Bio 328. Spring 2003                                                                                          Name

Test #3

Provide concise answers in the space provided after each question, or, if more space is needed, continue on the back side of the page. The potential value of each answer is 4 points unless otherwise noted in the margin.

1. (a) When stimuli induce a calcium change in plant cells the change typically happens in the form of a spike, with a rapid rise in [Ca²⁺]_cyt, followed by a rapid fall. Explain what causes the rise, and why this is typically followed by a rapid fall. 

 Rise is due to calcium entry into cytoplasm through channels from outside the cell or from internal stores. Fall is due to calcium activation of calmodulin, which activates Ca pumps that move Ca from cytoplasm to outside of cells or into internal stores.

(b) An increase in [Ca²⁺]_cyt, can induce stomata opening and can induce stomata closing. When a stomatal cell experiences an increase in [Ca²⁺]_cyt, how does it know whether to open or close?

The frequency of Ca spikes can tell the stomates whether to open or close.

(c) A recent finding revealed that there was a calmodulin binding domain as part of a calcium channel, and that this domain was important for conveying the calcium signal to the nucleus.  Relate the relevance of this finding to the observation that there are multiple calmodulin isoforms and to the hypothesis that not all induced increases in [Ca²⁺]_cyt are equivalent in what they lead to.

Channels that have CaM binding domains can bind specific CaM isoforms, and specific channel-CaM pairings can lead to specific outcomes. This kind of specificity would not be possible if all CaMs were equivalent.

2. (a) Auxin receptor activation often leads to the activation of SCF and proteosome complexes. Give an example of a protein whose destruction by an auxin-induced proteosome complex would be needed for an auxin-induced response.

 (i) Protein suppressor of IAA-induced genes (e.g. IAA genes); (ii) PIN3

(b) The Figure below is relevant to the topic of gravity-regulated differential auxin transport. What method is used to visualize auxin distribution before (B) and after (D) gravistimulation? Use an arrow to point to the lower side of the root on Fig D (right panel), and indicate what changes auxin induces on that side to mediate curved growth.

 Method: Use plants transformed with reporter construct made up of DR5 (auxin-responsive) promoter linked to GFP reporter gene. Where IAA is present at significant levels GFP is turned on. Arrow should point to the right side of panel D, and the label should say: "Auxin inhibits growth on this side".

 

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3.  (a) What are PIN3 and AUX1 and how do they mediate polar auxin transport?

PIN3 and AUX1 are intrinsic membrane proteins that function as auxin transporters. AUX1 imports auxin into cells; PIN3 exports auxin from cells.

(b) How does gravistimulation alter the distribution of PIN3 in columella cells of the root, and what inhibitor would prevent this distribution change and why?

 Gravistimulation causes the distribution of PIN3 to change from relatively uniform to primarily along the new bottom of the cells. Brefeldin A, which inhibits Golgi-mediated secretion, would prevent this distribution change, because directional secretion is needed for the redistribution of PIN3.

(c) When the concentration of auxin is such that it stimulates growth, what cell activity does it activate to promote growth?

 Auxin turns on proton-pumping ATPases that acidify the wall, and this activates wall expansins, which promote wall extensibility and cell growth.

4.  (a) Initially scientists thought ethylene directly stimulated the growth of deepwater rice. What was the evidence that this was an indirect effect, and that another hormone was more directly involved?

 GA inhibitors block the ability of ethylene to promote growth.

(b)  How is ABA involved in the flooding-stimulated growth of deepwater rice?

Ethylene production, which is induced by flooding, suppresses ABA levels, and this increases the sensitivity of internodal cells to GA, which causes them to expand greatly.

5. (a) Where is the aleurone layer in a seed, and how does its physiology and anatomy change in response to GA?

The aleurone layer surrounds the endosperm of the seed. GA induces aleurone cells to make hydrolytic enzymes (such as amylases and proteases) and to secrete them into the endosperm. Cells become more vacuolated when they switch to the secretory mode.

(b) What does the slender gene encode, and how is its expression affected by GA?

 The slender gene encodes a suppressor of a transcription factor, GAMyb, that turns on GA-regulated genes (such as gene for alpha amylase). GA stops the slender protein from being able to inhibit GAMyb expression.

(c) What does the GAmyb gene encode, and describe the evidence that it is needed for a specific GA response in barley seeds.

 GAMyb encodes a transcription factor that turns on GA regulated genes. If the level of mRNA for GAMyb is suppressed by RNAi, GA can no longer induce alpha amylase gene in aleurone layer of barley seeds.

