Experiment 2 transport of materials across cell membranes and plant cell water relations

transcript

TRANSPORT OF MATERIALS ACROSS CELL MEMBRANES &

PLANT-CELL WATER RELATIONS

Alcantara. Catindig. Ignacio. Kim.GROUP 2

DIFFUSION OF SELECTED PLANT PIGMENTS

4 plant specimens

A B C D

dH2O dH2O+

H2O bath

veg. oil heated veg. oil

plant specimen

*a total of 16 test tubes were used

Methodology

Set aside for 30 minutes

Shake test tubes

Compare color intensities. Record results.

Methodology

Results

Test tube 1 (w/ dH2o) +++

Test tube 2 (w/ heated dH2o) ++++

Test tube 3 (w/ veg. oil) +

Test tube 4 (w/ heated veg. oil) ++

Bixa orellana

Results

Test tube 1 (w/ dH2o) +

Test tube 2 (w/ heated dH2o) ++

Test tube 3 (w/ veg. oil) +++

Test tube 4 (w/ heated veg. oil) ++++

Zingiber officinale

Results

Test tube 1 (w/ dH2o) +

Test tube 2 (w/ heated dH2o) ++

Test tube 3 (w/ veg. oil) +++

Test tube 4 (w/ heated veg. oil) +++

Solanum toberosum

Results

Test tube 1 (w/ dH2o) ++

Test tube 2 (w/ heated dH2o) +++

Test tube 3 (w/ veg. oil) +

Test tube 4 (w/ heated veg. oil) +

Allium cepa

Discussion

Diffusion: directed movement of molecules from a region of high concentration to a region of lower concentration random thermal motion

Affected by: Concentration and size of diffusing particles

Discussion

Bixa orellana contain the pigments bixin and orelline Carotenoid pigments Lipid-soluble due to long hydrocarbon chain

Zingiber officinale contain flavonoids (quercetin, rutin,

catechin, epicatechin, kaempferol and naringenin)

Lipid-soluble due to the ring-like carbon structures.

Discussion

Red Onion Anthocyanin: water-soluble Quercetin: lipid-soluble

Potato skin Contains carotenoid pigments (neoxanthin,

violaxanthin and lutein) Lipid-soluble

Discussion

Bixin and orelline were able to diffuse much faster than the others

Carotenoids are able to reach high concentrations within chromoplastids and may actually form crystals

Large amount of bixin and orelline increased the rate of their diffusion throughout the medium

OSMOSISCELL CHANGES IN PLASMOLYSIS

Osmosis: Cell Changes in Plasmolysis

OSMOSIS Diffusion of water across a semi-

permeable membrane

•A Tradescantia spathacea leaf was obtained and strips of its lower epidermis were prepared using a blade.

•A wet mount was made using the lower epidermis and the cells were observed under the microscope.

•Water was drawn off the slide with tissue paper and was replaced with a drop of 5% NaCl.

•The cells were again observed under a microscope and changes were noted.

•The procedure was repeated using white onion and then apple skin.

Discussion of Results

Tradescantia spathacea

Wet mount 5% NaCl

5% NaClWet mount

Allium cepa

5% NaClWet mount

Turgid cell happens when cell is hypotonic to the

surrounding solution optimal for plants

Plasmolyzed cell happens when cell is hypertonic to the

surrounding solution; plasma membrane lysis may cause cell death cell wall still intact

Anthocyanin water-soluble pigment discoloration in plasmolysis

Conclusion

Osmosis is the diffusion of water through a semi-permeable membrane and this can be observed using different epidermal cells with pigments

Cells in hypotonic solutions become turgid and cells in hypertonic solutions become plasmolyzed as water goes in and out of the cell, respectively.

