Science and Plants for Schools – Student Project Starter
Changes in water potential . . . during ripening and
storage of fruits and vegetables
This is a project starter, suitable for Advanced Higher biology investigations or
A-level extended projects. Don’t forget to credit this resource in your
bibliography by including the title, the website, the web address and the date
you accessed it.
Do you like your lettuce limp or crunchy . . . your apples shrivelled or crisp
(and juicy!)? How do we keep fresh fruits and vegetables in just the right
condition, from harvest to the supermarket shelf, especially if kept a long time
in storage? How much water do they lose, why do they lose it . . and how can
we minimise these losses?
Much of the mechanical support required by fleshy parts of a plant is provided
by the turgid state of the cells within. The parenchyma tissue which forms the
bulk of the flesh of a fruit or vegetable is characterised by the presence of
thin-walled cells, packed together in such a way that their walls press upon
one another, providing a stiffness to the tissue as a whole. A wilted (or
shrivelled) fruit or vegetable is usually the result of a loss of turgidity of the
tissues, as a consequence of excessive water loss during storage.
The normal turgid state of the plant cells is the result of osmosis. This involves
the movement of water by diffusion into the cell, from a region of higher water
potential outside the cell to the vacuole of the cell, which has a lower water
potential. The increasing volume of liquid in the vacuole causes it to expand
and press the cell contents harder against the cell wall (wall pressure).
Eventually a point is reached when the cell wall cannot stretch any more, and
at this stage there will be no further net uptake of water by osmosis - a
balance is reached in which the wall pressure offsets further osmotic inflow of
water.
The lower the water potential of a plant cell vacuole (i.e. the greater the
concentration of the solutes dissolved in it), the greater the osmotic force
which causes water to be drawn in, and the greater the wall pressure that
develops. Some materials, such as starch, are relatively insoluble and
consequently have little effect on water potential. But when starch is broken
down to glucose, which is soluble, the water potential is affected. As part of
the process of ripening, there is often a dramatic change in starch reserves as
it is mobilised to form glucose.
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Another change that affects the water relations of ripening fruit and vegetable
cells, is the modification (and weakening?) of cell walls as result of enzyme
attack by pectin-dissolving enzymes such as polygalacturonase.
Given the enormous biochemical and structural changes that occur during
ripening, it would be very surprising if the water potential and biomechanical
properties of the fruit or vegetable did not change. Some possible
investigations include:
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the changes in starch and / or glucose concentration during ripening
how the water potential changes during ripening
how the water potential changes during storage or drying out
how the stiffness of the fruit changes during ripening
how the stiffness of the fruit changes during storage or drying out
the relationship, if any, between polygalacturonase activity and firmness
A method for measuring the water potential of some fruit or
vegetable tissues
This procedure involves placing weighed samples of plant tissue in solutions
of varying water potential, and then re-weighing them after an number of
hours. This can be done successfully only with fairly solid tissues, such as
apple and potato, which can be cut with a cork borer. By plotting a graph of
the gain or loss of mass against the concentration of the bathing solution you
can determine the water potential of the plant tissue cells.
This technique depends upon the effects of osmosis between the tissue
sample and the solution that bathes it. If the water potential of the tissue is
greater than that of the bathing solution, then water is lost from the tissue, and
this is reflected as a loss in tissue mass. If, however, the water potential of the
tissue is less than that of the bathing solution, then the tissue gains water and
this is reflected as an increase in the mass of the tissue.
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Use a cork borer to remove a series of cylinders of tissue from the fruit
or vegetable. The cylinders should be cut of the same diameter (e.g. 1
cm), and from comparable regions of the plant organ.
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Place the cylinders on a tile and use a scalpel or razor to remove any
remaining skin. Then carefully cut them to exactly the same length (e.g.
2 cm).
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Make up a series of bathing solutions of different concentrations, say
0.1, 0.2, 0.3, 0.4, 0.5, 0.6 0.7 and 1.0 M sucrose in water.
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Weigh each cylinder and place in a labelled test tube or boiling tube with
sufficient bathing solution to cover the cylinder to a depth of at least 5
cm.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
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Leave the cylinders to soak for several hours, or better still, overnight in
a refrigerator (to avoid the growth of microbes in the sucrose solution).
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Remove each cylinder from its bathing solution. Blot briefly on a paper
towel then re-weigh.
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Calculate the loss or gain in mass by the cylinders. Plot a graph with the
loss / gain of mass on the y-axis and sucrose concentration on the xaxis.
