Deciphering molecular mechanisms for differentiation and irreversible cell fate commitment of guard cells in plants
- The generation of multicellular organisms from single cells involves the establishment of developmental programs to produce specialized cells, tissues and organs. Many of these programs rely on early pluripotent stem cells and later lineage specific stem cells. Transcription factors, typically acting in cascades, are key to execute programs in a spatiotemporal manner and coordinate cell proliferation and differentiation events. To reach a mature fate, cells must exit the stem cell program and switch to a highly specialized cell fate program that will make it into a functional cell type. In my thesis I investigate how transcription factors (i) drive cell fate transitions in a developmental pathway, (ii) guarantee that terminal differentiation is stable and irreversible, (iii) intersect with the cell cycle machinery, and (iv) diversify their function in plants with divergent evolutionary histories and morphological adaptations. I investigated these questions in the stomatal lineage in the model dicot plant Arabidopsis thaliana and also in the monocot model plant Brachypodium distachyon. The stomatal lineage is an epidermal lineage whose final products, stomata, are composed of two cells - named guard cells - that control the aperture of pores and the exchange rate of water and gas between the plant and the atmosphere. Stomatal precursors include a population of stem cells dispersed on the plant leaf epidermis that undergo asymmetric self-renewing cell divisions before stably differentiating into guard cells. The related bHLH transcription factors SPEECHLESS, MUTE and FAMA are expressed sequentially and modulate major cell fate transitions in the pathway. The lineage is located on the epidermal surface and provides a powerful system to dissect how transcription factors drive the programing and reprograming of stomatal cells in vivo. In the first data chapter (CHAPTER II), I investigate novel molecular mechanisms and the extent to which FAMA drives differentiation of guard cells in Arabidopsis. In particular, FAMA processes a canonical RETINOBLASTOMA-RELATED (RBR) interacting motif; RBR (or RB in animals) is a ubiquitously expressed tumor suppressor player controlling cell cycle and differentiation processes. By disrupting the physical interaction of FAMA with RBR in vivo, I demonstrated that FAMA requires RBR to permanently shut down earlier stem cell like genes via recruitment of RBR to specific locations in the genome. The results presented here shed light into how terminal cell fates are established and how mature cells are locked into them. In CHAPTER III, I extend the characterization of how FAMA drives stable differentiation of guard cells by identifying its direct targets by ChIP-seq. FAMA binds to a large number of target genes. Importantly, among these are genes normally expressed during early stomatal development, consistent with my hypothesis in Chapter II that FAMA would repress these genes during terminal differentiation of guard cells. FAMA also binds to genes expressed or functioning in mature guard cells, suggesting that also works to activate genes to make guard cells. In a preliminary comparison of the FAMA ChIP-seq with a SPCH ChIP-seq, I found that around 60% of the targets are bound by both related transcription factors, and this shared class is enriched for early stomatal genes, but lacks the mature guard cell genes, which are exclusively FAMA targets. In CHAPTER IV, I turn my attention to the cell division event preceding guard cell formation in Arabidopsis. In order to form 2-celled valves, guard mother cells need to divide one and only once. I investigated how this single cell division is triggered and restricted. CYCDs are known triggers of cell division and we found that CYCD7 is expressed in guard mother cells. By performing expression and co-expression analysis I refined the expression window for CYCD7, and demonstrate with ChIP-qPCR and ChIP-seq profile that FAMA binds to its promoter and coding region. Furthermore, by exploring the gain-of-function phenotypes I demonstrated that division requires interaction with RBR. Proposed future experiments include investigation of a cycd7 knockout mutant phenotype, analysis of how CYCD7 is controlled at the transcriptional levels by stomatal transcription factors, and transcriptome profiling of CYCD7 expressing cells. Lastly, in CHAPTER V, I change model organisms to explore the functional diversification of stomatal transcription factors in the stomatal developmental pathway in Brachypodium. Besides building genetic and molecular tools, I investigate the function of MUTE, a transcription factor that works before FAMA. With transcriptional and translational reporters, and with gain and loss-of-function analysis I demonstrated that BdMUTE is required for the acquisition of subsidiary cells, the pair of cells flanking the guard cells that provides chemical and mechanical assistance to guard cell function, and comprise an important innovation in the stomatal complexes in monocots.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Matos, Juliana de Lima
|Stanford University, Department of Biology.
|Simon, Michael, (Biology professor)
|Simon, Michael, (Biology professor)
|Statement of responsibility
|Juliana de Lima Matos.
|Submitted to the Department of Biology.
|Thesis (Ph.D.)--Stanford University, 2016.
- © 2016 by Juliana de Lima Matos
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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