Stem cell organization in Arabidopsis : from embryos to roots

Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a highly predictable and relatively simple model to study several developmental processes. <strong>Chapter 1 </strong>discusses the Arabidopsis embryo as a model for development and morphogenesis and describes the currently known factors involved in these processes. Molecular cloning is a vital technique of today’s plant biological research. The ability to quickly produce reliable constructs for follow-up analyses can greatly accelerate biological research. In <strong>Chapter 2</strong>, we describe the optimization of a highly efficient Ligation Independent Cloning method. This method makes use of sticky overhangs that enable <em>in vivo </em>ligation of cloning products. We present a step-by-step protocol that enables generating plant transformation-ready constructs in a semi-high-throughput manner, within two to three days. This method can for example facilitate follow-up analysis of genome-wide approaches. Proteins regularly function as part of larger protein-complexes and their interaction partners can often be indicative of functionality. Unbiased, <em>in vivo</em> analysis of protein complexes can therefore be very informative for the functional characterization of a protein of interest. In <strong>Chapter 3</strong>, we describe an optimized method for immunoprecipitation followed by tandem mass-spectrometry. By performing mass-spectrometry measurements on at least three biological replicates, relative abundance of proteins in GFP-tagged sample compared to background controls can be statistically evaluated to identify high-confidence interactors. In this step-by-step protocol we detail the entire procedure from plant material to data analysis and visualization. The establishment of distinct cellular identities is of critical importance for multicellular organisms. The first step that leads to cell identity is the activation of a unique set of transcripts and this often exploited in order to infer cell identity. In <strong>Chapter 4</strong>, we have generated 12 gene expression marker lines and describe their expression domain in the Arabidopsis embryo. We divided them into four different categories based on their expression domain: (I) ground tissue; (II) root stem cell; (III) shoot apical meristem; and (IV) post-embryonic. In addition, we used two stem cell markers to show their use as marker lines in genetic studies. A central player in development of the Arabidopsis root meristem is the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP). Several downstream targets of this transcription factor have been characterized, but the main focus has been on targets that were themselves transcription factors. An open question remains, therefore, how MP can orchestrate cellular responses during development. <strong>Chapter 5</strong> describes the in-depth functional and biochemical characterization of a group of IQ-domain proteins. We show that their expression is regulated by the hormone auxin and that they bind microtubules and Calmodulins, <em>in vivo</em>. In addition, we show that the subcellular localization of IQD18 is cell cycle dependent. Loss- and gain-of-function analysis resulted in differential auxin- and calcium-signaling output, suggesting these proteins may form a bridge between these two major signaling pathways. Furthermore, this indicates a mode for how MP may be affecting cellular responses, during root development. In <strong>Chapter 6</strong>, we take a step back and re-evaluate the currently prevailing model for stem cell organization in the Arabidopsis (embryonic) root. Using different gene expression markers, we were able to generate non-cell type specific and cell type specific transcriptomic datasets from systematically obtained ontogenetic cell populations in the root meristem. Follow-up analyses give support for an extended model for stem cell organization in the root. Finally, in <strong>Chapter 7</strong>, we discuss the novel findings of this thesis and suggestions are made for future research directions.