Understanding fate transitions in the stomatal lineage of Arabidopsis thaliana

Development, the process by which a single cell gives rise to an entire organism, requires that organisms balance cell proliferation with cell differentiation. While similar themes are found in plant and animal development, multicellularity evolved after the split of the animal and plant lineages 1.6 billion years ago. Thus, few of the actual proteins or pathways are conserved between animals and plants. Plants are known for their developmental flexibility, changing the size and compositions of organs such as leaves in response to the environment. Whole and partial genome duplications in plants have allowed for the expansion of many gene families, leading to subfunctionalization. The increase in the number of cyclins, for example, has led to speculation that different cyclins allow for integration of developmental and environmental information into the cell cycle. Stomata are pores on the surface of the plant epidermis flanked by two guard cells, which allow plants to balance gas exchange with moisture loss and are thus essential for life on land. In addition to the physiological importance of stomata, the stomatal lineage of Arabidopsis thaliana is a microcosm of development. The major regulators of stomatal development are three bHLH transcription factors SPCH, MUTE and FAMA. In stomatal development, first a subset of epidermal precursor cells begins to express SPCH and divide asymmetrically to form stem-cell-like meristemoids, then these meristemoids continue to divide asymmetrically to produce the majority of cells in the leaf epidermis. Next the meristemoid stops expressing SPCH, begins expressing MUTE and transitions into a guard mother cell (GMC). GMCs express FAMA before dividing once, symmetrically, to produce the two guard cells of the stoma. Major cell fate transitions, from meristemoid to GMC and GMC to guard cells, occur and are coordinated with cell divisions. During this process, cell-cell signaling is key for proper patterning of the epidermis, and the lineage responds to signals from the environment and from other tissues. Thus, the stomatal lineage can be used to study many different aspects of development in a tissue that is accessible and amenable to live imaging. While my research in this thesis is focused on Arabidopsis it is worth noting that stomata are found in all higher plants, and my research may be relevant to other plant model species, as well as crop species. In this dissertation, Chapter 1 is an introduction to the literature and is followed by three data chapters (2-4) and a final fifth chapter providing an overall perspective on my work and potential future directions. In Chapter 2, I analyzed SOL1 and SOL2, two stomatal lineage expressed genes identified as direct targets of the transcription factor SPCH. These genes encode proteins containing putative DNA-binding proteins with a distinctive CXC-Hinge-CXC (CHC) domain. I found that sol1 sol2 double mutants display phenotypes consistent with defects in cell fate transitions as well as defects in the cell cycle. Both SOL1 and SOL2 display a striking cell cycle dependent expression pattern and time-lapse imaging revealed that they consistently begin expression sometime in G1, before disappearing 1 -- 2 hours before the new cell plate is detected. Perhaps most surprisingly I found that their homolog TSO1, which is not a SPCH target but expressed in the epidermis nonetheless, opposed the action of SOL1 and SOL2 at the final symmetric division. While SOL1 and SOL2 restrain this division, TSO1 promotes it. In Chapter 3, I identified a segregating background mutation in the sol1 sol2 line, which was causing a more severe phenotype. The more severe phenotype included greater numbers of small cells than sol1 sol2 at the 7 dpg stage and additional divisions at the guard mother cell stage, resulting in extended tumors of undifferentiated guard cells reminiscent of fama and flp myb88 mutants. Whole genome sequencing seedlings with the severe phenotype revealed a mutation in HOMEODOMAIN GLABROUS 2 (HDG2). HDG2 was confirmed as the causal mutation by constructing a triple sol1 sol2 hdg2 mutation using a previously characterized SALK line and rescuing the severe phenotype using a HDG2pro:HDG2-GFP transgene. In Chapter 4, I explored the idea of a plant version of the DREAM complex including the CHC and MYB3R proteins introduced in Chapter 2 as well as other plant homologs of proteins found in the animal complexes. Using publicly available datasets, I examined expression patterns of DREAM complex components throughout the stomatal lineage. I also screened available mutants for stomatal lineage phenotypes and constructed reporter lines. I did not identify any stomatal lineage phenotypes in these mutants likely due to redundancy among the members of these multi-gene families. Of particular interest were the ALWAYS EARLY family, which in animals are direct binding partners of the CHC proteins. Despite multiple attempts, I was unable to identify aly1/2/3 triple mutants from a segregating population which could indicate linkage of alleles due to a chromosomal rearrangement or early embryo lethality.

Keywords: Biology.
Advisor: Bergmann Dominique 
Fuller Margaret T.
Simon Michael (Biology professor)