Bio 328. Spring 2002                                                                                         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 4 points unless otherwise noted in the margin.

1. By genetic crosses it was shown that all the genes for self-incompatibility in a plant were always clustered in a small region of a specific chromosome. This gave rise to the hypothesis that self-incompatibility was a single-gene trait. Discuss how following the 3rd rule of Strong Inference would help to formulate alternatives to the single-gene hypothesis.  In your answer you should state what is the 3rd rule of Strong Inference.

Ans. The third rule of Strong Inference is Carry out the experiment so as to get a clean result. This implies that the conclusion from an experiment should take into account the resolution power of the method being used. The method of genetic crosses says that the Self Incompatibility (SI) locus has a restricted locus, but it cannot predict how many genes are in that locus. Gene knockout and chromosome sequencing methods have potentially higher resolving power, and these methods have revealed multiple genes in the SI locus.

2. (a) Pollen whose genome encodes genotype 1 of the self-incompatibility locus elicits an incompatible reaction when it lands on a stigma carrying genotypes 2 and 3 of the self-incompatibility locus. Describe where this incompatible reaction probably took place, and explain what evidence made you conclude this.

Ans.: The incompatibility reaction probably took place on the stigma surface, because this is where sporophytic incompatibility reactions occur. Since the pollen itself did not code for the incompatibility determinant, that determinant must have been placed there by the parent sporophyte plant, thus this SI reaction must have been of the sporophytic type.

(b) For the self-incompatibility reaction described in 2(a), name two proteins involved and give the function of each.

Ans.: (1) SRK (S-locus receptor kinase), a transmembrane protein which functions to bind on the outside of the cell to the determinant coming from the pollen and then, after the binding, phosphorylate factors inside the cell needed to promote the signal transduction chain leading to the incompatibility reaction.

2) SCR (S-locus cysteine-rich protein), a small soluble protein that moves from the exine layer of the pollen to the stigma surface, where it binds to and activates SRK.

(c) In another self-incompatibility reaction, a certain pollen failed to be fertile when its RNA was destroyed. Where did this incompatible reaction probably take place, and what evidence made you conclude this?

Ans.:This reaction probably took place in the style of the stigma on which the pollen germinated., because destruction of pollen tube RNA is the incompatibility reaction characteristic of gametophytic self-incompatibility, and this reaction typically occurs in the style of the pollinated flower..

2. (a) As of November 2001, what was the main gene encoding a determinant of self-incompatibility that remained unknown?

  Ans.: The gene encoding the male determinant of SI in gametophytic self-incompatibility.

(b) What three features of this gene can you reliably predict, even before it is discovered?

Ans.: This gene will be located at the S-locus, it will encode a protein with a hypervariable region, and it will be expressed in haploid pollen cells.

4.  Regarding Figure 1 below,

(a)    Define P_SAG12, and IPT, and state what would activate P_SAG12.

Ans.: P_SAG12 is the promoter for Senescence-Activated Gene #12.. IPT Is isopentyl transferase, the rate-limiting enzyme for the synthesis of cytokinins. P_SAG12 would be activated by the developmentally regulated or environmentally induced onset of senescence.

(b)    State what is the question addressed by the experiment that generated these data, and what is the answer.

Ans.: The question is whether the developmentally regulated onset of senescence in older leaves would activate the P_SAG12 promoter and turn on the IPT gene, and whether the enhanced expression of IPT (and enhanced production of the anti-senescence hormone cytokinin) would delay the senescence of older leaves. The answer is yes.

(c)    When the P_SAG12-IPT construct was used in lettuce leaves, transgenic plants carrying this construct had a distinctly different distribution of LSU and chlorophyll over a range of younger and older leaves compared to the azygous (control) plants.  What was this difference, and what caveat does this suggest about using the P_SAG12-IPT construct for commercial lettuce production?

Ans.: The difference was that in the transgenic plants the distribution of chl and LSU was uniform among older and younger leaves, at a level lower than found in the control plants in young leaves and higher than found in control plants in older leaves. This suggests that the transgenic expression of P_SAG12-IPT can reduce the protein content and greenness (and thus the nutritive value) of younger lettuce leaves, which would reduce the commercial value of the transgenic lettuce.

5.   (a) Describe the assay that reveals the effects of expansin on wall extension.

 Ans.: Plant cell walls are  isolated, the proteins in them are denatured by boiling, and then purified expansin is added back to the walls and the effects of this addition on the extensibility of the walls are mechanically tested.

(b). What is the pH dependence of expansin effects, and what is the significance of this relative to physiological conditions in the wall that promote growth?

Ans. In the assay described above, expansin promotes wall extension only at acid pH values. Environmental and hormonal factors that promote wall extension almost always do so by first promoting the acidification of the wall.

(c)  What is the distribution of expansins in graviresponding maize roots, and  what is the effect of Brefeldin A on this distribution?

Ans.: Graviresponding maize roots curve downward, and in these roots there is more immunostain for expansin on the expanding convex side of the root than on the slower growing concave side.  Brefeldin A blocks this asymmetric distribution pattern.

(d)   The distribution pattern of expansins in graviresponding roots could be explained by two alternative hypotheses, and the more likely of these two hypotheses was revealed by the brefeldin A experiment. What are the expected effects of  brefeldin A on cells, and what alternative hypothesis was rendered unlikely by the results of the brefeldin A experiment? 

