we tracked the developing epidermis of M82 (WT) and mutant tomato seedlings. We found that, like in Arabidopsis, tomato undergoes a series of asymmetric and symmetric cell divisions to produce stomata. However, we found one type of asymmetric cell division (ACD) was missing in the tomato epidermis, and other ACDs could be used to generate non-stomatal cells. These data suggest differences in ACD strategies that control stomatal production between plant species. Since SPCH serves as the major integrator of environmental information in stomatal development, we targeted the tomato SPCH promoter for CRISPR-based mutagenesis. By screening lines in response to light and temperature, we found putative SPCH cis-regulatory elements that indicate complexity in the regulation of developmental flexibility. Taken together these results further our understanding of the species-specific cellular and genetic pathways plants use to adapt to their environment.