Investigation to look at the water potential of Celeriac

The aim of this investigation is to look discover the water potential of the root vegetable Celeriac. To do so, the effect of varying concentrations of sugar solutions on Celeriac will be observed and a conclusion reached. As preliminary work, the effects of sugar solution on potato has been investigated to give a general idea of what to expect and to discover any problems with the, method before the larger Celeriac experiment. Background research has also highlighted properties of Celeriac that are different to potato and will therefore affect the water potential of the plant.

Relevant background knowledge

The generalised plant cell is as in the diagram below:

The vacuole and cytoplasm contain the solution that is being investigated.

The cell wall is fully permeable and therefore does not effect the movement of substances into and out of the cell. It does however contain 20-40% cellulose which applies a pressure potential that is important in the water potential of the cell.

The cell membrane and tonoplast (membrane surrounding the vacuole) are partially permeable, therefore allowing certain molecules through while prohibiting others. Non-polar and lipid soluble substances can pass through since they are not effected by the phospholipid bi-layer. Molecules that are polar and ions however cannot pass through the membrane unless ‘carried’ across by specialist proteins, large insoluble substances cannot enter and do not affect Osmosis (only soluble substances affect solute potential). Although water molecules are polar, they are not affected by the membrane due to their small size. This allows Osmosis to take place without being affected by the membrane.

Diffusion is the spreading out of a substance from a high concentration gradient to a low one (without the use of energy). There are many examples of diffusion, such as orange squash – when it is added to water it all turns orange due to the ‘squash molecules’ dispersing throughout the water.

Osmosis is the name given to the specific diffusion of water. It is the most common form of diffusion in the cellular environment and also the means by which the water potential of Celeriac can be obtained. Osmosis across a semi-permeable (selective) membrane occurs when the water potentials on either side of the membrane differ, the movement is from high concentration to low. Therefore Osmosis is:

The net movement of water molecules across a selective membrane from an area of high water potential to one of lower water potential

Water potential can be partially explained using this equation:

Water potential (?w) = Solute potential (?s) + Pressure potential (?p)

Inside the plant the solute potential (always negative) is affected by the soluble components of the cytoplasm/inside the vacuole, and the pressure potential is produced by the inward force of the cell wall.

If the water potential outside the cell drops dramatically, due to a vast increase in the solute potential, then water diffuses out of the cell. This can have very harmful affects; the cell membrane is pulled inwards away from the cell wall in an irreversible process called plasmolysis. The plasmolysed cell looks like the one below and can be easily observed with a light microscope using epidermis cells in varying sugar solutions.

In the other extreme when the water potential outside the cell drops well below that of inside the cell water diffuses in. Unlike animal cells however, the plant cells do not explode due to the pressure potential exerted inwards by the cell wall. As the increased contents of the cell applies a greater force on the cell wall, it pushes back with an equal force due to Newton’s 3rd Law. Water continues to diffuse in until the ?s equals the ?p and therefore the ?w equals zero. It is at this point that water stops entering the cell as it is said to be turgid.

Celeriac is root plant very similar to potato on the surface; it has a small round bulb about the same size as a potato (10-20cm) and lies close to the surface. There is however one major difference at cellular level between the two plants. When at cold temperatures, Celeriac does not leave the starch produced by photosynthesis in long, insoluble chains. Instead it transforms them into short monosaccharide chains that can be dissolved in the cytoplasm. This process has two advantages, one is that the freezing point of the cytoplasm increases to protect it from the frost (otherwise the cell will Lyse-cytoplasm expand and cell wall brake) and the second is that the plant will absorb any water available around the plant (that isn’t frozen). It is able to do this because as the monosaccharides dissolve they have an Osmotic affect (unlike the insoluble starch chains) causing the ?s to decrease thus lowering the ?w potential inside the cell. Since the Celeriac that is being used in the experiment has probable been stored in a cold environment, the levels of monosaccharides can be expected to be high and therefore the ?w to be lower than expected.