From the trend of data points it should be possible to interpolate the
concentration at which no gain or loss of mass might have been expected to
occur - at this (theoretical) concentration, the water potential of the bathing
solution is equivalent to that of the tissue.
As an alternative, you could shorten the period for soaking by cutting the
cylinders into a series of slices approximately 1 mm thick, and then placing
them on a long steel pin, so that they are all separated by a small space.
These pins are then immersed in the sucrose solutions as described above.
The osmotic equilibrium occurs within an hour or so.
Some vegetables may have water potentials outside those detectable by the
range suggested above, so pilot experiments may be needed. Also, for more
detailed measurements, additional concentrations of sucrose may be needed
within the range suggested.
Biomechanical tests on ripening fruits and vegetables
Discerning greengrocer customers often give the fruit they are about to
purchase a discreet squeeze. They are aware that the softness gives a clear
indication of how ripe the fruit is. But how might softness be quantified? In
addition, it is always easier to peel a ripe fruit. How can we measure the ease
of peeling?
Compression test
This depends on the principle that, when a load is applied, softer fruits
undergo greater distortions in shape than harder ones.
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Place the fruit on a level, hard surface and arrange for a simple light
platform to rest on its upper surface. A thin piece of stiff aluminium sheet
would be a suitable material for the platform. Measure the distance
between the platform and the surface on which the fruit is placed.
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Apply a known mass to the platform then re-measure the distance
between the platform and surface. Calculate the change as a result of
adding the mass.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
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It is important that the mass is not too heavy, so that it causes rupture of
the fruit. Some trial and error, therefore, is needed to judge what mass
works best. If the mass is distorting the fruit within its elastic limit, then
after removal of the mass the fruit should recover its shape.
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It would be best to take measurements within these elastic limits, as the
tissue 'failure' involved in more extreme distortions make the
biomechanical comparisons too complex to resolve.
It may be sufficient to carry out this test with a single mass, and then
make comparisons between fruits at different stages of ripeness.
Precautions need to be taken to control factors such as orientation of
the fruit (take several measurements, with the fruit in different
orientations) and the size of the fruit (express the distortion as a
percentage).
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A further elaboration of this technique would be to add successive loads,
then measure the increase in distortion, until the eventual 'failure' of the
tissues. Plot the percentage distortion versus applied load - the gradient
reflects the stiffness of the tissue (but only within the elastic limits of the
fruit).
Penetration test
This is based on the principle that it is easier to drive a nail into wood than into
concrete!
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Peel the skin from the fruit to be tested. Then prepare a block of the fruit
to be tested and place it under the 'penetration device'. This might be
anything, ranging from a mounted needle to a sharpened pencil.
Support the device by some kind of sleeve which allows vertical
movement.
Apply a load to the device, causing it to penetrate the flesh of the fruit.
Find the size of an appropriate load by trial and error. The load could be
applied by a force meter, thus allowing detailed information about the
force applied! Alternatively, use known masses applied to the
penetration device.
Measure the extent of penetration of the flesh by the device. (This could
be made easier by marking its sides with mm scale).
Possible complications include the fact that the flesh may vary in strength
according to the type of tissue being penetrated. Also, if serious fractures
develop in the fruit, then penetration may be the result of the propagation of
cracks rather than loosening of cell walls.
Peeling test
By measuring the force necessary to detach a piece of skin, the ease of
peeling can be assayed.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
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Use a scalpel to cut completely through the skin of the fruit, forming a 2
cm square. Leave the square in contact with the fruit flesh beneath.
Use 'Superglue' to fix a metal tag to the square (or perhaps attach a
fish-hook to the epidermal layers).
Attach a force meter to the metal tag and gradually increase the force
until the square of skin pulls loose. Note the force involved.
Repeat these measurements with some other areas of the fruit, to obtain
a mean value.
Some 'biotechnological tricks' have been played with fruits, such as injecting
pectinases under the skin of an orange to cause the skin to be more easily
removed. The techniques described above, might offer a way of quantifying
accurately any changes in the ease of peeling.
Thickness of skin
In fruits such as the banana, the thickness of the skin undergoes a noticeable
change during the ripening process. Perhaps this could be quantified, and
related to the changes in the strength of skin attachment to the underlying
flesh. There may be other macro or microscopic changes in structure that
could be followed during the ripening process.
Pectin assay
Pectins consist of a very diverse range of water-soluble polysaccharides, such
as homogalacturan, rhamnogalacturan and arabinogalacturan. Pectins are the
most 'hydrophilic' components of the cell wall and they are insoluble in
alcoholic solutions.
