Bio 328. Spring 2002 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 5 points unless otherwise noted in the margin.

1. Annexins are targets for Ca2+ signals in both animal and plant cells.
(a). When annexins bind Ca2+ what happens to their localization in cells?
Ans.: When they bind Ca2+, annexins change their localization from cytosolic to membrane bound. Sometimes annexins may also redistribute within a cell; e.g., move from a polar or asymmetric membrane localization to an evenly distributed membrane localization.

(b) Based on immunolocalization results name one cell type in which plant annexins are found and state a suggested function for them in this cell type.
Ans.: In tips of pollen tubes and rhizoids where they mediate the fusion of secretory vesicles to plasma membrane, or act as a calcium channel to maintain high concentrations at the growing tip.. Annexins are also in peripheral root cap cells, where they mediate secretion of mucilage.

(c) Describe experimental evidence that annexins can act as a novel type of Ca2+ channel.
Ans.:When annexins are incorporated into liposomes or membrane vesicles with calcium-indicator dyes inside their lumen they induce an increase in the internal calcium concentration. More indirect evidence is the localization of annexin at the site of the emerging rhizoid in ferns where there is also an influx of calcium ions, or at the tip of pollen tubes where there is also an influx of calcium ions.

2. (a) The plant cell is as differentiated into different domains as the earth is. How does this help explain how different signal transduction pathways can use the same amplification mechanisms and still lead to different adaptive outcomes?
Ans.: Signal transduction chains are initiated and propagated from specific microdomains within the cell, and these microdomains are differentially connected or “wired” to different effector molecules. Thus similar amplification mechanisms initiated from different microdomains can alter different effector molecules and thus result in different end responses.

(b) Many environmental and hormonal stimuli require changes in calcium concentration as intermediate steps in their signal transduction, but these calcium signals may not all be equivalent. Explain.
Ans.: Calcium signals initiated by different stimuli can differ in properties such as their amplitude, their pulse rhythm, and the extent and locale of their spread.

3. (a) For over 20 years most plant scientists believed that auxin did not need a protein carrier to get into cells, but did need a carrier to get out. What explanation did scientists give for this belief?
Ans.: Scientists believed that in the acid environment of the cell wall, IAA would be protonated and thus be neutrally charged and be able to diffuse into cells without a carrier. However, once inside the cell the IAA would be exposed to a neutral pH and become negatively charged, and so would not be able to diffuse across membranes and would need a carrier to exit the cell.

(b) NAA can inhibit root growth in the aux1 mutant, but IAA cannot. Explain.
Ans.: NAA can diffuse into cells and does not need the AUX1 transporter; IAA requires the AUX1 transport protein to enter cells efficiently.

(c) How does NPA affect the ability of NAA to rescue the gravitropism defect in the aux1 mutant, and what hypothesis does this result disprove?
Ans.: NPA, which is known to block auxin export, blocks the ability of NAA to rescue the gravitropism defect in the aux1 mutant. This disproves the hypothesis that the aux1 mutant has a defect in auxin export, because it shows that even though NAA can be polarly transported without AUX1, it needs the export machinery blocked by NPA to leave the cell. Thus NAA would not be able to rescue the aux1 mutant if that mutant was blocked in auxin export.

4. (a) Describe a signal transduction chain by which the transport activity of AUX1 promotes wall acidification without going through G-protein or Calcium-dependent intermediate steps.
Ans.: AUX1 promotes IAA uptake into the cytoplasm of cells, where it binds to ABP57, which allows ABP57 to bind to the proton-pumping ATPase and increase its activity, thus promoting wall acidification.

(b) How did knowledge of the primary structure of BSA aid an analysis of the signal transduction chain described in the answer to 4(a)?
Ans.: The primary structure of BSA contains a sequence identical to a sequence in ABP57, so antibodies to BSA can recognize and bind ABP57. So an affinity column containing anti-BSA antibodies can be used to easily and efficiently purify ABP57, which can then be used to show that purified ABP57 can bind to and activate the proton pumping ATPase.

5(a) What is DR5, and how could it be used to illustrate the effect of NPA on the asymmetric distribution of a chemical required for phototropism? Your answer should include a description of that NPA effect.
Ans.: DR5 is a DNA element that responds to IAA by promoting the transcription of mRNA sequences downstream of it. When linked to GUS the DR5-GUS can serve as a reporter gene for auxin presence. Using this construct scientists showed that NPA blocks the asymmetric distribution of IAA that is needed for phototropism.

(b) In the results shown below in Fig. 1, panel e and h, what is the question being addressed, and what is the answer?

Fig. 1


Ans.: The question is whether PIN3 changes its localization in response to a gravitropic stimulus, and the answer is yes.

(c) How could (i) a ubiquitin ligase complex and (ii) Golgi-mediated secretion promote the changes illustrated in Fig. 1?
Ans.: A ubiquitin ligase complex could add ubiquitin to the PIN3 proteins that are symmetrically distributed in Fig. 1, panel “e”, thus tagging them for proteolytic destruction. Golgi-mediated secretion could then insure that all the new PIN3 proteins inserted into the membrane were inserted asymmetrically at the site where IAA must exit cells to be laterally relocated, as shown in panel “h”.

6. (a) It is true to say that there are many gibberellins, but also true to say that there is really only one gibberellin. Explain.
Ans.: There are many intermediates in the GA biosynthetic pathway that can promote GA responses by promoting the synthesis of THE final active product of that pathway, which is GA1 in many plants.

(b) Describe a signal transduction chain in which Pfr leads to seed germination through a GA-dependent step.
Ans.: Pfr induces the transcription of the mRNA encoding a hydrolase enzyme that converts GA20 to GA1. Increase in GA1 in seeds stimulates their germination.

7. (a) What are two biological/biochemical functions of ETR1?
Ans.: ETR1 binds ethylene and has protein kinase activity.

(b) How were yeast used to discover one of the functions of ETR1?
Ans.: Wild-type version of ETR1 was expressed in yeast and conferred on them the ability to bind ethylene.

8. (a) Theoretically the systemin system helps plants defend themselves against insect feeding. What is systemin, and by what process is it thought to help in plant defense. Your answer should include the words jasmonic acid and inter-plant communication.
Ans.: Systemin is a peptide hormone that helps in plant defense by moving from the site of insect injury through the vascular system to the rest of the plant, where it induces a signal transduction pathway that leads to the production of protease inhibitors that can interfere with the protein-digesting ability of insect feeding on plants. This pathway includes the increased production of jasmonic acid that can readily be converted to methyl jasmonate which can serve as a volatile signal to induce protease inhibitors in surrounding plants that have not (yet) been attacked by the insects. .

(b) What is the experimental evidence that the systemin system does in fact help plants defend themselves against insects.
Ans.: Transgenic plants expressing the antisense of systemin are suppressed in systemin production, and insects feeding on these plants feed more and grow much bigger than insects on wild-type plants.

(c) Plants can enlist the aid of animals in their defense against insect feeding. Explain.
Ans.: A signal (volicitin) from the saliva of a feeding army worm can induce plants to produce volatile compounds that attract to the plant parasitoid wasps that attack the feeding worms and lay eggs in them. The larvae hatched from these eggs feed on and kill the army worms, thus serving as a kind of animal defense of the plants.