Ans.: Brefeldin A would be expected to block Golgi-mediated secretion of proteins and other materials to the wall. The fact that it apparently disrupts the gravity-induced asymmetric pattern of expansin distribution, makes it unlikely that the asymmetry of expansin expression is an asymmetry of expansin activation, and more likely that the asymmetry is due to asymmetric secretion of expansin, preferentially to the side that will end up growing faster.

6..(a) The expression of what two proteins would be expected to be increased by the exposure of plants to heavy metals, and what are the functions of these proteins?

Ans: (1) Metallothioneins, which chelate heavy metals, and (2) phytochelatin synthase, an enzyme important for the synthesis of phytochchelatins, which chelate heavy metals.

(b) Contrast symplastic and apoplastic pathways of mineral uptake, and indicate how each deal with the Casparian strip barrier.

Ans.: In the symplastic pathway, minerals are actively taken up into root cells (typically root hair cell) membranes and move through the symplasm across plasmodesmata from cell to cell all the way across the Casparian strip into the interior vascular cells of the root. In the apoplastic pathway, minerals diffuse passively into the apoplast (ECM or wall space) of the outer epidermal cells (including root hair cells) of the root, and from there diffuse passively in the ECM up to the Casparian strip. Here no further passive diffusion toward the vascular bundle in possible, and further transport requires that the minerals be taken up into cells, from which they move through the symplasm into the central vascular bundle.

7. (a) In Fig. 2 below the results of an historic experiment that led to the discovery of two photosystems are described.  From what we now know about the  wavelength sensitivities of PSII and PSI and about the interactions of PSII and PSI, explain the results shown in Fig. 2.

Ans.: The results show that Red and far-red wavelengths individually support the same low level of photosynthesis, but the two together support a level much higher than what would be expected from adding the two individual results together. .We can explain these results today by saying the red light preferentially activates PSII and the far-red light PSI, and when the light energy going into both is balanced photosynthesis operates at maximal efficiency, because electron flow between the two photosystems is optimized. 

 (b) The results shown in Fig. 2 above suggest that when the light energy flowing into PSII and PSI are not balanced, photosynthesis is relatively less efficient. Explain the mechanisms that plants use to solve this imbalance problem when (1) PSII is receiving much more light energy than PSI, and (2) PSI is receiving much more light energy than PSI. Your explanation should include the role of enzymes and of redox intermediates in the electron transport chain.

Ans.: (1) When PSII is receiving relatively more light energy, the plastoquinone intermediate in the electron transport chain stays in the reduced state much more than in the oxidized state, and this high ration of PQ reduced to PQ oxidized triggers the activation of a protein kinase that phosphorylates proteins in the LHCII complex. This induces a significant percentage of the antennae chl proteins in this complex to move away from PSII into PSI, thus decreasing the light harvesting capacity of PSII and increasing that of PSI and restoring the activity balance between the two photosystems.

8. (a) What is photorespiration, and why is it favored under high light intensities?

Ans.; Photorespiration is the series of reactions that occur when Rubisco catalyzes the addition of oxygen to RuBP to produce a  product that is eventually broken down into carbon dioxide.  It is favored under high light conditions because these conditions can result in the rapid depletion of CO2 from the intercellular air space in leaves, to below the compensation point needed for Rubisco to function as a carboxylase.

 (b) Contrast the position and functions of outer mesophyll cells and bundle sheath cells in C4 plants.

Ans.: The outer mesophyll cells are those leaf cells located closest to the epidermal layers (and stomata) of the leaf. They use PEP carboxylase to fix CO2 and produce ultimately a malate product that is transported into the bundle-sheath cells, where the malate is decarboxylated. The released CO2 from this reaction assures a high CO2 concentration for the Rubisco enzyme in the bundle-sheath cells, which functions as a carboxylase there and fixes the CO2 by the standard Calvin cycle.

9. (a)  Regarding phloem loading,, use anatomical criteria to contrast plants that are symplastic loaders from those that are apoplastic loaders.

Ans.: In symplastic loaders the SE-CC complex is symplastically connected by many plasmodesmata to the surrounding mesophyll cells. In apoplastic loaders, the SE-CC complex is symplastically isolated , with few if any plasmodesmatal connections to the surrounding cells. 

(b) Name two proteins that would play major roles in apoplastic loading, but not symplastic loading, and describe what those roles are.

Ans.: Sucrose transporter  (SUC)and proton pumping ATPase (H+ATPase) would play major roles in apoplastic loading. SUC would serve as the transmembrane transporter, co-transporting in a symport process both protons and sucrose  from the apoplast into the companion cells .. The H+ATPase on the PM of the CC cells would pump protons out into the apoplast , creating the proton gradient that powers the uptake of sucrose through SUC.

(c) Describe a knockout experiment used to test whether Arabidopsis was an apoplastic loader or a symplastic loader, detail two key results of that experiment, and state the conclusion that was drawn.

Ans.: Scientists selected Arabidopsis plants that had a T-DNA insertional mutation in a SUC gene that is expressed in phloem cells, and documented that these cells could not express a functional version of this SUC gene. They then showed that the plants carrying the SUC mutation were deficient in their ability to transport sucrose out of source leaves (that were fed labeled CO2), did not deliver sucrose made in the source leaf  to roots, which would be an expected sink, and had aberrant patterns of transport to other tissues in the plant.  They concluded that the SUC gene must be important for sucrose transport in Arabidopsis, and that, therefore, Arabidopsis must be an apoplastic loader.