Bio 328. Spring 2002 Name
Test #2

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) The difference in water potential between two cells can predict which direction water will move. Explain.
Ans.: Water always moves to the cell that has the relatively lower water potential.

(b) The title of an article published in January 2001 in Science was “Xylem may direct water where it is needed”. How does xylem do this? Your answer should have the words hydrogel, flow rates, and salt.
Ans.: Between adjoining xylem cells there is a kind of thin wall or hydrogel which swells and shrinks as the salt concentration of the xylem sap changes. When it swells the pores of this gel become smaller, and the flow across this gel (wall) slows down; when it shrinks the pores get larger, and the flow can go faster. By controlling salt content of xylem, plants can selectively make water flow faster through xylem cells radiating in one direction than in another.

(c) The same article noted in (b) above used the term “pit membrane”, but this structure is not a lipid bilayer. What is it?
Ans.: It is a thin region of the wall (with a pectic matrix that can swell or shink in response to salt changes),

2. (a) What is the earliest gravity response measured so far in Ceratopteris, and what is the evidence that this response is related to some subsequent gravity response in this fern?
Ans.: Earliest response is a calcium current that flows from the bottom to the top of the cell. If this current is blocked, the ability of gravity to direct the polarity of the cell is blocked.

(b) A laboratory at Cornell University found that the application of proteases to Chara cells disrupted their gravity response. In what cell compartment did the proteases have their effect and what experiment helped to reveal the likely target of protease action in this compartment?
Ans.: Proteases digested protein in the wall of the cell. Because RGDS, which dissociates integrins from their wall binding site, can also block gravity response, Cornell group concluded that this protein and the protein achors in the ECM to which it binds are important for the gravity response, so these are likely targets of protease action in the wall when the protease treatment disrupts the gravity response.

3, (a) Changing the density of the medium in which a submerged rice seedling grows can help resolve two alternative hypotheses about what structure senses gravity in rice cells. What are the two hypotheses, and how does changing the medium density help resolve which of these alternatives is correct?
Ans.: Hypothesis 1; The falling of amyloplasts (statoliths) initiates the gravity response. Hypothesis 2: the settling of the whole protoplast initiates the gravity response. If the density of the medium is adjusted so that it equals that of the protoplasm the protoplasm will float, but the amyloplasts will still sink. Such an adjustment blocks the gravitropism of rice seedlings submerged in this medium, a result that favors Hypothesis 2.

(b) Experimentally, how could you make a downward growing root bend to the left without moving the root to a different position?
Ans.: You could treat the left side of the root with calcium (or auxin), which would decrease the growth on that side and make the root bend toward the left.

(c) Describe a lab experiment that would allow you to make a crayfish swim upside down, and state one important conclusion about gravity sensing that would follow from the results of this experiment.
Ans.: When the crayfish molts provide it with iron filings as the only option for it to choose when it moves small particles into its inner ear. After the molting is complete and the iron filings are in the inner ear, place a magnet above the crayfish. The iron filings will move up and the crayfish will swim upside down. Conclusion: The movement of particles in the inner ear play a major role in determining how the crayfish orients itself relative to the gravity vector.

3. (a) To document the global changes in gene expression that occur in single-celled germinating fern spores in microgravity, what would be the preferred method today? Explain.
Ans.: Microarray, because this method allows you to simultaneously survey the expression level of thousands of genes.

(b) What are ESTs, and why would a library of ESTs from you brain differ from one from your liver?
Ans.: ESTs are Expressed Sequence tags, or partial sequences of cDNAs made by reverse transcription from the mRNAs that were being expressed in the tissue source from which the mRNAs were isolated. Because each EST library reflects the unique composition of mRNAs being expressed in the tissue source, the ESTs from brain would differ from those based on mRNA from liver.

(c) What is RNAi, and why is it thought to have arisen as a defense mechanism against viruses?
Ans.: RNAi is RNA interference, a mechanism of suppressing the expression of specific mRNAs that have become double-stranded by complexing with antisense strands. In RNAi the cell recognizes and destroys double-stranded RNA molecules. Because the main natural source of double-stranded RNA in biology is from viruses, it is thought that this mechanism arose as a defense against viruses.

4, (a) What response does the NPH1 protein regulate, where is it localized in cells, what is its main enzyme activity, and what is one substrate of this activity?
Ans.: NPH1 is the photoreceptor for phototropism, it is localized on the plasma membrane, its main enzyme activity is that of a protein kinase, and one of its substrates is itself (it autophosphorylates).


(b) What is one growth response regulated by the CRY1 protein, what is one of the molecular functions of this protein when it is activated by blue light, and where does it carry out this function?
Ans.: CRY 1 is the blue-light photoreceptor for suppressing hypocotyls elongation; one of its molecular functions is to bind to and inactivate COP1, and it carries out this function in the nucleus.

