Characterization of Organelles in the Vacuolar-Sorting Pathway by Visualization with GFP in Tobacco BY-2 Cells

Abstract

We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

“…Accordingly, actin and its motor protein myosin could contribute the force required for auxin-dependent vacuolar fission and fusion events. Conversely, other studies have shown that several plant cells show interconnected vacuolar structures (26)(27)(28)(29)(30)(31). Accordingly, the actin-myosin system could generate or release vacuolar constrictions.…”

Overexpression of theArabidopsisSyntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo

“…As expected from published findings, the majority of the protoplasts expressing YFP-SYP22 show clear tonoplast localization in the absence of SYP21 overexpression ( Figure 6C). The presence of internal subregional structures, previously named bulbs, exhibiting a stronger fluorescence than the outer vacuolar membrane are also a typical feature of tonoplast staining (Mitsuhashi et al, 2000;Saito et al, 2002;Uemura et al, 2002).…”

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

“…Day 3 BY-2 cells have been used in our drug treatment studies because these cells are at their log phase (Matsuoka et al, 2004) and XFP BP-80 fusions remain in PVCs as punctate patterns, which will be targeted to vacuoles as diffusion patterns at day 6 and day 7 (Mitsuhashi et al, 2000;Lo and Jiang, 2006). As shown in Figure 2, in cells expressing the Golgi marker GONST1-YFP, typical enlarged compartments were observed in all cells treated with BFA at tested concentrations from 5 mg mL 21 to 100 mg mL 21 ( Fig.…”

“…First, cells at these 2 d are at their log phase, thus representing physiologically healthy stages, whereas cells at day 6 or day 7 represent stationary stages (Matsuoka et al, 2004). Second, in transgenic BY-2 cells expressing the PVC marker XFP BP-80, typical punctate fluorescent patterns representing PVCs/MVBs (Tse et al 2004) were observed in day 2 or day 3 cells, but such PVC patterns gradually disappear during later stages of cultures with fluorescent signals detected in vacuoles at day 6 and day 7 cells (Mitsuhashi et al, 2000;Lo and Jiang, 2006).…”