Posted: May 31st, 2023

Plant Physiology – BIOL415

Plant Physiology – BIOL415 name

Acid Growth Hypothesis
The cell wall must be strong to withstand the relatively large pressures that can develop. Yet, at the same time, the wall must be capable of growth. This presents an interesting paradox – how is the wall able to be strong, yet allow for cell growth and elongation. One answer is that the wall is “loosened” or undergoes “stress relaxation” to permit wall components to slide past one another. The following experiments are designed to test the hypothesis that wall loosening is moderated by acid (acid growth hypothesis).

Question: Does acid treatment loosen cell walls?

Prediction #1: IF cell walls can be loosened by acid, THEN treating stem or hypocotyl sections with acid buffers should stimulate elongation. The graph below plots the length of etiolated cucumber hypocotyl sections versus time. Initially the pH is 7 and at the arrow is changed to pH 5. Answer the following questions:

Hypothesis #1: Cell walls are loosened by acid.

1. What is a hypocotyl?
2. What does etiolated mean?
3. Suggest a reason why etiolated hypocotyls were used in this experiment.
4. True or False: Hypocotyls elongate faster at pH 7 than at pH 5
5. True or False: Cell elongation is associated with acidic pH
6. What do you conclude about the hypothesis?

Prediction #2: IF cell walls are loosened by acid, THEN regions of cell growth/elongation should be associated with acid production. The diagram below shows maize and sunflower seedlings mounted in a petri dish containing agar. A pH indicator has been incorporated into the agar. Acidic regions are indicated by an “X”.

1. Why does the agar become acidified near the tip of a vertically-mounted maize seedling?
2. Why is the acidified region of the vertically-mounted seedling not at the very tip, but a few millimeters behind the tip?
3. Why is the agar acidified only on the side of the sunflower seedling away from the light?
4. Why is the agar acidified only on the upper side of the root of the horizontally-mounted maize seedling?
5. What do you conclude about the hypothesis?

Hypothesis #2: Auxin, a plant growth hormone, is associated with cell elongation and wall acidification.

Prediction: IF protons stimulate elongation and wall acidification, THEN treating seedlings with auxin should stimulate elongation in a manner similar to acids. The graph below shows the length of a stem section in various pH buffers, before and after treatment with auxin. The initial pH in both experiments is pH 7. From these data we can conclude:

1. True or False: Acid stimulates cell elongation
2. True or False: The rate of elongation is slow but constant at pH 7.
3. True or False: The response to auxin is rapid.
4. True or False: Auxin stimulates the rate of elongation at pH 7.
5. True or False: The rate of auxin-stimulated elongation is equal to that of acid-stimulated elongation.
6. What do you conclude about your hypothesis?

Hypothesis #3: Auxin stimulates cell elongation, in part, by stimulating protein synthesis.

Prediction: IF auxin stimulates protein synthesis, THEN treating cells with antimycin, an inhibitor of eukaryotic protein synthesis, will block its action. The graph below shows the fresh weight increase in artichoke root disks after treatment with water, water + auxin, or water + auxin + antimycin.

1. True or False: Auxin stimulates root disk growth
2. True or False: The effect of auxin is abolished by a protein synthesis inhibitor
3. True or False: Protein synthesis is required for growth of root disks

Hypothesis #4: Auxin has two roles in cell elongation: (a) Auxin has a short-term effect that involves activating a proton pump and (b) Auxin has a longer-term effect that is involved with stimulating protein synthesis (transcription and translation).

Prediction: IF auxin stimulates protein synthesis, THEN treating coleoptiles with a protein synthesis inhibitor should prevent expansion. IF auxin stimulates a proton pump directly, THEN this should not be affected by a protein synthesis inhibitor. The graph below shows the rate of coleoptile expansion after treatment with either: (a) auxin alone; (b) the same level of auxin plus cycloheximide (a known inhibitor of eukaryotic protein synthesis); or (c) acid (HCL).

