Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that mediate protein transport from late Golgi or trans-Golgi network (TGN) to vacuole via prevacuolar compartment (PVC) in plant cells. The N-terminus of a VSR is believed to be important for cargo binding while its transmembrane domain (TMD) and cytoplasmic tail (CT) are essential for its correct subcellular localization. In this study, I first developed and tested an expression system using transgenic tobacco BY-2 cells to produce truncated VSR proteins (VSRNT) lacking the TMD/CT into the cultured media. The secreted VSRs bind specifically to the vacuolar sorting determinants (VSDs) of known vacuolar proteins and such binding is calcium dependent in vitro. Thus, VSR cargo proteins are likely secreted into the cultured media along with the truncated VSRs, which enable the identification of various VSR cargo proteins from the cultured media of transgenic cells. I then identified these putative VSR cargo proteins through liquid-chromatography with tandem mass spectrometry (LC-MS/MS) and Fourier transform mass spectrometry (FT-MS) using transgenic Arabidopsis cell suspension cultures PSB-D expressing these truncated VSRs. Among the 17 unique proteins found in the cultured media of transgenic Arabidopsis PSB-D cell line expressing VSRNT, an Arabidopsis glycosyl hydrolase family 3 protein At5g10560 (GH3) was chosen for further study on VSR-cargo protein interaction. GFP-tagged GH3 fusion protein was found to co-localize with VSR-RFP marker protein in PVC, whereas GH3 was also shown to interact with a VSR protein BP-80. Loss-of-function analysis demonstrated that the GH3 contained a vacuolar sorting determinant (VSD) for PVC targeting. / Suen, Pui Kit. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-84). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In this study we applied ultra-short laser pulses on a biological sample (Arabidopsis), in order to cut it precisely in a square pattern and subsequently use it for studying stress generation mechanisms. For this purpose, we utilized femtosecond laser pulses at 100 fs pulse width and 80 MHz repetition rate. We took two processing parameters into consideration such as laser power, laser exposure time which is related to the stage speed. Therefore, we were able to find the laser optimum conditions for ablation of biological tissues. The mutant and wildtype (control) obtained from laser cutting with a size of 500 µm × 500 µm were directly transferred (in-situ with laser cutting) into a microfabricated chamber containing ~500 nanoliters deionized water for measuring ion conductivity. The ion conductivity is a signature of cell-death mechanisms caused by various stresses. A light with intensity of 100 µmol was exposed to the samples for 2 hours and 20 minutes as a source of stress. A quantitative electrical analysis with high accuracy was assured by utilizing a microchamber, which enables a measurement in nanoliter volume. We measured the impedance which is reciprocal of conductivity using a lock-in amplifier and a precise current source at frequency of 130 Hz. Initially high impedance of mutant sample tended to drop within 2 hours and finally approached the constant value which signified that the cell death mechanism was complete. However, the wildtype sample demonstrated approximately constant impedance (conductivity) during the experiment.