Vacuole transport lab?
Scott T. Meissner
smeissne at PRAIRIENET.ORG
Fri Nov 8 14:41:33 EST 1996
Sorry, I feel compelled to point out a possible flaw.
Recently helm at siena.edu asked
"Does anyone out there know of a good lab to illustrate
And Russ Koning (koning at ecsu.ctstate.edu) answered with information
".... beet root cylinder project.... The betacyanin
is located in the vacuole and leaks out under some of these
He gave some of the classical conditions that have been
used to run this test. However the assumption is that the
betacyanins do cross the tonoplast and then the plasma membrane
and out into the solution to be detected. That assumption may
Stiles, in 1927, noted that red beet tissue placed in
water produces an "exosmosis of dissolved substances from
storage tissue into water." This implies that beet storage
tissue is not isotonic with water, and when placed in water
will take up the water, swell, and produce such high turgor
that it may damage itself or even burst cells. I have
observed this process. You can too by taking a slice of
red beet storage parenchyma tissue and placing it in water
and other slices in up to 0.8 M sorbitol solutions in say
0.1 M steps. At one point you will see plasmolysis, and
and at the other you will see cells with such high turgor
that they swell, and many will burst over time releasing
their contents. The process of cell rupture due to high
turgor pressure is called plasmoptysis.
So placing beet storage tissue plugs is not
examining movement across membranes, it is examining
plasmoptysis! Actual cell rupture. So just like with
animal cells that need the proper saline, some plant
tissues may function best in an isotonic solution.
In my measurements red beet is isotonic in
0.2-0.5 M sorbitol. It varies from beet to beet.
Perry etal. (1987) noted that the leakage of
sugars out of beet storage tissue varied with the
osmoticum of the solution. And Zamski (1986) has shown
that sucrose uptake into storage tissue discs was
higher in a 400 mM mannitol solution than in water. Both
of these works may be consistent with plasmoptysis of
the tissue in water.
The betacyanins are indeed in the vacuole. This
makes the use of red beet storage tissue great for
examining plasmolysis, and you might have students look at
the cells just for this. They are ideal osmometers! You
can shrink them further and further by placing them in
higher concentrations of sorbitol and calculate that the
amount of osmotica in them is held constant and it is just
the water that is leaving by measuring their diameter and
modeling their shape as a sphere.
This use of cut disks for studying membrane
transport is a classical approach. But to be correctly done
it must be done at the proper water potential to avoid
cell rupture. It has been used in many papers as relating
to membrane properties (Remington 1928, and Van Stevenick 1964).
But none of this early work checked for cell rupture, so much of
it may be questioned. Of course this could be used as a
great example for students of how new knowledge forces us to
revise our views of previous data. ;)
Also, the vacuoles of red beet are extremely easy to
get out. Just a few minutes. Anyone who is interested in
some isolation solutions can see my paper (Meissner etal. 1992).
But basically slice a 4 mm cube of tissue in ten drops of
1 M sorbitol, push the debris to one side with a probe, pour
the eluate on a slide, and .... perfect vacuoles every time!
You get dozens to hundreds of these. Patch-clampers love
them as it is a nicely exposed membrane, and easy to get.
You can get more at pH 7.25, and other items can be added to
help the vacuoles last longer...
I have tacked on the citations at the end for those who
Beets are the best!
Scott T. Meissner, Division of Science and Mathematics
McKendree College, 701 College Rd, Lebanon, IL 62254
Tel: (618) 537-6934
E-mail: smeissne at a1.mckendree.edu
I study beets because I ain't got no rhythm... . .
Meissner etal. 1992 Plant Science 82: 251-260
Use of stability of vacuoles isolated from the storage
parenchyma cells of dormant and freshly harvested red
beets (Beta vulgaris L.) to test for population
Perry etal. 1987 in- Plant Vacuoles: Their importance in
compartmentation in cells and their applications in plant
biotechnology. B. Marin editor. Plenum Press, N.Y.
Osmotic factors affecting the mobilization of sucrose
from vacuoles of red beet storage root tissue.
Remington 1928 Protoplasma 5: 338-399
The high frequency wheatstone bridge as a tool in
cytological studies: With some observations on the
resistance and capacity of the cells of the beet root.
Stiles 1927 Protoplasma 2: 577-601
The exosmosis of dissolved substances from storage tissue
Zamski 1986 Bot. Gaz. 147: (#1) 20-27
Structure and function of Beta vulgaris parenchyma cells:
Ultrastructure and sugar uptake characteristics of tissue
and cell suspension.
More information about the Plant-ed