1. What is a coleoptile?
2. Why are coleoptiles used in these types of experiments?
3. Which of the following plants would produce a coleoptile? sunflower, oat, maize, wheat, bean, barley, lettuce, RCBr, turnips.
4. What does cycloheximide do? Would cycloheximide be a good antibiotic to take if we get an infection?
5. Do you think antimycin could have been used in place of cycloheximide?
6. True or False: Auxin causes coleoptile elongation by a mechanism that involves acidification of the cell wall.
7. True or false: Dilute acid mimics the initial, but not the longer-term, effect of auxin on the coleoptile cells.
8. True or False: Auxin stimulates elongation by two different mechanisms, with an initial acid- induced passive phase followed by a longer-term response that requires protein synthesis.
9. True or False: The initial phase of auxin induced elongation can occur in the absence of protein synthesis
10. True or False: Auxin-induced acidification of the cell wall does not require protein synthesis.
11. What do you conclude about your hypothesis?
12. Write a summary that explains the mechanism by which cell walls are loosened. Be sure to include the impact of auxin, acid, proton pumps, protein synthesis, short-lived and longer-term effects of auxin.

Reference: This exercise is adapted, in part (esp. hypothesis #4), from an unpublished course handout from Dr. W Becker, U of Wisconsin.

A hypocotyl is the part of the stem of a young plant embryo that lies between the cotyledons and the roots.

Etiolated means the plant has grown in the dark, lacking exposure to light.

Etiolated hypocotyls were used in this experiment because they grow quickly and uniformly in the dark.

False. Hypocotyls elongate faster at pH 5 than at pH 7, as seen in the graph.

True. Acidic pH is associated with cell elongation, as seen in the graph.

The hypothesis that cell walls are loosened by acid is supported by the results of this experiment.

Prediction #2:

The agar becomes acidified near the tip of a vertically-mounted maize seedling because the cells in this region secrete protons, which acidify the cell wall, making it more extensible.

The acidified region of the vertically-mounted seedling is not at the very tip, but a few millimeters behind the tip because the wall at the very tip is already elongated and therefore less extensible.

The agar is acidified only on the side of the sunflower seedling away from the light because the cells on this side are elongating more rapidly than those on the shaded side, leading to more acidification of the cell wall.

The agar is acidified only on the upper side of the root of the horizontally-mounted maize seedling because this is the side of the root that is elongating more rapidly due to gravity.

The hypothesis that auxin is associated with cell elongation and wall acidification is supported by the results of this experiment.

Prediction:

True. Acid stimulates cell elongation, as seen in the graph.

True. The rate of elongation is slow but constant at pH 7, as seen in the graph.

True. The response to auxin is rapid, as seen in the graph.

True. Auxin stimulates the rate of elongation at pH 7, as seen in the graph.

False. The rate of auxin-stimulated elongation is not equal to that of acid-stimulated elongation, as seen in the graph.

The hypothesis that auxin stimulates cell elongation and wall acidification is supported by the results of this experiment.

Prediction:

True. Auxin stimulates root disk growth, as seen in the graph.

True. The effect of auxin is abolished by a protein synthesis inhibitor, as seen in the graph.

True. Protein synthesis is required for growth of root disks, as seen in the graph.

Hypothesis #4:

A coleoptile is a protective sheath covering the emerging shoot of a germinating seed.

Coleoptiles are used in these types of experiments because they elongate rapidly and uniformly.

Maize would produce a coleoptile.

Cycloheximide inhibits protein synthesis by binding to the ribosome and preventing elongation of the peptide chain. It would not be a good antibiotic to take if we get an infection because it specifically inhibits eukaryotic ribosomes and would not target bacterial ribosomes.

Antimycin could not have been used in place of cycloheximide because antimycin inhibits oxidative phosphorylation in mitochondria, which is not related to protein synthesis.

True. Auxin causes coleoptile elongation by a mechanism that involves acidification of the cell wall.

True.

True. Auxin stimulates elongation by two different mechanisms, with an initial acid-induced passive phase followed by a longer-term response that requires protein synthesis.

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