Investigating a core family of bHLH transcription factors in the regulation of stomatal development in Brachypodium and Arabidopsis

Plants are essential players in the global cycling of carbon and water. The structures central to this function are stomata—adjustable epidermal valves formed by two guard cells (GCs) flanking a pore. Through these pores, atmospheric gases like carbon dioxide or ozone are able to enter the plant, while water vapor and oxygen exit. In addition to playing a role in global ecology, stomata also exhibit differences in morphology, distribution, and patterning which can greatly impact plant water use efficiency and drought tolerance. With rising global temperatures, these attributes are especially appealing for crop development. Therefore, it is necessary to understand the mechanisms behind stomatal development and regulation. Selected for centuries as staple crops, grasses have demonstrated an aptitude for growth even in dry climates where other plants falter, and their distinctive stomatal morphology likely provided the change necessary to give grasses the evolutionary edge in their establishment across the globe. Additionally, grass stomatal development is characterized by distinct asymmetric cell divisions that, when leveraged against knowledge from the dicot species, Arabidopsis thaliana (Arabidopsis), offers an optimal system in which to study the evolution of regulatory strategies involved in cell cycle regulation, division orientation, and fate decisions. In this dissertation, I study aspects of stomatal complex formation and patterning in the genetic model grass, Brachypodium distachyon (Brachypodium) and explore the strategies that grasses used to create efficient stomata. Closely related to both wheat and barley, Brachypodium's small size, faster generation times, and amenability to genetic manipulations make it an ideal model to study grass-specific stomatal development. Five basic-helix-loop-helix (bHLH) transcription factors, FAMA, MUTE, SPCH, ICE1, and SCRM2, regulate specific stages of stomatal development in Arabidopsis, and their homologues have also been found to regulate stomatal development in more crop-relevant grasses such as rice and maize. In Arabidopsis, FAMA activity is required to promote differentiation of GCs and enforce cell fate commitment and a single symmetric division. My work focused on characterizing the role of the Brachypodium ortholog, BdFAMA, in the stomatal lineage, using genetics and protein interaction assays to examine the divergent regulation of stomatal development between dicots and grasses by leveraging the functional differences of FAMA between Arabidopsis and Brachypodium. I show that BdFAMA is necessary and sufficient for specifying GC fate, but bdfama does not exhibit the loss of cell division control seen in Arabidopsis FAMA mutants. I found BdFAMA has a broader temporal expression than expected given its terminal role in stomatal development, and, using further genetic assays within the Brachy stomatal lineage, I show BdFAMA is able to partially compensate for BdMUTE and drive the guard mother cell (GMC) to GC fate transition, in addition to recruiting subsidiary cells in bdmute plants. The compensatory ability of BdFAMA is distinct among the grasses, and I provide evidence that it is due to a difference in FAMA regulation. Despite this divergence within the grasses, BdFAMA was still able to rescue stomatal development in the Arabidopsis fama mutant, demonstrating functional conservation between monocots and dicots. My characterization of the final member in the Brachypodium stomatal bHLH transcription factor family highlights the importance of developing a model system for temperate grasses and reveals we still have much to learn about these master regulators by leveraging genetic analyses across evolutionary timescales. I also discuss innovations and future directions that can build upon the stomatal developmental framework established in Brachypodium and briefly demonstrate the advantages of extending stomatal work in the available sequenced ecotypes. Finally, I take a step back from grasses and review another important aspect of graduate careers: mentorship and DEI work, and offer advice for ways all members of the scientific community can contribute to improving inclusivity in STEM

Keywords: Genetics.
Advisor: Bergmann Dominique
Baker Julie (Professor of genetics)
Fire Andrew Zachary
Lipsick Joseph Steven