The RootScope: A Simple High-Throughput Screening System For Quantitating Gene Expression Dynamics In Plant Roots

Background: High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots.Results: The promoter of Hsp17.6 was used to build a Hsp17.6(p):GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p: GFP response dynamics. Hsp17.6(p):GFP signal increased with temperature increases from 28 degrees C to 37 degrees C. At 40 degrees C the kinetics and localization of the response are markedly different from those at 37 degrees C. This suggests that different mechanisms mediate heat shock responses above and below 37 degrees C. Finally, we demonstrate that Hsp17.6(p):GFP expression exhibits wave like dynamics in growing roots.Conclusions: The RootScope system is a simple and powerful platform for investigating the heat shock response in plants

Quantitative 4D tracking analysis and chemical induction of heat shock granules during cytosolic misfolded protein stress

Heat Shock Granules (HSGs) are subcellular structures composed of small Heat Shock Proteins (sHSPs) and misfolded proteins that form in response to heat stress in plants. While sHSPs are found in other organisms, HSGs have only been reported in plant cells and only in response to heat stress. This thesis examines the signaling pathways that regulate the transcription of sHSPs and the formation of HSGs and investigates whether heat is the only stress that could activate these pathways. By visualizing HSGs in an Arabidopsis thaliana BOBBERl:GFP reporter line using still and 4-D confocal microscopy, we characterize HSG formation and HSG structural qualities such as volume and shape. 4D tracking is used to describe dynamic behavior. We also show that inducing protein misfolding by treating live seedlings with amino acid analog L-Azetidine2-Carboxylic Acid (AZC) or proteasome inhibitor MG132 induces granule formation. We propose that the term Heat Shock Granule is a misnomer, since HSG formation can be catalyzed by misfolded protein stress in the absence of heat treatment