Regulation of stomatal development initiation and cell fate transitions by the bHLH transcription factor SPEECHLESS [electronic resource]
Keywords: Biology.

Developmental programs are carefully regulated to generate organized, functional multicellular organisms. Beginning with the single celled zygote, transcription factors regulate gene expression to modulate proliferative and differentiation processes. The spatiotemporal regulation of transcription factors themselves, in turn, is necessary in order to execute programs in the correct spatiotemporal contexts. The plant epidermis is a useful post-embryonically derived organ to study how developmental programs are orchestrated. In Arabidopsis, leaf primordia provide a common pool of precursors from which pavement cells, trichome cells, and guard cells are all specified. As guard cells develop through a series of divisions and cell fate transitions, the stomatal developmental program allows for a dissection of both proliferation and differentiation mechanisms. Previous work has identified three closely related transcription basic helix-loop-helix transcription factors, SPEECHLESS (SPCH), MUTE, and FAMA that sequentially regulate developmental decisions during lineage progression. SPCH initiates the developmental program and controls the early asymmetric proliferative divisions of the lineage while MUTE and FAMA promote the fates of later stage precursors. SPCH, MUTE, and FAMA are closely related and their different functions in stomatal development are attributable to their biochemical function rather than their different expression patterns. Cell-to-cell signaling molecules likely converge on SPCH phospho-regulation to impose proper epidermal patterning. In my dissertation work presented here, I use various approaches to better define the role of SPCH in stomatal development on several levels, considering how sequence motifs confer different developmental behaviors, what genes SPCH regulates, and how developmental modules with common signaling molecules that converge on SPCH function are partitioned during development. To determine how SPCH differs from the later acting stomatal bHLHs, I use a structure-function approach focused on SPCH to identify how alteration of sequence motifs alters stomata developmental outputs. Based on theses studies, I find that stomatal bHLHs differ in their requirement for DNA-binding and that phosphorylation has both quantitative and qualitative effects on SPCH activity. To assess what genes are under SPCH transcriptional control, I generated inducible hyperactive SPCH lines and used RNA-sequencing to identify SPCH targets on the genome-wide level. Combined with ChIP-seq experiments performed by Dr. On Sun Lau, the RNA-sequencing results identify several previously characterized positive and negative stomatal regulators among SPCH targets. Follow-up characterization of two CXC domains genes identified in the RNA-sequencing screen, revealed a role of these genes in both early and late stage precursors to restrict extra cell divisions. To analyze the coordination of developmental programs on a larger scale, I analyzed ERf receptors and CHALLAH family ligands, which are cell-to-cell signaling molecules that negatively regulate early stomatal lineage progression. In collaboration with Emily Abrash, our results indicate that CHAL family ligands mediate the non-stomatal roles of ERECTAf receptors and that the TMM receptor allows for tissue specific signaling specificity by inhibiting CHALf signaling. Lastly, I propose follow up experiments to further understand the diversification of stomatal bHLHs and the role of CXC domain proteins through the identification of binding partners and expansion of transcriptional regulation experiments.