To apply this to the experiment is relatively simple. If the ?w in the solution is equal to the ?w inside the cell the net movement of water molecules will be zero. This will therefore not affect the size or mass of the Celeriac since the net gain/loss is nothing. By finding out the relevant ?w to sugar concentration figures, the ?w of the inside of a Celeriac cell can be deduced. The following table shows the sugar concentration to ?w ratios:

Molarity of sucrose solution

Solute potential of plant cell

(mol /dm�)

(kPa)

0.05

-130

0.10

-260

0.15

-410

0.20

-540

0.25

-680

0.30

-860

0.35

-970

0.40

-1120

0.45

-1280

0.50

-1450

0.55

-1620

0.60

-1800

0.65

-1980

0.70

-2180

0.75

-2370

0.80

-2580

0.85

-2790

0.90

-3000

0.95

-3250

1.00

-3500

Preliminary work

For preliminary work an experiment to find the ?w of potato cells was carried out and using the table above this can be achieved. When the mass gain was zero then the ?w inside and outside the cell was equal, by converting the sugar concentration at this time, the ?w of the cytoplasm inside the potato can be discovered.

Results

Concentration

Mass before

Mass after

Change in

Change

Average

of solution

Mass

(% of 1M)

(g)

(g)

(g)

(%)

(%)

0

2.5

2.7

0.2

8.00

8.00

2.5

2.7

0.2

8.00

10

2.3

2.5

0.2

8.70

8.51

2.4

2.6

0.2

8.33

20

2.4

2.6

0.2

8.33

8.33

2.4

2.6

0.2

8.33

30

2.4

2.6

0.2

8.33

6.25

2.4

2.5

0.1

4.17

40

2.4

2.6

0.2

8.33

4.17

2.5

2.5

0.0

0.00

50

2.4

2.4

0.0

0.00

0.00

2.4

2.4

0.0

0.00

60

2.5

2.4

-0.1

-4.00

-4.00

2.5

2.4

-0.1

-4.00

70

2.4

2.1

-0.3

-12.50

-12.50

2.4

2.1

-0.3

-12.50

80

2.4

2.3

-0.1

-4.17

-8.33

2.4

2.1

-0.3

-12.50

90

2.1

1.8

-0.3

-14.29

-12.14

2.0

1.8

-0.2

-10.00

100

2.4

2.2

-0.2

-8.33

-6.34

2.3

2.2

-0.1

-4.35

The mass change was zero when the molarity of the sugar solution equalled 0.05M (50% of 1M). This is equal to a ?w of -1450 (using the table previous).

During the preliminary work 5cm cylinders of potato were used, it was noticed that the tops of these were out of the solution. Therefore in the Celeriac experiment the cylinder will be 3cm to make sure they are totally submersed.

Also the experiment will be left longer to make sure the Osmosis has finished taking place.

Prediction

The prediction for the ?w of Celeriac must take into account two factors. Firstly the ?w of potato and secondarily the fact that Celeriac converts insoluble starch molecules into soluble monosaccharide chains (at low temperature) which will have an Osmotic effect. Therefore the prediction for the ?w of Celeriac is:

The ?w of Celeriac is going to be less than that of potatoes (-1450) but not by a huge amount

Variables

Independent

* Sucrose solution molarity: The molarity of the sucrose solution will vary according to the concentrations stated in the method. They will be made up by mixing varying amounts of 1M sucrose solution and distilled water (?s of zero). To avoid errors the amounts of each will be measured carefully and they will not be left open for long since evaporation will lower the water content and therefore increase the concentration.

Measuring devise: Pipette 1ml divisions

Dependant

* Mass of the chip: This will vary depending on whether the ?w potential is higher inside or outside the cell. If it is higher outside, then due to Osmosis, water will enter the plant, increasing its mass. This change can be measured to work out a total percentage loss/gain so that starting mass will have a minimal affect; when there is no change the ?w must be equal and therefore the ?s of the solution equals the ?w of the Celeriac.

Measuring devise: Electronic weighing scales 0.01g accuracy

* Length of the chip: Similar to the mass change, the movement of water molecules into/out of the plant due to Osmosis will affect the length of the chip. This can also be recorded and the ?w calculated. This however is not as accurate since the weighing equipment is more accurate than the measuring equipment available.

Measuring devise: Ruler 1mm divisions

Constants

* Temperature: The temperature must be constant since an increase in temperature would cause an increase in the kinetic energy that the water molecules have. This would speed up the rate in which they brake through the cell membrane, effectively increasing the rate of Osmosis. Although the ?w potentials inside and out should be equal at the end, this might not be the case and an experiment at a higher temperature would be nearer this equilibrium (thus affecting the end mass).

