Plants 10 - BIOLOGY JUNCTION

Chlorophyll Fluorescence

Chlorophyll Fluorescence

INTRODUCTION

When a pigment absorbs light, electrons of certain atoms in the pigment molecules are boosted to a higher energy level. The energy of an absorbed photon is converted to the potential energy of the electron that has been raised to an excited state. In most pigments, the excited electron drops back to its ground-state, or normal orbit, and releases the excess energy as heat. Some pigments, including chlorophyll, emit light as well as heat after absorbing photons.
In the chloroplast, these excited electrons jump from the chlorophyll molecule to a protein molecule in the thylakoid membrane, and are replaced by electrons from the splitting of water. The energy thus transferred, is used in carbohydrate production.
This release of light is called fluorescence. Chlorophyll will fluoresce in the red part of the spectrum, and also give off heat. In this lab, you will observe this fluorescence by separating the chlorophyll from the thylakoid membrane.

MATERIALS

Spinach leaves	Flashlight or small lab light
Mortar and pestle	Test tube
Acetone	Filter paper
25-mL graduated cylinder	Funnel
Ring stand or funnel rack	Safety goggles

PROCEDURE

1. Grind the spinach leaves using a mortar and pestle.

2. Add acetone to the ground leaves, using enough acetone and spinach leaves to get between 10 and 15 mL of extract.

3. Set up your filtering apparatus, and using proper filtering technique, filter the extract to a test tube. NOTE: Use a small amount of acetone to wet the filter paper, to hold it into place, instead of water.

4. Shine a flashlight, or other similar light source, through the test tube and extract.

5. Observe the fluorescence of the chlorophyll at a 90 degree angle to the flashlight.

Chromatography Lab

Chromatography Lab

Problem: How do you separate the different pigments in a plant?

Materials:

Cone-type (size 4) coffee filter paper (or Whatman #1 chromatography paper)
large glass jars
acetone
distilled water
capillary tubes
fresh spinach
mortar and pestle
clean sand

Introduction:

In this activity you will be experimenting with a technique called chromatography which will allow you to visually demonstrate that the pigment in leaves is a combination of several different colored pigments.

This technique is useful in that it can separate and identify the various components of mixtures, such as those contained in plant pigments. A pigment is a substance that absorbs light at specific wavelengths, chlorophyll is one of these pigments. Its green-yellow in color is due to the absorption of red, orange, blue, and violet wavelengths and the reflection of the green and yellow wavelengths. This occurs when white light (containing all of the light wavelengths, or the entire spectrum of colors) shines on the leaf surface, all of the wavelengths are absorbed except for the ones you see, which are green-yellow, those are the portions of the spectrum being reflected.

If the conditions are identical, the relative distance moved by a particular compound is the same from one mixture to another. This is why chromatography can be used to identify a compound. The actual identification requires a simple calculation as shown below:

Rf = distance moved by compound from original spot divided by the distance moved by solvent from original spot

It is important to remember that several factors can influence the reliability of the Rf value, these include humidity, temperature, solvent, pigment extract preparation, and the amounts of the material present. Values are comparable only when the extracts are prepared in the same way and the chromatograms are prepared identically and developed together in the same container.

Acetone is flammable (even the amount found in nail polish remover), keep it away from sparks or open flames. Wear eye protection, especially if using pure acetone.

Procedure

1. Each lab group (or individual if not working in groups) will need 4 strips of filter paper, approximately 6 inches long and 1 inch wide, 2 chromatography development containers (500 ml beakers or large fruit jars work well), 2 large rubber bands (able to stretch around the vessels from the mouth to the bottom of the vessel), 2 solvents, water and either pure acetone, or nail polish remover.

2. Do the following with both fresh spinach leaves; tear leaf material and place in a glass container, cover with acetone (this should be done the day before the actual lab activity). An alternative pigment extraction technique is to use a
mortar and pestle. Place plant material the vessel, add a little clean sand, some acetone and then grind until a dark green liquid appears. Both techniques yield very dark pigments with which to work. Be certain to keep the pigments apart throughout the entire activity.

3. Place one of each solvents (water and acetone, or nail polish remover) in the chromatography vessels and stretch a rubber band length-wise around each vessel. The rubber band will be the mechanism for hanging the chromatography strips.

4. Make a pencil mark on each of the 2 chromatography strips, in the center, directly above the point of the strip, about 1 inch from the tip of the paper. Using a capillary tube, or tooth pick, apply the plant pigment to each filter paper strip. This is done by touching the tooth pick or capillary tube which has been dipped in the pigment, to the pencil mark. Make an application, then wave the paper gently to dry it a little before the next application. Be patient, you will need 12 to 15 applications.

5. By now you should have 2 strips with spinach pigment. Suspend one of each in each of the chromatography development vessels. You can attach them with paper clips, or simply fold over a portion of the end and it should hang in place. The tip of each strip should just touch the solvent.