FACTORS AFFECTING INTEGRITY OF CELL MEMBRANE

Methodology

Red apple peel

In test tubes

A: Distilled + Room

B: Distilled +

Refrigerator

C: Distilled + 60°

Under the microscop

D: 50% Chlorofor

E: 50% Acetone

F: 0.1M NaOH

G: 0.1M HCl

Results

Test Tube Intensity of Color

A (room temp.) +++

B (refrigerator/cold) ++

C (water bath/ 60C) +

D (Chloroform) +

E (Acetone) ++

F (NaOH) +++

G (HCl) ++++

Discussion

Red violet pigment in apples: ANTHOCYANIN

Found at the vacuole

Too big to exit cell membrane and tonoplast

Discussion

Heat: Denatures proteins; destroys membrane

Cold: fatty acid tails rigid; less permeability

Organic Solvents interact with bilayer causing disruption of membrane

Low and High pH: destroys tertiary and quaternary structure of pigments

DETERMINATION OF SOLUTE CONCENTRATION OF CELLS

(PLASMOLYTIC METHOD)

10Drops of sucrose solution + Tradescantia spathacea

epidermal strips(0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M and 1.0M)

30 minutes

Methodology

Wet mount

Observed under the microscope

The number of PLASMOLYZED and UNPLASMOLYZED cells were recorded as well as the concentration that caused INCIPIENT PLASMOLYSIS.

The OSMOTIC POTENTIAL value was also calculated.

Methodology

OSMOSIS: diffusion of water across a semi-permeable membrane

Water potential (Ψw) Important in determining the direction of

osmosis High to low Ψw

PLASMOLYSIS: shrinking of a cell due to water loss happens when a cell is submerged in a

hypertonic solution

Source: http://www.excellup.com/interbiology/planttransportquestion.aspx

The cell wall is permeable to water and sucrose.

The plasma membrane is permeable to water but not to sucrose.

Sucrose + Water

Sucrose Concentrati

on (M)

Osmotic Potential

(bars)

Plasmolyzed Cells (#)

Unplasmolyzed Cells

Total # of Cells

Counted

% Plasmolyze

0.1 -2.5 6 145 151 3.97

0.2 -5.0 20 174 194 10.31

0.3 -7.5 41 84 125 32.8

0.4 -10.0 90 105 195 46.15

0.5 -12.5 99 88 187 52.94

0.6 -15.0 186 85 271 68.63

0.7 -17.5 112 83 195 57.44

0.8 -20.0 76 54 130 58.46

0.9 -22.5 89 61 150 59.33

1.0 -25.0 172 73 245 70.20

Data showing the osmotic potential, number of plasmolyzed and unplasmolyzed cells, total number of cells counted, and

the percentage of plasmolyzed cells found under the microscope for each concentration of sucrose solution used.

Using the data from petri dish 1,

Sucrose Concentrati

on (M)

Osmotic Potential

(bars)

Unplasmolyzed Cells

Total # of Cells

Counted

% Plasmolyze

0.1 -2.5 6 145 151 3.97

0.2 -5.0 20 174 194 10.31

0.3 -7.5 41 84 125 32.8

0.4 -10.0 90 105 195 46.15

0.5 -12.5 99 88 187 52.94

0.6 -15.0 186 85 271 68.63

0.7 -17.5 112 83 195 57.44

0.8 -20.0 76 54 130 58.46

0.9 -22.5 89 61 150 59.33

1.0 -25.0 172 73 245 70.20

percentage of plasmolyzed cells increased as the concentration of sucrose in the solution increased.

sucrose concentration of 0.6M - 68.63% of plasmolyzed cells

INCIPIENT PLASMOLYSIS osmotic potential of the cell is the same as

the solution’s the protoplast just fills the cell volume and

neither exerts pressure to the cell wall nor withdraws from it

50% of plasmolyzed cells

Sucrose Concentrati

on (M)

Osmotic Potential

(bars)

Unplasmolyzed Cells

Total # of Cells

Counted

% Plasmolyze

0.1 -2.5 6 145 151 3.97

0.2 -5.0 20 174 194 10.31

0.3 -7.5 41 84 125 32.8

0.4 -10.0 90 105 195 46.15

0.5 -12.5 99 88 187 52.94

0.6 -15.0 186 85 271 68.63

0.7 -17.5 112 83 195 57.44

0.8 -20.0 76 54 130 58.46

0.9 -22.5 89 61 150 59.33

1.0 -25.0 172 73 245 70.20

Concentration where incipient plasmolysis occurred

is 0.5M with a 52.94% of plasmolyzed cells.

