Vacuole transport lab?

[Scott T. Meissner]

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
transport...."

And Russ Koning (koning at ecsu.ctstate.edu) answered with information 
about the
	".... beet root cylinder project....  The betacyanin
is located in the vacuole and leaks out under some of these 
conditions."

	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
be wrong.

	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
are interested.


Beets are the best!


Aure Entuluva!
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 
	differences.

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.
	pgs 89-94
	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
	into water.

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.