6. Wait 20 to 30 minutes for the chromatograms to develop. Remove the chromatograms. Mark with a pencil (NOT a pen) where the solvent stopped as it moved up the chromatogram. This is called the solvent front. Mark also where each pigment stopped moving up the chromatogram. Using the equation below, determine a reference number for each pigment on the chromatograms. Depending on which chromatogram you are viewing, you should see greens, yellow/yellow orange, and red. All measurements should be in mm. (Any material which did not move from the
pencil dot is insoluble).

Rf = distance moved by compound from original spot divided by the
distance moved by solvent from original spot

Note: each pigment has a special name,
green = chlorophyll a or b
yellow/yellow orange = carotene
red = anthocyanin
brown = xanthophyll

The reference numbers for the chlorophylls in this activity are:
0.28 = chlorophyll a, 0.18 = chlorophyll b (spinach). You need these
numbers so that you can determine one chlorophyll from the other.
Calculate reference fronts for all of your pigments.

See if your calculations come close to those above for chlorophyll a and b.

Note: You can use different solvents such as mixtures involving petroleum ether
to do this sort of paper chromatography.

To view notes and a graphic showing a separation of plant pigments involving
paper chromatography, click here. Can you calculate the Rf values for the
pigments separated in this graphic?

Conclusion Questions

1. What reference numbers (Rf) did you calculate for chlorophyll a and chlorophyll b?
2. With what you have discovered about pigments, what conclusions can you
make regarding the changing color of leaves in autumn?
3. What adaptive purpose do different colored pigments serve for a plant?
4. Why do some pigments move farther up the chromatogram than others?
5. What are some possible sources of error in this lab?

Paper chromatography is a technique used to separate a mixture into its component molecules. The molecules migrate, or move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper.

For a simple, beautiful example of this technique, draw a large circle in the center of a piece of filter paper with a black water-soluble, felt-tip pen. Fold the paper into a cone and place the tip in a container of water. In just a few minutes you will have tie-dyed filter paper!

Separation of black ink pigments

The green, blue, red, and lavender colors that came from the black ink should help you to understand that what appears to be a single color may in fact be a material composed of many different pigments —and such is the case with chloroplasts.

chromatography setup

In paper chromatography the pigments are dissolved in a solvent that carries them up the paper. In the ink example, the solvent is water. To separate the pigments of the chloroplasts, you must use an organic solvent

pigment separation

Analysis of Results I

If you did a number of chromatographic separations, each for a different length of time, the pigments would migrate a different distance on each run. However, the migration of each pigment relative to the migration of the solvent would not change. This migration of pigment relative to migration of solvent is expressed as a constant, Rf (Reference front). It can be calculated by using the formula:

Chapter 29 AP Objectives

Chapter 29 Plant Diversity I: Colonization of Land

Objectives

An Overview of Land Plant Evolution
1.	Describe four shared derived homologies that link charophyceans and land plants.
2.	Distinguish among the kingdoms Plantae, Streptophyta, and Viridiplantae. Note which of these is used in the textbook.
3.	Describe five characteristics that distinguish land plants from charophycean algae. Explain how these features are adaptive for life on land.
4.	Define and distinguish among the stages of the alternation of generations life cycle
5.	Describe evidence that suggests that plants arose roughly 475 million years ago.

	Bryophytes
6.	List and distinguish among the three phyla of bryophytes. Briefly describe the characteristics of each group.
7.	Distinguish between the phylum Bryophyta and the bryophytes.
8.	Explain why bryophyte rhizoids are not considered roots.
9.	Explain why most bryophytes grow close to the ground.
10.	Diagram the life cycle of a bryophyte. Label the gametophyte and sporophyte stages and the locations of gamete production, fertilization, and spore production.
11.	Describe the ecological and economic significance of bryophytes.

	The Origin and Diversity of Vascular Plants
12.	Describe the five traits that characterize modern vascular plants. Explain how these characteristics have contributed to their success on land.
13.	Distinguish between microphylls and megaphylls.
14.	Distinguish between the homosporous and heterosporous condition.
15.	Explain why seedless vascular plants are most commonly found in damp habitats.
16.	Name the two clades of living seedless vascular plants.
17.	Explain how vascular plants differ from bryophytes.
18.	Distinguish between giant and small lycophytes.
19.	Explain why whisk ferns are no longer considered to be “living fossils.”
20.	Describe the production and dispersal of fern spores.