Bio 328, Test # 3 continued—page 3                                                      NAME: ________________

6. (a) How does ABA antagonize the promotive effect of GA on seed germination?

 ABA induces a signal transduction chain that blocks GAMyb activity.

 (b) In Ceratopteris, RNAi is induced by direct uptake of dsRNA into the spore cells, but scientists use a different method to get dsRNA into barley cells to achieve the RNAi-mediated suppression of specific genes. What is this method?

The method is to transform cells with a gene that encodes a hybrid mRNA for a specific gene that includes both the sense and antisense orientation of that gene. When this hybrid mRNA is transcribed it forms adsRNA for that gene, because the sense and antisense strands pair together.

(c) In the Figure below, what is the main question being addressed, what is the answer, and what is the significance of this answer relative to the mechanism by which ABA suppresses alpha-amylase expression?

The main question is whether suppressing the mRNA for slender by RNAi will (1) allow amy-GUS to be expressed in the absence of GA (ANSWER is YES), and (2)have any affect on amy-GUS expression in the presence or absence of ABA (ANSWER is NO). Significance: ABA's ability to inhibit Amy-GUs expression does not depend on slender, and GA is not needed to induce GA-genes if slender is not expressed.

7. (a) Why would a lipase inhibitor block a plant’s defense response to insect attack, and what specific defense mechanism would it block?

 A required step in the signal transduction chain leading from insect attack (plant injury) to the production of a defense compound is the enzymatic breakdown of membrane phospholipids by a lipase to produce linolenic acid, a precursor of jasmonic acid, which can induce protease inhibitors, which can retard the growth of insect predators.

(b) Regarding the defense mechanism noted in your answer to 7(a), what is the experimental evidence that this mechanism actually confers some defense against insect predation?

 Transgenic plants expressing the antisense of systemin, a hormone induced by injury, are suppressed in their ability to transduce insect-inflicted injuries into protease inhibitors, and they support the growth of Manduce larvae better than wt plants do.

(c) A famous movie star, who is paid big bucks to market various perfumes, notices that the more intimate time she spends complimenting her plants the less they are bothered by aphids and other insect pests. She is sure this is because her plants have a better self-image and are stronger as a result of the positive attention she gives them. Propose an alternative explanation, and an experiment with alternative possible outcomes to test it, and indicate what result would favor your proposal. 

 Alternative proposal: Her perfumes include a methyl jasmonate component, which induces defense compounds in her plants. Experiment: She should wear no perfumes when she talks to her plants. Alternative possible outcomes: Her plants are still less bothered by insects when she talks with them, or not. If her not wearing perfumes results in her plnats now no longer having any special resistance to insects, this result would favor the alternative proposal.

Bio 328, Test # 3 continued—page 4                                                      NAME: ________________

8. (a) Volicitin is involved in both an attraction and a repulsion process. What is volicitin? Describe the attraction and repulsion processes.

 Volicitin is a compound in the saliva of army worms that induces the production of volatile compounds that attract parasitoid wasps to the army worms but repel the parent moth that lays the eggs that hatch into army worms.

(b) Compare and contrast volicitin and systemin, listing two points of similarity and two points of difference.

 Systemin is a protein; volicitin is not; systemin is a plant product induced by the injury stimulus; volicitin is produced by the army worm while it is eating. Both compounds induce plants to produce volatile signaling agents; both compounds induce defense responses.

9. (a) What is the evidence that MDR helps to mediate ATP release from plant cells?

Overexpression of MDR in Arabidopsis results in more ATP accumulating in the walls of these plants than would accumulate in the walls of WT plants.

(b) If MDR transports both toxins and ATP, discuss a model on how these two transport processes could be linked, and describe a prediction of this model that would include the enzyme ectoapyrase.

 The movement of toxins out of cells could be driven by a symport process with ATP moving down its inside-out concentration gradient, allowing toxins to move out against a gradient. Since ectoapyrase activity (which destroys extracellular ATP) helps to maintain the steepness of the inside-out concentration gradient of ATP, inhibition of ectoapyrase activity should decrease the efficiency of MDR in removing toxins, and overexpression of ectoapyrase should make MDR more efficient and make plants more resistant to toxins.

(c) Describe genetic and inhibitor data consistent with the model you proposed in your answer to 9(b).

 Genetic: Overexpression of ectoapyrase in yeast or Arabidopsis makes these organisms more resistant to toxins. Inhibitor:  Inhibition of ectoapyrase activity by chemical inhibitors makes plants more sensitive to herbicides.