It is impractical to conduct the experiment in a controlled environment, but by making sure all the containers are close together and none are in direct sunlight, the temperatures should be close enough not to give a noticeable affect.

* Time: Time must also be kept constant since if Osmosis had not fully finished then an increased amount of time would allow more water molecules to enter/leave the plant which would affect the results.

Measuring devise: Clock accurate to 1 second

* Concentration of 1M solution: Since all the solutions are made up from the sucrose solution, it must be accurately 1M and also constant (all of it made up at the start of the experiment). Not applying this could lead to inaccurate concentrations which would lead to inaccurate results of ?w.

To make sure the solution is constant, it will all be made up at the start of the experiment and not left out for long since evaporation of water would lead to an increase in the strength of the concentration.

* Starting length/mass of chips: At the start of the experiment the shape, volume and length of the chips will be as close to each other as possible. Although it would be impossible to cut each cylinder to exactly the same size, it is possible to be fairly accurate. By using a cork borer the cross-sectional area will be constant, cutting it with a scalpel and using a ruler will help to keep the length constant.

* Same Celeriac plant: All the cylinders will be cut from the same plant since different plants will have grown in slightly different environments. The variation in the environments will have affected the amount of starch produced (due to photosynthesis-light and water availability) and therefore the conversion into monosaccharides and the ?s. If the ?s of all the cylinders were different it would be much harder to find a value for ?w.

* Same part of the plant: The cylinders will also be cut from the same area of the cortex (as is physically possible). Different areas will have varied densities, concentrations of sugars and therefore ?w. A constant ?w makes the results obtained more accurate.

Apparatus

Cork borer size 4

Scalpel

Tile cutting slate

Ruler 30cm accurate to 1mm

Electronic weighing scales accurate to 0.01g

Celeriac bulb

Sucrose solution 1M 1litre

2 Syringe 25ml with 1ml divisions

11 McAndrew Bottles & Tops 50ml

Distilled water 1 litre

Clock 12 hours accurate to 1 second

Tissue paper

Chinagraph pencil

Experimental safety

* Take care while cutting with the cork borer and the scalpel, making sure you cut away from the body on the tile

* Take care not to get any of the chemicals in your eye – safety glasses should not be necessary

Method

1. Using the 2 syringes, measure out the correct amounts of 1M sucrose solution/distilled water for each of the different solutions, pouring each into a different McCartney bottle. Use the table below for quantities

Strength of sucrose solution

Distilled water

1M sugar solution

(% 1M)

(ml)

(ml)

0

20

0

10

18

2

20

16

4

30

14

6

40

12

8

50

10

10

60

8

12

70

6

14

80

4

16

90

2

18

100

0

20

2. Label each of the McCartney bottles using the Chinagraph pencil

3. Using the cork borer, cut out 11 cylinders (use a pencil to get the Celeriac out of the borer)

4. Measure the cylinders using the ruler and cut each to 3cm using the scalpel (on the cutting tile)

5. Weigh each of the cylinders and record the results

6. Place the Celeriac cylinders in the different solutions and leave for an 3 hours

7. After an hour take them out of the cylinders, dry them off on some tissue paper and weigh them

8. Record your results and wash out the McCartney bottles for the next experiment

9. Repeat the experiment twice more

Results

Concentration in solution

Mass before

Mass after

Change in

Change

Average

mass

(M)

(g)

(g)

(g)

(%)

(%)

