TY  - GEN
Y1  - 2017///
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/23502/
AV  - public
ID  - heidok23502
N2  - All higher plants form leaves and roots, organs specialized to efficiently absorb carbon dioxide, collect energy from the sunlight and take up water as well as minerals. These tissues usually have a large surface area made up of big cells. To fill the space most resourcefully plant cells developed unique compartments, central vacuoles, which can occupy almost the entire cell volume. The transport of water into vacuoles is much more economical than to generate a similar amount of cytosol, decreasing the metabolic cost of plant growth substantially. Besides, vacuoles fulfill vital functions in storage of nutrients, detoxification of the cytosol, and protein degradation. The physiological roles of vacuoles are well studied but detailed knowledge about how they develop is limited. Due to their gigantic size vacuole biogenesis in plants is not only a matter of balancing incoming vesicle trafficking and membrane turnover, but also requires enormous amounts of membrane in order to enable proper vacuole expansion. The consensus model states that central vacuoles arise by fusion of multiple small organelles, initially formed by a combination of late endosomes and autophagy. However, the mechanistic details are mostly reasoned from 2D electron microscopy images and lack any quantification. In this thesis we analyzed the biogenesis of lytic vacuoles in root tips of Arabidopsis thaliana in more detail. By computing 3D surface renderings of vacuoles we reconstituted their complex tubular network in meristematic cells and discovered that in contrast to the existing model vacuoles develop as a single compartment. This was further substantiated by FRAP-based measurements of vacuolar volumes. Instead of growing by fusion of smaller volumes vacuoles inflate over time until they reach their final size in differentiated cells. Still, we found that vacuole development depends on CORVET/HOPS mediated membrane fusion as indicated by vacuole fragmentation upon induced knockdown of the subunit VPS16. Also, calculating the number and lifetime of late endosomes in elongating cells revealed that their membrane contribution does not allow for the growth rate of vacuoles, suggesting an additional membrane donor. We identified a so far undiscovered pathway that delivers V-ATPases and V-PPases to the tonoplast via an intermediate compartment we termed provacuole. By using different genetic and chemical means to block known vacuolar transport routes we found that provacuole trafficking bypasses Golgi and post-Golgi compartments and does neither require COPII-mediated ER export nor the core autophagy machinery. Most importantly, with a custom-built inducible expression system and 3D super-resolution confocal laser scanning microscopy we were able to calculate that in elongating cells provacuoles provide the majority of the required tonoplast material. In conclusion, the evidence provided in this thesis redefines the model of vacuole biogenesis in Arabidopsis thaliana.
TI  - Vacuole biogenesis in Arabidopsis thaliana
A1  - Krüger, Falco
CY  - Heidelberg, Deutschland
ER  -