	Student Misconceptions
21.	Many students have difficulty in understanding the significance of derived characters that are shared between two extant groups. Just as many members of the general public have the mistaken notion that humans evolved from chimpanzees, some students will think that charophyceans are in some sense ancestral to plants or that charophyceans are identical to the last common ancestor that plants and charophyceans shared.
22.	It is important to make sure that your students understand alternation of generations in bryophytes and seedless vascular plants. Plant life cycles are challenging for all students. Without a good understanding of the life cycles of plants with recognizable gametophytes and sporophytes, students will have great difficulty with gymnosperm and angiosperm life cycles.
23.	Students tend to think of derived traits as “advanced.” Be careful to avoid this term. Point out that organisms have a combination of primitive and derived traits, and that all living organisms have an equally long evolutionary history, dating back to the origin of life on Earth.
24.	Many students are not very familiar with or knowledgeable about plants. Some of the terminology of plant life cycles can be confusing to such students. Clarify for students the meaning of these pairs of terms: a. homosporous and heterosporous b. bryophyte and phylum Bryophyta c. rhizoid and root

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Chapter 30 AP Objectives

Chapter 30 Plant Diversity II: The Evolution of Seed Pants

Objectives

Key Terrestrial Adaptations Were Crucial to the
Success of Seed Plants

Name five terrestrial adaptations that contributed to the success of seed plants.

Compare the size and independence of the gametophytes of bryophytes with those of seed plants.

Describe the ovule of a seed plant.

Contrast the male gametophytes of bryophytes with those of seed plants.

Explain why pollen grains were an important adaptation for successful reproduction on land.

Explain how a seed can be said to include contributions from three distinct generations.

Compare spores with seeds as dispersal stages in plant life cycles.

Gymnosperms

Explain how climatic changes with the formation of the supercontinent Pangaea favored the spread of gymnosperms.

List and distinguish among the four phyla of gymnosperms.

10.

Describe the life history of a pine. Indicate which structures are part of the gametophyte generation and which are part of the sporophyte generation.

Angiosperms (Flowering Plants)

11.

Identify the following floral structures and describe a function for each:

a. sepal		f. anther
b. petal		g. stigma
c. stamen		h. style
d. carpel		i. ovary
e. filament		j. ovule

12.

Define fruit. Explain how fruits may be adapted to disperse seeds.

13.

Explain why a cereal grain is a fruit rather than a seed.

14.

Diagram the generalized life cycle of an angiosperm. Indicate which structures are part of the gametophyte generation and which are part of the sporophyte generation.

15.

Describe the role of the generative cell and the tube cell within the angiosperm pollen grain.

16.

Explain the process and function of double fertilization.

17.

Explain the significance of Archaefructus.

18.

Explain the significance of Amborella.

19.

Distinguish between monocots and eudicots.

20.

Explain how animals may have influenced the evolution of terrestrial plants and vice versa.

Plants and Human Welfare

21.

Name the six angiosperms that are most important in the diet of the human species.

22.

Describe the current threat to plant diversity caused by human population growth.

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Chapter 35 AP Objectives

Chapter 35 Plant Structure and Growth

Objectives

The Plant Body
1.	Describe and compare the three basic organs of vascular plants. Explain how these basic organs are interdependent.
2.	List the basic functions of roots. Describe and compare the structures and functions of fibrous roots, taproots, root hairs, and adventitious roots.
3.	Describe the basic structure of plant stems.
4.	Explain the phenomenon of apical dominance.
5.	Describe the structures and functions of four types of modified shoots.
6.	Describe and distinguish between the leaves of monocots and those of eudicots.
7.	Describe the three tissue systems that make up plant organs.
8.	Describe and distinguish between the three basic cell types of plant tissues. For each tissue, describe one characteristic structural feature and explain its functional significance.
9.	Explain the functional relationship between a sieve-tube member and its companion cell.

	The Process of Plant Growth and Development
10.	Distinguish between determinate and indeterminate growth. Give an example of each type of growth.
11.	Distinguish among annual, biennial, and perennial plants.
12.	Explain this statement: “In contrast to most animals, which have a stage of embryonic growth, plants have regions of embryonic growth.”
13.	Distinguish between the primary and secondary plant body.
14.	Describe in detail the primary growth of the tissues of roots and shoots.
15.	Describe in detail the secondary growth of the tissues of roots and shoots.
16.	Name the cells that make up the tissue known as wood. Name the tissues that comprise the bark.

	Mechanisms of Plant Growth and Development
17.	Explain why Arabidopsis is an excellent model for the study of plant development.
18.	Explain what each of these Arabidopsis mutants has taught us about plant development: a. fass mutant b. gnom mutant c. KNOTTED-1 mutant d. GLABRA-2 mutant
19.	Define and distinguish between morphogenesis, differentiation, and growth.
20.	Explain why (a) the plane and symmetry of cell division, (b) the orientation of cell expansion, and (c) cortical microtubules are important determinants of plant growth and development.
21.	Explain how pattern formation may be determined in plants.
22.	Give an example to demonstrate how a cell’s location influences its developmental fate.
23.	Explain how a vegetative shoot tip changes into a floral meristem.
24.	Describe how three classes of organ identity genes interact to produce the spatial pattern of floral organs in Arabidopsis.

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