The osmotic potential (Ψs), in bars, of the sucrose solutions were computed using this formula:

where:m = concentration of the solute expressed as molality (moles solute/ kg H2O)i = ionization constantR = gas constant (8.314 J/mol∙K)T = absolute temperature (C + 273)

Sample computation:Osmotic potential of a 0.1 M sucrose

solution

Ψs = -(0.1 mol/L)(1)(8.31 J/K-mol)(300K)Ψs = -249.3 J/L (0.01 bars/ 1 J/L) = -2.493 barsΨs ~ -2.5 bars

Sucrose Concentrati

on (M)

Osmotic Potential

(bars)

Unplasmolyzed Cells

Total # of Cells

Counted

% Plasmolyze

0.1 -2.5 6 145 151 3.97

0.2 -5.0 20 174 194 10.31

0.3 -7.5 41 84 125 32.8

0.4 -10.0 90 105 195 46.15

0.5 -12.5 99 88 187 52.94

0.6 -15.0 186 85 271 68.63

0.7 -17.5 112 83 195 57.44

0.8 -20.0 76 54 130 58.46

0.9 -22.5 89 61 150 59.33

1.0 -25.0 172 73 245 70.20

Conclusion

As solute concentration increased, osmotic potential became more negative along with the water potential

% of plasmolyzed cells also increased as water potential became more negative Water diffuses to a region with a more negative water

potential To equilibrate the concentration of water inside of cell

to that of the surrounding solution, water moved out of the cell

ESTIMATION OF THE WATER POTENTIAL OF STORAGE TISSUE

(VOLUME CHANGE METHOD)

Methodology

•Eleven sets of five potato cylinders (each potato cylinder 1cm long) were cut off from a large potato and immediately placed in 50 mL beakers

•20-ml of one concentration of sucrose solution (0.1 M-1.0 M, with 0.1 graduations) were placed in the 10 separate beakers respectively.

•The remaining beaker contained 20 mL distilled water

Methodology

•The fresh weights of each set were recorded. The potato cylinders were removed after 90 minutes and weighed again.

•The difference between the initial and final weights were divided by the initial weight, and then multiplied by 100 to get % weight change.

∆weights of the potato cylinders=caused by the presence of sucrose (this stimulated the cells to generate an osmotic potential (Ψs))

Osmotic potential reduces the free energy of the system.

The effect of osmotic potential is countered by hydrostatic pressure.

Sucrose Contentration

Initial Weight(go)

Final Weight (g)

∆ Weight (g - go)

% ∆ Weight

0 2.7930 2.8600 0.0670 2.40

0.1 2.8373 2.8325 0.0048 0.169

0.2 2.7395 2.5479 0.1916 6.99

0.3 2.6992 2.3307 0.3685 13.65

0.4 2.6900 2.0258 0.6642 24.69

0.5 2.8865 2.1195 0.7670 76.70

0.6 2.8773 3.0325 0.1552 5.39

0.7 2.9200 3.0635 0.1435 4.91

0.8 2.9564 3.0940 0.1376 4.65

0.9 2.8681 2.9964 0.1354 4.72

The initial, final, and change in weights and the Percent Weight Change of potato cylinders placed in different concentrations of sucrose solutions for 90 minutes.

0 0.2 0.4 0.6 0.8 1 1.20

Sucrose Concentration (in M, moles/L)

Percent Change in Weight (g)

Fig 5. Plot of Percent Change in Weight (in grams) vs. Sucrose Concentration (in M, moles/L).

Experimental data: failed to present the expected trend and failed to show the concentration of sucrose where there is 0% ∆ in weight

Theoretical data would show that the higher the concentration of sucrose, the higher the percent change in weight.

Conclusion

Theoretically, the sucrose concentration between 0.2-0.3M should have registered the zero percent change in weight.

Experiment 2 transport of materials across cell membranes and plant cell water relations

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