Bio 328. Spring 2003                                                                                          Name

Test #1

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 3 points unless otherwise noted in the margin.

1.   (a) Scientists have observed that in gametophytic self-incompatibility,  RNase secreted by the style induces the incompatibility reaction in pollen.  Propose two alternative hypotheses to explain the observation, an experimental design that could resolve which of the two alternatives was more likely to be correct, and a credible method for carrying out the experiment.

Ans.: Alternatives: RNase gets into only incompatible pollen; RNase gets into both incompatible and compatible pollen. Experimental design: Assay whether RNase enters compatible pollen. Method: Use an antibody to RNase with immunolocalization methods to test whether RNase enters compatible pollen.

(b)  What observation has led scientists to propose these two alternative explanations:

The male incompatibility factor binds to the RNase and activates it, or

The male incompatibility factor binds to a repressor of the RNase and inactivates it. ?

Ans.: RNase enters both compatible and incompatible pollen, so its interaction with the male incompatibility factor must determine whether or not it is active inside the pollen tube, not whether or not it enters the pollen tube.

2.  (a) In the Table below, explain why the stigma parent col::SRKb has an incompatible reaction with pollen from the col:SCRb parent, but a compatible reaction with SaSa pollen from A. lyrata. Your answer should define SRKb, SCRb, and should explain what is the main difference between the A. lyrata and the col (Columbia ecotype) plants relevant to this experiment. 

Ans.: Stigma parent col::SRKb has a self-incompatible (SI) reaction with pollen from the col:SCRb parent,, because the binding of the male SI factor SCR (SI-cysteine rich protein) with the female incompatibility factor, SRK (SI-receptor kinase) induces the incompatible reaction. The “b” after SCR and SRK indicates that these two proteins are from the same (self) plant. Col is self-compatible; A. lyrata is self-incompatible. Col:SRKb is compatible with pollen from A. lyrata because the SRKb is from a different plant than the “a”-type pollen.

(b) A key conclusion of the results shown in the Table above is conveyed by the title of the article which presented those results. Please complete in your own words this partial title of the article:  Generation of Self-Incompatible Arabidopsis thaliana by transfer of two self-incompatible genes (SRK and SCR) from A. lyrata,

Bio 328, Test # 1 continued—page 2                                                      NAME: ________________

3.  (a) In what cells and what subcellular location would SRK protein be highly expressed?

Ans.: The SRK protein would be expressed on the plasma membrane of stigma epidermal cells.

(b) In what cells and what subcellular location would the SCR protein be highly expressed?

Ans.: SCR protein would be expressed in the exine layer (wall) of pollen.

(c) Describe one structural feature of SRK and one structural feature of SCR, and for both features explain how it helps the protein carry out its function.

Ans.: SRK is integral membrane protein with its binding site on the outside and its kinase site on the inside. This allows it to detect an external signal (male SI factor) and transduce that binding event into a cellular signaling step. SCR is small, soluble and cyteine rich. Its cysteine richness helps it form cystine linkages which help it retain its shape better at the high temperatures pollen often encounters.

4. (a) What is a key feature of the dormant state of a seed?

Ans.: It is relatively dry and relatively inactive metabolically.

(b) What survival benefit does the dormant state provide to seeds?

Ans.: It helps it survive extremes of environmental conditions.

(c) What is the endosperm cap and how does it change during seed germination?

Ans.: The endosperm cap is a region of the endosperm covering the tip of the embryonic radicle. It must soften in order to facilitate the emergence of the radicle from the seed at germination.

5. (a) ORE9 has a key structural feature on its N-terminal end. What is that feature, what does it allow ORE9 to do, and what is the evidence that it functions in this way?

Ans.: ORE9 has an F-box at its N-terminal end that allows it to bind to ASK-1. In a co-immunoprecipitation assay ORE-9 will bind to ASK-1 as long as it has the F-Box, but modified versions of ORE-9 that are missing the F-Box will not bind to ASK-1.

(b) ORE9 appears to regulate leaf senescence in Arabidopsis. Give one line of evidence for this.

Ans.: Plants knocked out in ORE9 have delayed onset of senescence.

(c) By what hypothetical mechanism is ORE9 thought to regulate leaf senescence? What is the main evidence in favor of this hypothesis?