0

1.34

1.50

0.16

11.94

13.96

1.67

1.92

0.25

14.97

1.27

1.46

0.19

14.96

0.1

1.23

1.32

0.09

7.32

12.98

1.48

1.79

0.31

20.95

1.31

1.45

0.14

10.69

0.2

1.37

1.49

0.12

8.76

10.37

1.63

1.82

0.19

11.66

1.31

1.45

0.14

10.69

0.3

1.31

1.45

0.14

10.69

10.29

1.62

1.83

0.21

12.96

0.97

1.04

0.07

7.22

0.4

1.35

1.45

0.10

7.41

5.16

1.70

1.76

0.06

3.53

1.32

1.38

0.06

4.55

0.5

1.33

1.39

0.06

4.51

3.48

1.70

1.75

0.05

2.94

1.34

1.38

0.04

2.99

0.6

1.22

1.30

0.08

6.56

1.69

1.80

1.74

-0.06

-3.33

1.09

1.11

0.02

1.83

0.7

1.06

1.00

-0.06

-5.66

-6.16

1.70

1.59

-0.11

-6.47

1.42

1.33

-0.09

-6.34

0.8

1.10

1.03

-0.07

-6.36

-6.19

1.57

1.50

-0.07

-4.46

1.29

1.19

-0.10

-7.75

0.9

1.16

1.16

0.00

0.00

-6.78

1.71

1.53

-0.18

-10.53

1.02

0.92

-0.10

-9.80

1.0

0.92

0.84

-0.08

-8.70

-10.22

1.67

1.49

-0.18

-10.78

1.25

1.11

-0.14

-11.20

Conclusion

From the results obtained and circumstantial support from the background knowledge (and preliminary work) it can be concluded that

As the Concentration of the solution increases, the percentage change in mass decreases

The percentage change in mass is largest when the concentration of the sucrose solution is 0M. At this point the change is +13.96%, as the concentration increases by 0.5M intervals, the change drops to +12.98, +10.37, +10.29, +5.16, +3.48 and +1.69. It then crosses the x-axis when the concentration is 0.62M, thus allowing the water potential to be calculated (below). The percentage change continues to drop (losing mass), -6.16 at 0.7M, then -6.19, -6.78 and -10.22 when the concentration is 1M. This appears as a steep, relatively straight line graph of concentration against percentage change.

Also from the results, a figure for the water potential of Celeriac can be discovered

The Water Potential of this particular Celeriac bulb was -1872kPa

This conclusion has been obtained from the graph of solution concentration against change in mass; the point in which the line crosses the x-axis is when the net movement of water into and out of the cell due to osmosis is zero. Since the net movement is zero, the water potential inside and outside the cell must be equal. By calculating the water potential of the sucrose solution, the water potential of Celeriac can be discovered.

The line crossed the x-axis at 0.62M. Using results taken from a separate source (should in the background knowledge), the water potential of 0.62M sucrose solution is -1872kPa. Therefore the water potential of the root vegetable Celeriac is (approx.) -1872kPa.

Interpretation

This conclusion supports the original prediction that the water potential of Celeriac is going to be lower than that of potatoes. The preliminary work gives the water potential of potatoes to be -1450kPa, since Celeriac is -1872kPa the prediction is correct.

Since the cellular structure of Celeriac is practically identical to that of potatoes, there is only one explanation for this difference in water potential. As stated in the background knowledge, when Celeriac is in a cold environment it brakes down long, insoluble starch chains into shorter, soluble monosaccharide chains (such as sucrose). These shorter chains dissolve in the cytoplasm and decrease its solute potential, because of the equation:

Water Potential = Solute Potential + Pressure Potential

The decrease in solute potential (and the constant pressure potential) also causes a decrease in the water potential. This explains why Celeriac has a lower water potential than potatoes that are unable to perform this process.

Evaluation

The results might have been slightly inaccurate. This could have been due to several different factors:

1. The length of the cylinders might not have been exactly constant throughout all of the chips. The ruler only measured to millimetres and human error could have led to slight variation to the lengths of the cylinders which would have affected the volume:surface area ratio and therefore Osmosis speed.

2. The time in which the cylinders were in the sugar solutions were not exactly the same. It would be impossible for all of the cylinders to be put into their solutions at precisely the same time. They were however put in and taken out in the same order which should minimise the variations. Also since the experiment went on for an hour, a few seconds will have a minimal affect.

3. The weighing of the cylinders could have also created inaccuracies in two separate ways. Firstly the electronic weighing scales could have been calibrated wrongly which would have given constant inaccuracies throughout the measurements. Secondly the scales only weigh to 0.01g which means that any amounts smaller than this would not have been recorded.

4. Sucrose solution concentration could also have lead to inaccuracies. The pipettes only measured to 1mm and combined with human error this will have caused slight variation. Also the 1M sucrose solution might not have been correct. It might have been made up inaccurately, which would have lead to a constant inaccuracy throughout all of the solutions; or if left out in the open, water would have evaporated increasing its strength.