(c) How would diffuse (non-directional) blue light affect the growth of a mutant cop1 plant. Explain. Ans.: Diffuse blue light would not have much of an effect on a plant defective in cop1, because COP1 is a suppressor of blue ligh effects, and in darkness the cop1 mutant would already have the phenotype of a plant grown in blue light.

6. (a) Fig. 1 (below) addresses a question related to CIP4, What is CIP4, what is the main question being addressed by this Figure, and how does dexamethasone (DEX) help provide the answer?
Note: 209-9 & 209-77 are 2 different lines of antisense CIP4 plants.
Ans.: CIP4 is a transcription factor that turns on light regulated genes. This Figure is asking whether plants suppressed in CIP4 show the light induced suppression of hypocotyls elongation when the light is white, blue, red, far-red. The antisense suppression of CIP4 expression is conditional, and occurs only when the promoter for the antisense construct is turned on by DEX.

(b) When plants grow in darkness they have abundant PhyA; when they emerge into the light they destroy PhyA. What is the survival benefit of the profligate production of PhyA in darkness and of the seemingly wasteful destruction of PhyA when the plant reaches the light?
Ans.: Making a lot of PhyA in darkness makes the plants more sensitive to light. This allows them to begin synthesizing the photosynthetic machinery (a response trigged by dim light) even while they are still hours away from emerging through the soil into full light, thus giving them a head start toward becoming autotrophic. The destruction of PhyA after light is abundant allows them to recycle the amino acids of the Phy protein when sensitivity to light is no longer an advantage.

7. (a) What enzyme activity does Phy have, what is one substrate of that activity?
Ans.: Phy is a protein kinase, and it autophosphorylates itself as well as PKS1 (and CRY and NDPK)

(b) What are 2 lines of evidence favoring the conclusion that G-proteins help mediate Phy responses?
Ans.: (1) Microinjection of activated G proteins can induce Phy responses in transgenic plants missing Phy. (2) Agents that activate G-proteins (chlolera toxin) can trigger Phy responses in Phy- plants; agents that block G-protein activity (pertussin toxin) can block some Phy responses.

(c) If G-proteins mediate Phy responses, what is a likely target of Ga in the transduction chain, and how could activation of this target lead to a cellular response that would reduce stem elongation rates?
Ans.: The likely target is PLC. Activation of PLC generates IP3, which releases calcium which would activate calmodulin which would turn on a calcium ATPase pump on the plasma membrane, which would increase the calcium concentration in the wall, which could decrease wall extensibility and reduce stem elongation rates.

8. (a) If Phy regulates gene expression in part by direct interaction with COP and/or transcription factors, how fast would you expect this response to occur? Cite experimental evidence for your answer.
Ans.: Probably 5-15 min, because according to GFP-Phy experiment, it takes that long for Pfr to move to the nucleus after its photoactivation, and COP functions in the nucleus.


(b) It might be faster for Phy to turn on gene expression via calmodulin. Explain, and include in your explanation a hypothetical transduction chain in which calmodulin links Pfr to altered gene expression.
Ans.: Phy-induced increase in cytosolic calcium takes place in seconds. This would rapidly activate calmodulin. One of the targets of calmodulin action is a transcription factor that binds to and activates promoters of light-regulated genes.

(c) Both plant and animal cells have mechanisms for rapidly removing calcium from the cytoplasm. What would be the disadvantage of letting calcium levels remain high in the cytoplasm, and what is the advantage for signal responsiveness to maintain calcium levels low in the cytoplasm?
Ans.: The disadvantage of letting calcium levels remain high is that this calcium would associate with cell phosphate and form insoluble precipitates with it, thus reducing phosphate availability in cells, which would have a very negative impact on the synthesis of crucial molecules such as ATP, DNA, RNA, etc. The advantage of low calcium for signal responsiveness, is that it would take very little input energy (i.e., a very subtle environmental signal) to allow a small amount of calcium ions to leak into the cytoplasm, which would suffice to activate calcium-binding proteins involved in signaling.

9. (a) One interpretation of the acronym CDPK is “Calmodulin-domain protein kinase”. Explain.
Ans.: CDPK has within its primary structure a domain very similar to the calcium-binding domain of calmodulin.

(b) CDPK is thought to be involved in the signal transduction chain leading from water stress to stomata closing. State the steps in this pathway.
Ans.: Water stress leads to increase in [ABA] in guard cells; this promotes an increase in cytosolic calcium, this activates CDPK, which phosphorylates K+ channels on the plasma membrane (closing them) and on the vacuolar membrane (opening them). These K+-flux changes are critical for the stress induced net flow of K+ and water out of the guard cells, which is what makes the stomates close.