Molecular Evolution in Plants
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Molecular Evolution in Plants

Barbara W. Heavers, Jane Y. Meneray,
Jane E. Obbink, and Harry J. Wolf

Target age or ability group: Introductory Biology through AP Biology level.
Class time required: Paper Chromatography Lab: 2 periods
Absorption Spectrum Lab: 2 periods
Gel Electrophoresis: 3 periods or more
Materials and equipment:
Materials
test tubes
beakers
microcentrifuge tubes
staining trays
serological pipet w/ bulb
distilled water pH meter

Equipment
Gel Electrophoresis Chamber
Electrophoresis Power Supply
5-50 µl Micropipette
Microcentrifuge
Electronic balance
pH meter

Chemicals

Tris/Glycine buffer
  (see recipe in methods section)
Tris/HCL
agarose
loading dye (DNA loading dye works fine)
100 mM NaOAc (Sodium Acetate)
1M NaOAc (Sodium Acetate)
alpha-Naphthyl phosphate
Fast Garnet stain
True Blue Peroxidase Substrate
Naphthylacetate
DMSO (dimethylsufoxide) OR DMF
  (dimethylformamide) NAD
Malic Acid (DL)
NBT(Nitro blue tetrazolium)
PMS( phenzaine methosulfate)
Amido Black

*Optional chemicals*
3.0% Hydrogen Peroxide
4-chloro-1-naphthol
Methanol
MTT ( tetrazolium thiazolyl blue)
Liquid detergent
Adolph's Tenderizer
Ethanol
Tris-EDTA
non-iodized NaCl

*may be substituted in some cases throughout methods

The chemicals listed above pertain to both the gel preparation and staining for several enzymes. Check the materials for each enzyme stain to determine your specific needs.

Summary of activity: Paper Chromatography: A variety of simple studies with paper chromatography allow students to study and specify the chlorophylls and other light absorbing pigments in a variety of plants. Several comparative studies are suggested. Absorption spectra for extracted pigments can be determined spectrophotometrically as well.

Gel Electrophoresis: Studies of plant proteins and isozymes are suggested utilizing gel electrophoresis. Investigators may select particular enzymes to compare plants, observe the effects of environmental stress, or compare enzymes in C3 and C4 plants. Electrophoresis may also be used to compare DNA in related species.

Prior knowledge, concepts or vocabulary necessary to complete activity: The paper chromatography studies are suitable for introductory biology students. Absorption spectra studies and gel electrophoresis studies are appropriate for students who have complete both introductory biology and chemistry. These projects are not an introduction to protein separation, but some aspects can be introduced in a first year biology course, perhaps as a demonstration. Some knowledge of electrophoresis is necessary for the students to run successful separations and to be able to make testable hypotheses.
Teacher instructions: Successful work with gel electrophoresis requires that the teacher be familiar with the system and have some experience with staining. A teacher workshop is an excellent way to gain familiarity with this technology. Try simple separations before attempting more complex ones. Work with more advanced students initially and develop your own modifications to these experiments which are suitable to the materials you have available. Independent projects done by senior students can provide valuable experiences which can then be adapted as labs for younger students. Many of these molecular studies are natural extensions to current studies of plant morphology and help students to distinguish true phylogenetic relationships from similarities resulting from convergent evolution.


Molecular Evolution in Plants

I. INTRODUCTION

A variety of molecules may be studied in plants which will allow us to detect physiological adaptations within groups of plants, variation within populations, speciation, and historical continuity in phylogenetic relationships.

The methods of study to be discussed here include paper chromatography and gel electrophoresis. The first method, paper chromatography, is one of the easiest and most common and has been successfully employed to separate chlorophylls and auxiliary pigments in a wide variety of plants. Usually, a drop of the sample to be separated is applied to a sheet of absorbent material. This material can be paper, plastic, or glass. In the case of plastic or glass these are covered with the absorbent. A mixture of solvents is placed so that it will move across the sheet as it is absorbed. As the mixture moves across, it separates the molecules. Different components of the sample move differently according to their size and/or relative solubility. The various chlorophylls and other plant pigments can easily be separated by this method showing bands of different pigments. The use of paper chromatography is especially suitable as an introduction to the logic and methods of separation technology. It can be utilized as a technique for the novice and generally produces an excellent visualization of different plant pigments.