The assay
The principle of this assay is to boil the fruit (or vegetable) in alcohol to
remove most of the alcohol-soluble carbohydrates of the cells, and then to boil
in water followed by alcohol-precipitation of the pectins.
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Cut the fruit (or vegetable) into thin slices (no more than a few mm
thick), and place a weighed amount into boiling industrial methylated
spirits (IMS) in a boiling tube. The volume of IMS should be
approximately 5 cm3 per g of fruit.
Safety note - Inflammable solution - heating must be done using a
thermostatic water bath, NOT a naked flame.
Boil in IMS for at least 5 minutes, then dispose of the IMS safely.
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Place the slices in a mortar and grind with silver sand and distilled water
to form a smooth, loose slurry. Depending upon amounts being
prepared, a blender could be used.
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Decant the slurry into a glass beaker and make the volume to a
measured total (e.g. 150 cm3 for an initial mass of fruit = 50 g). Boil the
mixture for 10 minutes, using a hot-plate (or bunsen flame only if no
boiling ethanol is in laboratory). Pay careful attention to the boiling
mixture, to avoid it foaming over the sides of the beaker.
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After boiling, cool the beaker to room temperature and add a little more
water to restore any volume evaporated during boiling.
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Use a pH meter in the slurry and add 1M NH4OH dropwise until the
mixture is pH 6.5.
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Separate the solids using a Buchner funnel or centrifuge. Dispose of the
solids but keep the liquid extract (which contains the dissolved pectins).
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Mix the pectin solution with ethanol in the ratio 1 part pectin : 4 parts
ethanol. The pectins should now precipitate out of solution.
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Filter off the pectins, or better still, centrifuge the mixture. Then dispose
of the supernatant and keep the pectin pellet, which is either weighed
wet, or dried to a constant mass.
The pectins thus extracted can have their identity checked by being redissolved in water. Pectins form a milky solution, and, when pectinase is
added, the milkiness clears after incubation, say at 35 °C for one hour.
Starch grain assay
The following procedure can be used to determine the mass of solid starch
grains present in fruit or vegetable tissues. The procedure as described is for
analysis of a potato, but it can be applied to any vegetable or fruit. The
blender is not essential, and can be replaced with a mortar and pestle, or the
latest small hand-blender (usually used for pureeing vegetables for soups) is
very effective at disrupting tissues. The violence of the blending process is
effective at liberating the maximum number of starch grains from the cells.
The assay
The method depends upon the insolubility of starch in saline and the resulting
sediment gives a measure of starch grains present in the material.
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Peel and cut the potato into small pieces (less than 1 cm cubes).
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Weigh the pieces, and put them in a blender, adding 2 cm3 1% NaCl for
each gram of potato. Blend the mixture, to produce a loose slurry. Try
blending for about 30 seconds, or more if needed.
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Pour the slurry through two layers of muslin, stretched over the mouth of
an appropriate sized beaker. Dispose of the solids left in the muslin.
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Leave the filtrate in the beaker to stand for some minutes, to allow the
grains of starch to form a solid sediment at the bottom of the beaker.
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When the sediment has formed, carefully pour away the liquid above it,
and re-suspend the sediment in fresh 1% NaCl. Once again, allow time
for the starch grains to settle to the bottom.
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Repeat the re-suspension / re-washing procedure twice more.
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Finally rinse the starch layer in a similar fashion, using 0.01 M NaOH.
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Then assess the 'amount' of sediment (containing the starch grains),
either by its volume or by its mass. (Volume could be useful in terms of
simplicity.)
To measure volume of sediment, pour a known volume of the starch
suspension into a tapered, calibrated centrifuge tube. Allow it to stand
vertically for a few minutes. The volume of starch grains can then be directly
read off the side of the tube. If you do not have a pre-calibrated tube, then
make one by adding small aliquots of water to a suitable tube, and using a
marker pen to indicate the volumes on the side of the tube.
As an alternative, to assess the sediment in terms of mass, weigh to find the
wet mass, or dry to a constant mass and then weigh to find the dry mass of
starch grains obtained.
Remember you can always check the quality of your extract by adding iodine
solution, which should colour the grains blue-black, and / or examining a
sample under the microscope. You might be able to distinguish the unique
features of starch grains belonging to particular species of vegetables. Oh,
and don't forget to add a drop of amylase solution to such a sample, and see
the effects of amylase on the structure of the grains.
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