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Hi Christian,

There are a few papers out there with FPs in the vacuole lumen. Some have
said the vacuole is too acidic for good FP fluorescence, but we've seen it
reasonably often - GFP, YFP and CFP but not RFP (haven't looked). The
vacuolar location in each case above was unwanted! - thought to be
incorrect targeting.

A couple of published examples:
Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S., and Neuhaus, J.-M.
(2001). Regeneration of a lytic central vacuole and of neutral peripheral
vacuoles can be visualised by GFP targeted to either type of vacuoles.
Plant Physiol, 126, 78-86
Flückiger, R., De Caroli, M., Piro, G., Dalessandro, G., Neuhaus, J. M.,
and Di Sansebastiano, G. P. (2003). Vacuolar system distribution in
Arabidopsis tissues, visualized using GFP fusion proteins. Journal of
Experimental Botany 54, 1577-1584


These guys have an explanation, but the problem with this and similar
papers is assuming all plant vacuoles are the same - they are not, and
different cell types will also accumulate vacuolar FPs differently:
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi,
S.-i., Watanabe, M., Nishimura, M. and Hara-Nishimura, I. (2003), Why
green fluorescent fusion proteins have not been observed in the vacuoles
of higher plants. The Plant Journal, 35: 545­555


cheers,
Rosemary

On 27/01/12 4:32 AM, "Christian" <[log in to unmask]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>As we're on plants...  has anyone imaged GFP or RFP in the vacuole of
>plants?  Tonoplast is quite routine, but what about the lumen of the
>vacuole?  I have a user who believes there is a cleavage event which
>should lead to the build up of GFP inside the vacuole, my concern was
>degradation of the signal due to pH issues.
>
>Does anyone have any experience with imaging inside the vacuole of
>plants?  This will be in tobacco until the stable Arabidopsis lines are
>available.
>
>Thanks
>
>Christian
>