The second method has been applied to the separation of a wide variety of proteins as well as nucleic acids in plants. Electrophoresis separates proteins, isozymes (multiple molecular forms of enzymes which share a common substrate, but differ in electrophoretic mobility), and nucleic acids by making use of their overall positive or negative charge. If an electric field is applied to a solution containing the molecules to be identified, the molecules will move through the solution at a rate that depends on charge, size, and shape. This may be used to separate proteins or nucleic acids in aqueous solutions or in a solid gel such as starch or agarose. They can then be stained and their presence detected by the bands formed by their migration.

The beauty of plants as organisms of study lies not only in the ease of collecting them, but also in the opportunity for teachers and students to explore the plant biomes of their locales. These techniques allow the scientist to examine variation in plant populations in their own backyards, as well as to investigate more complex evolutionary questions among species in the unique biomes of their regions. Plants which have been developed for agricultural needs also provide rich opportunities for evolutionary comparisons and have the added advantage of simple propagation in the laboratory during winter months in the colder climes.

II. APPLICATIONS

A. Paper Chromatography

1. Study of chlorophylls in spinach as a model for separation.

2. Comparison of chlorophylls and auxiliary pigments of marine algae (green, red, and browns) based on habitat distribution.

3. Comparison of chlorophylls and auxiliary pigments of tropical herbaceous plants (better known as house plants) and temperate zone herbs such as wildflowers.

B. Gel Electrophoresis

1. A comparison of plant proteins from the family: Brassicacae (broccoli, cauliflower, turnips, and Fast plants, etc.) to assess the level of similarity within these cultivated plants.

2. A comparison of proteins in native or commercial grasses and herbs to assess levels of variation within the species.

3. A comparison of DNA or RNA sequences in C3 and C4 plants such as beans vs. corn.

4. A study of corn root proteins grown under drought and flood conditions.

5. Study of DNA in local plant populations.

III. METHODS

A. Paper Chromatography

Materials:

15-20 grams plant material of each kind
Mortar and pestle (pinch of clean fine sand if available)
Centrifuge or Thin filter paper
Ice
Small centrifuge tubes or regular test tubes (100 mm long)
Large test tubes (200 mm long) and corks
Acetone
Petroleum ether (8 ml) + acetone (92 ml) for chromatography solvent
210 mm strip of Whatman paper (2 cm wide)
Pasteur pipettes or capillary tubes
Beakers
Test tube rack

Methods:

Grind plant material to fine consistency with 2-3 ml of acetone. Transfer to centrifuge tube or filter with cheese cloth. Place 3-5 drops with fine pipette of green extract 1.5 cm at bottom end of filter paper. Let each drop dry before applying next one.

Place 3-5 ml of chromatography solvent in large test tube. Make sure end of strip is wet, but the spot is not immersed. Place tube in rack and allow to remain undisturbed for approximately 10-20 minutes. Do not let chromatography front run off top of strip. Dry and then mark spots detected. Measure the distance from the original spot to top of spot as well as the distance the front traveled. The ratio of the spot distance to front distance is the

Rf value. The Rf value allows comparisons between the molecules on separate chromatographs. Make notes on colors of each pigment molecule.

This separation system can be used for all the applications suggested.

Comments: The separation of the various chlorophylls and other pigments allows the student to observe the wide variety of differences in plants living in various light conditions. Spinach, a long season plant flourishes in spring and fall plantings when the daily light availability is diminished. The marine algaes live at different depths in the ocean, typically greens are limited to surface waters, browns extend to deeper waters, and reds approach the limits of photosynthetic activity at about 50 m. Tropical ground plants live in greatly diminished light on the floors of rain forest environments. It is expected that they will also possess a range of photosystem pigments which allow them to more efficiently obtain energy under these conditions. A comparison with annuals typical of the temperate zone should demonstrate differences in geographic distribution.

The Absorption Spectra of Light Absorbing Pigments

If the student wishes to determine the light absorption range of the pigments separated by chromatography, she can cut out the dried spot on the strip and elute the pigment from the paper with 20 ml of acetone. The pooling of 4-5 individual chromatograms increases the concentration of each chlorophyll or pigment. The absorption spectra for each pigment can be easily determined with a spectrophotometer. A blank of 20 ml of acetone should be used to set the absorbency to zero at each wavelength. Record the absorbency of each sample between 400 nm-700 nm. Intervals of 20 nm work well. This additional study is particularly informative when examining the relationship between pigment occurrence and habitat distribution for tropical and temperate herbaceous plants and the marine algaes. Students should plot the spectrum for each pigment and compare it to a published spectrum for identification of the specific pigment molecule.

