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Abstract

Eukaryotic cells can have a plethora of different shapes. To develop these, cells have to grow polarly and therefore concentrate their resources at a defined domain at the plasma membrane. We have a great understanding how such polar growth is achieved and regulated. However, the early processes that initiate the formation of a new polar domain at the plasma membrane, especially the timing of protein recruitment, remain largely elusive. The aim of this thesis was to understand how a polar domain is established and how the machinery for polar growth is assembled. As a model system I used Arabidopsis thaliana root hairs, which grow tube-like protrusions out of epidermal cells and I established a timeline of protein recruitment to the root hair initiation domain (RHID). To enable sensitive measurements of protein localization, I quantified properties of 10 fluorescent proteins in planta. Using the brightest fluorophore, I was able to detect low amounts of protein and quantitatively measure accumulation at the RHID at endogenous levels. The combined analysis of timing and localization of 30 different markers showed that different proteins accumulate specifically at different phases of root hair development and led to the identification of four novel candidates that act during root hair initiation, along with the established RHID markers RhoGTPases OF PLANTS (ROPs). For ROP2 I could show that its activity, as well as the interaction domain and the C-terminus are important for membrane association and recruitment to the RHID, while posttranslational S-acylation had no effect in this process. Two of the candidates found during RHID initiation, the actin-regulating SCAR/WAVE complex and the undescribed POLLEN RECEPTOR-LIKE KINASE 7 (PRK7,) both led to the establishment of independent projects. Additionally, I identified two ROP activating GEFs, GEF3 and GEF14, that precede ROP accumulation at the RHID. I found that GEF3 is necessary for root hair initiation and sufficient to form polar domains at the plasma membrane, which recruit ROP2. After RHID assembly, tip growth is initiated. This process is regulated by an oscillating apical [Ca2+]cyto gradient. In a collaboration we could show that at least three CNGC Ca2+ channels are regulating these Ca2+ oscillations in root hairs.