Live cell analysis in Arabidopsis thaliana using randomly generated markers

This thesis describes the use of the green fluorescent protein (GFP) to explore cellular organization by marking subcellular structures using random fusions between GFP and cDNAs. This approach was tested by conducting a large scale microscope-based screen of 5700 Arabidopsis seedlings transformed with random GFP::cDNA fusions. This yielded 120 markers of a wide array of known and novel subcellular structures, including the plasma membrane, endoplasmic reticulum (ER), peroxisome, vacuole and nucleus, efficiently yielding a collection of markers for live-cell analysis that was previously unavailable in Arabidopsis.; To characterize the markers isolated, the cDNA inserts for over 100 markers were end-sequenced. This yielded the surprising result that about half of the markers isolated were targeted to several subcellular structures (particularly peroxisomes) by fusions to out-of-frame cDNAs. Thus, “random” targeting, presumably created by the existence of a variety of low-information content targeting signals, was pervasive in our experiments.; By exploiting an observation made during the course of my experiments, the marker collection was used to create a descriptive model of plant cell death. These studies show that wounding induces a stereotyped mode of cell death in cells adjacent to wound sites that is marked by nuclear contraction, separation of the nuclear envelope, ER degeneration, cellular collapse and loss of plasma membrane integrity. Herbicide induced cell death triggers rapid cellular collapse but not nuclear contraction, demonstrating these events can be uncoupled. These dynamic multi-organelle portraits illuminate several previously unrecognized alterations in cellular architecture that accompany plant cell death and create a new framework for their analyses using genetic and biochemical approaches. In addition, they illustrate a previously unappreciated degree of similarity between the responses of plants to wounding and the hypersensitive response to pathogen attack at the cellular level.