B. Gel Electrophoresis

1. Separation of Plant Proteins Using Agarose Gels

The agarose gels can be used for a number of plant proteins. The first three applications suggested utilize the protocols presented here. The study of plants from the Brassicacae family, a comparison of native grasses and herbs, and the comparison of C3 and C4 plants all employ the agarose gel system. Students can employ a variety of stains to detect the presence of particular proteins (isozymes) in these plant tissues.

Follow the protocols presented below for the preparation of the gel, protein extractions, and loading of the gel. Then select the appropriate staining gel protocol for the enzyme you wish to study. You will be able to detect heterozygosity or variation in isozymes in plants from the same population. You will be able to observe the presence of particular isozymes in comparative studies.

Target age or ability group: Introductory Biology through AP Biology level.
Class time required: Paper Chromatography Lab: 2 periods
Absorption Spectrum Lab: 2 periods
Gel Electrophoresis: 3 periods or more
Materials and equipment:
Materials
test tubes
beakers
microcentrifuge tubes
staining trays
serological pipet w/ bulb
distilled water pH meter

Equipment
Gel Electrophoresis Chamber
Electrophoresis Power Supply
5-50 µl Micropipette
Microcentrifuge
Electronic balance
pH meter

Chemicals

Tris/Glycine buffer
  (see recipe in methods section)
Tris/HCL
agarose
loading dye (DNA loading dye works fine)
100 mM NaOAc (Sodium Acetate)
1M NaOAc (Sodium Acetate)
alpha-Naphthyl phosphate
Fast Garnet stain
True Blue Peroxidase Substrate
Naphthylacetate
DMSO (dimethylsufoxide) OR DMF
  (dimethylformamide) NAD
Malic Acid (DL)
NBT(Nitro blue tetrazolium)
PMS( phenzaine methosulfate)
Amido Black

*Optional chemicals*
3.0% Hydrogen Peroxide
4-chloro-1-naphthol
Methanol
MTT ( tetrazolium thiazolyl blue)

*may be substituted in some cases throughout methods

The chemicals listed above pertain to both the gel preparation and staining for several enzymes. Check the materials for each enzyme stain to determine your specific needs.

Methods:

One buffer will be used to grind the plants, run the samples on agarose gels, and to assist in the staining process at the end. This buffer is called:

Protein Extraction Buffer/Electrophoresis Running Buffer:

    Tris/Glycine pH ~ 6.0*
      3.0g Tris
      14.4 g Glycine
      up to one liter with distilled water

*The buffer Tris is normally at a pH of 8.0-8.3. For best results, use 1.0 M HCl to lower the pH of the extraction buffer. Better results have been obtained at pH 5 or 6 as compared to pH 8.3. If you are planning to view protein bands at a pH other than 8.3, be sure to change the pH of the extraction/running buffer NOW before proceeding with the lab.

HINT: Make the agarose gel first, before choosing plant samples to grind. As the gel is cooling, you can grind your plant samples. The gel will be ready when your plant extract samples are ready.

Casting the Agarose Gel:

1. Make a 1.5% agarose gel using the Tris/Glycine buffer:
For 100 mls: 1.5 g agarose
100 ml Tris/Glycine buffer
2. Microwave or heat the agarose and Tris/Glycine buffer in an Erlenmeyer flask until liquid boils and agarose is completely dissolved. Be careful, the glass is hot! Use a paper towel or mitt to handle the hot flask.

3. Allow the melted agarose to cool to the touch before pouring into the casting tray.

4. Place the gel comb in the middle of the gel tray, as enzymes will migrate towards both the negative and positive poles of the gel. Properly tape the gel tray in preparation for the agarose.

5. Once the melted agarose has cooled somewhat, pour it into the casting tray and allow the gel to harden. (NOTE: gel thickness should be about 0.3-0.5 cm: thinner gels seem to work better and are easier to stain and interpret). Take care not to bump or hit the gel box during this time.

Protein Extraction Protocol:

1. Obtain a small amount of plant matter such as grass, clover, herbaceous leaves, etc. (Tree and other woody plant leaves do not work well with this procedure as they are hard to grind).

2. Put plant matter into a mortar and pestle with a very small volume (1-2 ml) of Extraction Buffer. Grind the plant matter until the buffer is dark green. If the material is too dry, add another ml of buffer. You want the extract to be liquid, but concentrated, so use the grinding buffer sparingly.

3. Transfer 1 ml of dark green extract into a microcentrifuge tube and spin for three minutes to pellet the plant debris. (If no microcentrifuge is available, use a clinical centrifuge. More sample can be spun, and increase spin time to seven minutes on top speed).