5. The density of the Celeriac cut will also have an affect on its water potential. Since the densities will vary throughout the plant, and the same piece can not be used for every test, each different cylinder will have a slightly different density causing slightly inaccurate readings.

6. Temperature would have affected the rate of Osmosis. By keeping all of the cylinders out of the sunlight ad close together the temperature variations between cylinders should have been kept to a minimum.

7. Number of repetitions. Although three readings for each concentrations should be adequate for this investigation; to gain more accurate and reliable results, the experiment should be repeated many more times.

Errors

Although some of the inaccuracies cannot be measured such as human error, others can be. The possible inaccuracies due to sensitivity can be calculated to find the maximum possible percentage error, and see if it would have a noticeable affect on the readings.

The electronic measuring scales are accurate to 0.01g and therefore +/- 0.005g

Percentage error: 0.005 x 100 = 0.60%

0.84

The rulers are accurate to 1mm (0.1cm) and therefore +/- 00.5cm

Percentage error: 0.05 x 100 = 0.20%

30

The pipettes are accurate to 1ml and therefore +/- 0.5ml

Percentage error: 0.5 x 100 = 2.5% (5%)

20

Also since each solution is made up of a combination of 1M sucrose solution and distilled water, two pipette readings are made which will increase the maximum possible inaccuracy to 5%.

After looking at the relatively large variations between the readings for the same solution concentrations, it is unlikely that these errors would have had a noticeable effect on the analysis since they would only add up to a maximum error of about 6.5%.

The large variations between readings for each concentration does however question the reliability of the experiment although the averages do give results and a graph that is supported by the background research (decreasing change in mass as the concentration increases, and passing through the a-axis at approximately the right figure).

Limitations

There are several major limitations in this investigation, apart from the inaccuracies mentioned above, which if eliminated would allow much more accurate results to be obtained.

Firstly the time limit given for the experimental work. Since it was only possible to have a few hours practical time the number of readings that were obtained were less than adequate to make a detailed conclusion. Repeating the experiment more and more times would increase both the accuracy and reliability of the conclusions written on water potential in Celeriac.

Also the number of Celeriac plants available to use. Although when only repeating the experiment three times the same plant should be used, if the number of repetitions were to be increased so should the number of plants used. The question posed at the start of the investigation was not to find the water potential of one Celeriac plant, but an average reading for Celeriac. This could only be obtained by experimental work on numerous numbers of plants.

More accurate equipment would also allow more reliable readings to be taken; larger quantities of apparatus to reduce time taken and more people working together to cut out the time differences between the periods of time the chips of Celeriac were in the solution.

Anomalous results

The results obtained were relatively accurate considering all the inaccuracies mentions previously. There were however some anomalous results. A 21% increase at 0.1M and no change at 0.9M were way out from what was expected and the other two readings in their respective solution concentrations. This could have been due to a number of factors such as not waiting for the scales to finish calculating the weight, very inaccurate reading of the pipette or most likely a piece of Celeriac which was particularly more dense/less dense than the other cylinders.

Improvements

There are many improvements that could be applied to the experiment:

1. With more time the experiment could be repeating more times which would give an average results which would be far closer to the true value. This would be most noticeable in that the graph of solution concentration against percentage mass change would be a smooth curve or straight line.

2. More Celeriac plants would allow experiments on a variety of different plants which would give a more accurate reading for the water potential of Celeriac.

3. A team of scientists instead of just one. This would allow all of the chips to be cut and placed in the solutions at the same time and therefore cut out any inconsistencies in the timing of the experiment.

4. Making the 1M solution up closer to the time in which the experiment is going to take place would reduce the amount of water evaporating which increases the concentration of the solution.

5. Also mixing each solution using sucrose powder and water individually would cut out errors due to evaporating water and inaccuracies when measuring the sucrose solution in the pipettes. By calculating the ratio of sucrose powder to water for each concentration, the solutions could be prepared in each McCartney bottle.

Conclusion Validity

Taking into account the inaccuracies and problems mentioned above it can be concluded that although the results are not accurate enough to give a reliable figure for the water potential of Celeriac, they are accurate enough to show that it is noticeably lower than that of potato which supports the view that Celeriac brakes starch chaining down into short monosaccharides in cold weather.