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. 2001 May;126(1):78-86.
doi: 10.1104/pp.126.1.78.

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

G P Di Sansebastiano  1 , N Paris, S Marc-Martin, J M Neuhaus
Affiliations

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

G P Di Sansebastiano et al. Plant Physiol. 2001 May.

Abstract

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.

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Figures

Figure 1
Figure 1
Fluorescence patterns of GFPs targeted to a neutral vacuole (GFP5-Chi, A and B) and to an LV (Aleu-GFP6, C and D) 24 h after protoplast transformation. Confocal images (1-μm section) of chloroplast-rich tobacco mesophyll protoplasts. A and C, More frequent patterns; B and D, less frequent patterns (see Table I). Bar = 10 μm.
Figure 2
Figure 2
NR staining of a protoplast expressing Aleu-GFP6. A, Image in transmitted light. The black spots are chloroplasts and NR appears as a gray stain of the CV. B, Confocal image of the green fluorescence, which is partially quenched by NR. Bar = 20 μm.
Figure 3
Figure 3
Regeneration of vacuoles by miniprotoplasts. Confocal images (1-μm section) of tobacco miniprotoplasts at different times after evacuolation. A through C, Miniprotoplasts expressing the marker for neutral vacuoles 8, 36, or 52 h after evacuolation; D through F, miniprotoplasts expressing the marker for LVs 8, 36, or 52 h after transformation. Bar = 10 μm.
Figure 4
Figure 4
Model of vacuole biogenesis in miniprotoplasts and (chloroplast-rich) protoplasts. The marker for neutral vacuoles (GFP5-Chi) is indicated in gray and the marker for LVs (Aleu-GFP6) is indicated in black. The percentage of cells developing each pattern is indicated next to the arrows. A, Miniprotoplasts: There is no CV yet 8 h after evacuolation. GFP5-Chi labels ER, nuclear envelope, and peripheral (pre-) vacuoles, whereas Aleu-GFP6 labels smaller peripheral (pre-) vacuoles. Thirty-six hours after evacuolation the large CV is an LV labeled by Aleu-GFP6, whereas GFP5-Chi remains limited to PVs. Fifty-two hours after evacuolation the PVs have fused with the lytic CV in about one-third of the cells. B, Protoplasts: The CV is labeled by neither of our GFP markers 6 to 12 h after transformation. GFP5-Chi labels ER, nuclear envelope, and peripheral (pre-) vacuoles, whereas Aleu-GFP6 labels smaller peripheral (pre-) vacuoles. Then, within 24 h there are two possibilities depending on the nature of the pre-existing CV. Top, New peripheral LVs fuse with a lytic CV, whereas the neutral vacuoles remain peripheral. Bottom, The new peripheral neutral vacuoles fuse with a neutral CV, whereas the LVs remain peripheral.
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