4. Transfer the liquid extract to a fresh microcentrifuge tube. Discard the plant debris pellet.

NOTE: This is a stop point. The extract can be stored in the refrigerator overnight if desired.

5. Increase the density of the extract before loading into the agarose gel. You will want to fill the wells of the gel. Typically, a gel well will hold 15-25 µL. Choose ONE of the following methods to increase extract density:

    a. Dilute 3 parts extract with 1 part 50% glycerol/Bromophenol Blue.

    b. Dilute 3 parts extract with 1 part 40% sucrose.

    c. Dilute 3 parts extract with 1 part loading dye (i.e. DNA loading dye)

6. The sample is now ready to load on an agarose gel.

Loading Agarose gel:

Possible alternative gel loading options:

    a. When working in groups of four with an eight-well gel, make two identical gels on the same gel. Load samples 1, 2, 3, and 4 in wells 1-4. Then load the same samples 1, 2, 3, and 4 in wells 5-8. When the gel is completed, slice the gel carefully down the middle and stain in two different enzyme stains. NOTE: this will work when viewing different proteins of the same pH range.

    b. Make three identical gels on the same gel. Load samples 1 and 2 in wells 1-2, skip well 3 and load samples 1 and 2 in wells 4-5, skip well 6 and load samples 1 and 2 in wells 7-8. When the gel is completed, slice it into three pieces by slicing down through wells 3 and 6. The three pieces now contain the same samples and can be stained in three different enzyme stains.

1. Load 15-25 µL (fill wells) of increased density protein extract (from #5 above) into each well of gel.

2. Connect gel electrophoresis leads and run gel at 100 volts for approximately 30 minutes. Check to see that the dye has migrated about two-thirds down the gel.

3. When using stains that are pH sensitive (pH 5 or 6), allow gel to soak in 100 mM NaOAc for 10 minutes. This step is omitted when banding is not dependent upon pH.

Staining Gel/Stain Recipes:

Use one of the following recipes for each gel to be stained. Only one enzyme can be stained for each gel, therefore it may be desirable to use one of the alternative loading techniques described in the previous section. Each enzyme stain will form a non-soluble precipitate with the bands IN the gel. This means the gel can be stored in the refrigerator (minus the stain) for up to several weeks and the banding pattern will be distinct.

All gels should be bathed in stain for a minimum of 10 minutes. For best results, gels may be left in stain for longer periods of time (i.e., over night). (Special procedures follow for some stains.)

Enzyme: Acid Phosphatase

For a four-well gel:

1. Place gel in ~ 30 ml Tris/Glycine Buffer (use same pH as used for grinding and gel)

2. Dissolve 10 mg alpha-Naphthyl phosphate in 1 ml Tris/Glycine buffer and add to the gel.

3. Dissolve 50 mg Fast Garnet GBC (salt) in 2-3 ml of 1M NaOAc, pH 4.

4. Add small amounts of the Fast Garnet solution to gel; add more solution as needed.

5. View gel with white light box. Compare banding patterns of plants.

Enzyme: Peroxidases

1. Purchase True Blue Peroxidase Substrate, catalog # 710064 Kirkegaard & Perry. Add the pre-mixed substrate directly to the gel in the volume needed to cover and stain the gel.

OR

2. Make your own solution of True Blue Peroxidase Substrate with the following recipe:

    20-50 mg 4-chloro-1-naphthol dissolved in methanol (do this step first)
    200-600 µL of 3.0% Hydrogen Peroxide
    add to a final volume of 100 ml distilled water

3. View the gel with a white light box to see the banding pattern.

Enzyme: Esterases Same banding pattern regardless of pH used.

For a ~4 well gel,

1. Place gel in ~30 ml Tris/Glycine Buffer (use same pH as used for grinding and gel)

2. Dissolve 10 mg Naphthylacetate in 1 ml DMSO (dimethylsufoxide) OR in 1 ml DMF (Dimethylformamide). NOTE: DMSO is preferred over DMF for less toxicity.

