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Plant Physiol, May 2001, Vol. 126, pp. 78-86
Regeneration of a Lytic Central Vacuole and of Neutral Peripheral
Vacuoles Can Be Visualized by Green Fluorescent Proteins Targeted to
Either Type of Vacuoles1
Gian Pietro
Di Sansebastiano,2
Nadine
Paris,
Sophie
Marc-Martin, and
Jean-Marc
Neuhaus*
Laboratoire de Biochimie, Institut de Botanique,
Université de Neuchâtel, rue Emile-Argand 9, C.P. 2, CH-2007 Neuchâtel 7, Switzerland
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.
1
This work was supported by the Swiss National
Science Foundation (grant no. 31-46926.96).
2
Present address: Dipartimento di Biologia,
Università di Lecce, Prov. Lecce-Monteroni, 73100 Lecce, Italy.
*
Corresponding author; e-mail jean-marc.neuhaus{at}unine.ch;fax
4132-718-2201.
© 2001 American Society of Plant Physiologists
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