Analysis of the induction of autophagy during ER stress and the vesicle fusion machinery at the trans-Golgi network in Arabidopsis thaliana
The vacuole in plant cells occupies more than 80% of the cellular volume and is involved in development, detoxification, and degradation of proteins. In this dissertation, I focused on the two vesicle trafficking pathways, autophagy and vacuolar trafficking, which deliver cellular components to the vacuole.
Autophagy is an evolutionarily conserved mechanism in eukaryotic cells for degradation and recycling of cytoplasmic materials and damaged cell components during development or upon encountering stress conditions. Previous studies showed that autophagy is activated by endoplasmic reticulum (ER) stress and delivers ER fragments to the vacuole. ER stress is defined as accumulation of unfolded or misfolded proteins in the ER, which can cause programmed cell death ultimately. I examined whether the accumulation of unfolded proteins in the ER is a signal for activation of autophagy under ER stress. I found chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid reduced tunicamycin-, dithiothreitol- and heat- induced autophagy, but not autophagy caused by unrelated stresses. In addition, transient expression of an unfolded protein mimics zeolin or a mutated form of carboxypeptidase (CPY*) in protoplast of Arabidopsis leaves induced ER stress and autophagy. In this study, I demonstrate the accumulation of unfolded proteins in ER is the signal for activating autophagy during ER stress.
The trans-Golgi network (TGN) is one of the major sites for sorting proteins to the plasma membrane or the vacuole. TNO1, a putative long coiled-coil tethering factor, is a TGN-located protein in Arabidopsis. TNO1 interacts with SYP41 and is required for SYP61 localization. A tno1 mutant is hyper-sensitive to salt stress and has defective vacuolar trafficking. Here, I demonstrated that TNO1 is also required for maintaining the TGN and Golgi morphology. In addition, overexpressing SYP41 and SYP61 rescues the salt sensitivity, defective vacuolar trafficking, and disrupted TGN and Golgi caused by the loss of TNO1.
In addition, we applied a super-resolution fluorescence microscopy technique STORM (Stochastic Optical Reconstruction Microscopy) in intact roots of Arabidopsis. STORM has made substantial contribution to reveal detailed subcellular structures in mammalian cells. However, in plant cells, the application of STORM was limited. In this dissertation, microtubule array in Arabidopsis roots were visualized by STORM and a spatial resolution of 20-40 nm was successfully demonstrated. The high spatial resolution enabled quantification of microtubule density and orientation in intact roots.
Taken together, these results contributed to the understanding of the induction of autophagy during ER stress and the vacuolar trafficking machinery at the TGN in Arabidopsis. We also optimized the STORM with intact roots of Arabidopsis seedlings and achieved a spatial resolution of 20-40 nm.