3. Add the dissolved Naphthylacetate solution to the gel.

4. Dissolve 50 mg Fast Garnet GBC (salt) in 2-3 ml 1M NaOAc pH 4.

5. Add small amounts of the Fast Garnet solution to gel; add more solution as needed.

6. View the gel with a white light box to see the banding patterns.

Enzyme: Alcohol Dehydrogenase (for corn raised in drought and flood conditions)

1. Combine the following ingredients and pour over gel:

    50 ml 50 mM Tris-HCl pH <7
    1 ml NAD soln. (10 mg/1 ml)
    0.2 ml Ethanol
    1 ml NBT (Nitro blue tetrazolium) or MTT (tetrazolium thiazolyl blue) soln. (10 mg/ml)
    2 mg (0.4 ml) PMS (phenazine methosulfate) soln. (5 mg/ml)

2. Incubate at 37°C until bands are optimally developed. Rinse and store in water (if NBT is used) or fixative.

Enzyme: Ribulose-bisphosphate carboxylase (for C3 and C4 plants)

1. Dissolve the following:

    25 mg Amido Black
    50 ml Fixative consisting of: 5 parts water, 5 parts methanol, and 1 part acetic acid

2. Dissolve the Amido Black in the fixative solution. Pour over gel and stain for 15-30 minutes. Pour off stain and rinse three times with fixative for 15 minutes each to remove background staining. You may vary staining by adjusting time.

Enzyme: Malate dehydrogenase (for C3 and C4 plants)

1. Dissolve the following:

    50 ml 50 mM Tris-HCL, pH 8.5
    1 ml NAD soln. (10 mg/1 ml)
    1 ml Malic acid (150 mg/1 ml) Use L or DL, which is less expensive.
    1 ml NBT (Nitro blue tetrazolium) or MTT (tetrazolium thiazolyl blue) soln. (10 mg/ml)
    0.4 ml PMS (phenzaine methosulfate) soln. (5 mg/ml)

2. After ingredients above are dissolved pour over gel. Incubate until blue bands appear. Rinse and store in water ( if NBT is used) or fixative.

3. Note: Both C3 and C4 plants have these specific enzymes you will test, but as different isozyme forms.

Teacher Notes:

In order to visualize protein bands, Fast Garnet GBC is used as a coupler. The coupler binds to the enzyme/protein substrate and produces a color reaction, hence the bands become distinct. Any commercial dye containing dizonium salt can be used as a coupler. It is not recommended that Fast Green be used, but any other (color)Fast dye can be used. Check the Sigma catalog under "Fast" for other dye colors.

To order pre-mixed True Blue Peroxidase Substrate, Catalog Number 710064 (50 mls = $14.00)

    Kirkegaard & Perry
    2 Cessna Court
    Gaithersburg, Maryland 20879
    1-800-638-3167

To change the pH of the extract and visualize different bands at various pH's:

Mix up the Protein Extraction/Running Buffer as directed, but change to the desired pH by adding HCl. Use this "corrected pH" buffer for protein extraction, making of agarose gel, and gel running buffer. Remember to let the finished gel soak in 100 mM NaOAc for 10 minutes before staining. This step will ensure the gel is at a lowered pH and thus different protein bands will appear.

2. Extraction of Plant DNA and Separation by Gel Electrophoresis:

This method is an all-purpose technique for extraction of DNA from plants. Follow the steps outlined to obtain a DNA extract suitable for separation by gel electrophoresis.

a. Homogenization: Grind 12 g of finely cut plant leaves with a mortar and pestle with 1 ml distilled water. Add l 1ml of 20% liquid detergent (e.g., Dawn blue, Joy, etc.) and 9 g non-iodized NaCL. Place the mixture in a beaker or test tube and place in a hot water bath at 65° C for 10 minutes. Cool the mixture on ice to stop the process. Centrifuge the mixture to collect the filtrate. You may refrigerate the filtrate until the next day.

b. Deproteinization: Pour 6 ml of the filtrate into a tube and add 1 ml of either 15% solution of Adolph's Natural Tenderizer or one Contact Lens Cleaner (in tablet form). One ml of fresh papaya juice also works well. Gently invert the tube to mix.

c. Precipitation of DNA: Slowly pour 14 ml of cold 95% ethanol (or 7 ml 91% isopropanol) down the side of the test tube. Spool DNA into some sort of rod (glass) or wood splint by gently stirring the rod where the alcohol and filtrate meet.

d. The isolated DNA fragments or strands can be collected by separating the alcohol-DNA mixture with a pipette and evaporating the ethanol. It can then be reconstituted in TRIS-EDTA buffer and applied to an agarose gel as described for proteins in Part 1.

e. Additional techniques for further work with DNA can be found in DNA Science by D. Micklos and Greg Freyer (See references).

References:

Micklos, David A. and Greg A. Freyer, DNA Science, A First Course in Recombinant DNA Technology , 1990. Available from Carolina Biological Supply Company, Burlington, NC.

Soltis, Douglas E. and Pamela S Soltis, "Isozymes in Plant Biology." Advances in Plant Sciences Series, Vol. 4. T.R. Dudley, Editor, 1989.

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