Dissertation

Investigation of Early Signaling Mechanisms in Plant Osmotic Stress Response

Stecker, Kelly E

2016

Overview

How plants perceive and respond to water loss on a molecular level is a fundamental question in plant biology. Understanding the cellular signaling mechanism involved in sensing dehydration and initiating adaptive responses provides a tool for plant biologists to use in engineering crops with enhanced drought tolerance and water use efficiency as well as with developing new chemical approaches towards alleviating the negative effects of drought on crop yields. The increasing frequency and severity of drought, along with the depletion of fresh water resources, fortifies the need to understand the molecular networks that control drought response in plants. Currently the molecular mechanisms involved in the initial perception of dehydration are poorly understood. In this dissertation I present my work towards identifying previously unknown proteins involved in the initial dehydration response, using mass spectrometry based quantitative proteomic and reverse genetic approaches. Employing the model system Arabidopsis thaliana and hyperosmotic stress conditions to simulate water loss, I first identified proteins whose level of phosphorylation changes in response to short-term stress treatments, using quantitative untargeted mass spectrometry based phosphoproteomics technologies. From this work I identified proteins whose phosphorylation state changed within 5 minutes of stress treatment. To further characterize these proteins, I developed targeted proteomic assays to more routinely measure protein phosphorylated across many biotic and abiotic stress conditions. From this analysis I characterized a set of proteins that are uniquely regulated at the posttranslational level by rapid dehydration. These proteins are involved in cellular process such as mRNA degradation, microtubule restructuring, transcription factor activation, phospholipid signaling, and mitogen-activated protein kinase signaling and help elucidate the role that these processes play in the initial dehydration response. This work is presented in Chapter 2 of my dissertation. To validate the biological significance of proteins identified in my phosphoproteome analysis, I used reverse genetics to investigate Vac14, an uncharacterized protein in plants that displayed the largest change in phosphorylation under osmotic stress conditions. Vac14 is a highly conserved protein involved in the biosynthesis of the low abundant phospholipid phosphatidylinositol-3,5 bisphosphate, which regulates vacuole function and endomembrane vesicle transport in yeast and mammals. I demonstrate that Vac14 is an essential gene in plants and is responsible for regulating water homeostasis in cells. Vac14 overexpression mutants have increased drought tolerance and improved ability to germinate under water- limited conditions. This work is presented in Chapter 3 and supports the biological significance of proteins identified in my discovery phosphoproteomic work in Chapter 2. Finally, in Chapter 4 I describe my preliminary results in developing a new method for detecting proteins involved in cell signaling events through direct measurement of protein conformational changes. This method uses thermal denaturation profiling and untargeted quantitative mass spectrometry to identify proteins whose conformation has changed in response to in vitro or in vivo treatment conditions. Together, the data presented in this thesis demonstrate the utility of mass spectrometric based proteomic technologies for discovering previously unidentified proteins involved in cell signaling events and provides valuable insight into pathways activated during the initial few minutes of the osmotic stress response in plants.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry
• ISBN: 0355782146, 9780355782141