Ans.: ORE9 is thought to regulate senescence by mediating the proteolytic destruction of a factor that is a suppressor of the SAG genes that induce senescence. Knockout of the ORE9 gene delays the onset of senescence. The main evidence is that ORE9 forms a complex with ASK1, thus forming part of an SCF complex that is known to function primarily to promote ubiquitination of proteins, which leads to their proteolysis.

6. (a) Excised regions of stem were subjected to freeze-thaw cycles and then boiled in the experiments leading to the discovery of expansins. Explain the rationale for each of these two treatments and how, after these treatments, the resulting dead material was used in an assay to discover expansins.

Ans.: Freeze thaw kills the stem cells; boiling the dead stems denatures the wall proteins. Walls thus treated are missing proteins needed for wall extensibility. Adding back to these walls purified fractions of wall proteins allows the identification of which wall proteins can promote wall extensibility.

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(b) What method was used to examine the localization of LeEXP8 and LeEXP10 in tomato seeds?

Ans.; Blotting the cut surface of tissues onto paper allows the protein and RNA molecules from the cut cells to stick to the paper in the same arrangement and localization they had in the tissue. Then specific, labeled probes can be used to visualize where specific mRNA or protein molecules are located.

(c) At germination the expression of LeEXP8 was fairly restricted to a limited region of the embryo; the expression of LeEXP10 was more general (see Figure below). From these data propose different roles for LeEXP8 and LeEXP10 in tomato seeds,  describe an experimental approach to test your proposal, and indicate what result of this experiment would support your proposal.

Ans.: LeEXP8 has a more limited role in radicle growth; LeEXP10 has a more generalized role in overall embryo growth. This hypothesis would be supported by results in which Knock outs of LeEXP9 resulted in normal growth during embryogenesis, but poor radicle growth at germinatio, and KO of resulted in defective growth of embryos.

7. (a) What are phytochelatins, what enzyme regulates their synthesis and what stimulus increases their expression?

Ans.: Phytochelatins are compounds that chelate heavy metals. Their synthesis is catalyzed by phytochelatin synthase.  Exposing plants to certain heavy metals can induce phytochelatin synthesis.

(b) Why are some venture capitalists interested in how metallotheionein expression is regulated in plants?

Ans.: Plants engineered to overexpress metallotheioneins could, in principle, be especially useful for remediating soils heavily contaminated by heavy metals, and such plants could have significant commercial value, which would be attractive to venture capitalists.

8. (a) Why would red and far-red light have antagonistic effects on oxidizing cytochrome f in chloroplasts?  Your answer should explain how each color of light affects the oxidation state of cyt f.

Ans.: Red light would be especially effective in activating PSII which would favor donating electrons to (= reducing) cytf.. Far-red light would be  especially effective in activating PSI, which would favor drawing electrons away from (= oxidizing) cytf.

(b) Distinguish the different microenvironments of PSII and PSI on thylakoid membranes and indicate the functional logic of these differences.

Ans.: PSII is aligned mainly in the middle regions of grana stacks of thylakoid membranes; PSI is aligned mainly along the edges of these thylakoid membranes, in contact with stroma. PSI generates NADPH which is utilized in the stroma.

(c) If the ratio of reduced plastiquinone to oxidized plastoquinone in chloroplasts became very high what imbalance would this indicate, and how would this imbalance be corrected?

Ans.: High ratio of reduced to oxidized PQ would indicate that PSII is more active than PSI, and this imbalance would be corrected by the activation of a kinase that would phosphorylate proteins in the LHCII complex, inducing them to move from PSII LHC to PSI, thus making PSII less efficient in harvesting light and PSI more efficient.

9. (a) What is the relative involvement of PSII and PSI in temperature acclimation? Give evidence for your answer.

Ans.: PSII is more important for the acclimation process than PSI. PSII and PSI complexes can be separated from one another and separately tested for acclimation. By this test, PSII does show acclimation, PSI doesn’t.

(b) Explain the experimental origin of the terms C3 and C4 photosynthesis.

Ans.: C3 refers to the first detectible labeled product generated in the dark carbon fixation cycle by C3 plants,  3-phosphoglycerate, a 3-carbon compound. C4 refers to the first labeled product generated in the dark carbon fixation cycle by C4 plants,  oxaloacetate a 4-carbon compound.