PRENOTE: I am sure that you all can appreciate that this e-mail forum is best used to answer specific questions and clarify individual points of confusion. Because of limitations of space and time, this forum is less useful for answering global questions (like, What do we need to know about topic X?). I welcome ALL your questions, and, as I mentioned in class, those Q & A that I think will be useful to the class will be published on this web site. My thanks to all of you who submitted the questions below.

Questions on Self-Incompatibility

Q.: Regarding the hypervariable region of the RNase at the S-locus, in class you mentioned there was a theoretical problem of changing all four amino acids. I didn't quite understand what you meant by this--something about how the plants would lose the ablilty of self incompatibility if all 4 loci were changed.

Ans.: The value of Self-incompatibility to plants is that it helps protect against the genetic problems that arise from inbreeding and it promotes outcrossing. If in an individual all 4 amino acids in the hypervariable region change, that individual will lose its self incompatibility, so there would be evolutionary pressure against this happening. However, if none of these amino acids ever change, then this raises the question of how do new alleles of the self-incompatibility locus ever arise? The solution to this conundrum is that those individuals that change 3 amino acids both retain their original self-incompatibility AND develop a new allele of the self-incompatibility locus.

Questions on Cell growth and Expansins

Q.: This is regarding the handout that you passed out on 2/1 and we discussed on 2/3 entitled "Two endogenous proteins that induce cell wall extension in plants". What role does pH play in this experiment and in the mechanism of expansin? Why does the author mention in the abstract that "endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions..."? I am confused about the relevance of the figure (Figure 5) at the bottom of that first sheet as well.

Ans.: "Endogenous" wall extension activities refers to those activities that would be seen in native walls which contained their full complement of wall proteins. When these walls are exposed to low pH, their extensibility increases. Denatured walls (boiled in water to inactive all native wall proteins) do not show any change in extensibility in response to low pH. If these boiled walls are "reconstituted" with a purified fraction of wall proteins enriched for expansin, then these reconstituted walls show the same sensitivity to pH as do native walls; that is, low pH increases their extensibility. This is what Fig. 5 shows: at low pH the extension activity of walls reconstituted with expansin is higher than it is at pH values of 5.5 and above.

Q.: In the article "Modification of Expansin Protein Abundance..." that we discussed on 2/8, I am not sure of my interpretation of the figures. Figures 5 and 6 are titled Size Distribution of CDTA-Extractable Polyuronides from Fruit of Overexpresser and Suppressed plants. I drew the conclusion that in the Overexpresser vs. control there was not much difference in amount of polyuronide, so this meant there was no effect of overexpression of expansin. In the Suppressed plant vs. Control, there was a (albeit slight) difference so I drew the conclusion that when expansin is suppressed, polyuronide levels drop (expected result). Is this correct?

Ans.: In Fig. 4, you are correct, overexpression of expansin does not change the size distribution of cell wall polyuronides. In Fig. 5, suppression of expansin expression results in a shift in the size distribution toward the left (dashed line is displaced toward the left), which means that the cell wall polyuronides are larger as a result of the suppression. This means that when expansin expression is suppressed, polyuronides are less accessible to digestion by wall enzymes.

Q.: In the transformation experiments of the article "Modification of expansin protein abundance...", is the "mock transformation" plant simply used as a control?

Answer: Yes. It is theoretically possible that the various technical manipulations required to transform a plant might themselves induce some genetic expression change. To control against this, a control plant is transformed with an empty vector (i.e., a vector that does not contain the sense or antisense construct). The mock control should never show the same phenotype as the transgenics that overexpress or underexpress a gene. If it does, this means that the phenotype change was an artifact caused by the technical manipulations and not by the gene change.

Questions on Mineral Nutrition

Q.: In the handout of feb. 3rd we went over the zinc transport. Could you clarify the main point and significance of these experiments?

Ans.: The handout for the zinc experiment shows that vacuoles isolated from a zinc-sensitive cultivar take up zinc less efficiently than vacuoles isolated from a zinc-tolerant cultivar. Thus, another mechanism of heavy metal tolerance in plants is the direct uptake of zince into vacuoles, without the mediation of phytochelatins or metallothioneins.

Q.:In the case of symplastic and apoplastic transport, can you say that symplastic is active transport and apoplastic transport goes down a concentration gradient until it reaches the casparian strip at which point transport is active?

Ans.: The key issue is not so much active or passive, but discriminating or not discriminating. Apoplastic transport travels through the apoplastic space in a passive indiscriminate fashion up to the casparian strip, at which point it has to cross a membrane through transporters (either active or passive) that discriminate what goes across. Symplastic enters the cell across a membrane through a discriminating transporter (either actve or passive) and then passes from cell to cell through the continuous cytoplasm that links plant cells.

Questions on Gravity Responses

Q.: This question concerns the gravity response in the Chara system. What I understand from lecture is that the gravity response is the differential rate of cytoplasmic streaming in the upward and downward directions. How does the cell regulate this rate, after the initial signal is transduced, possibly by wall integrins and calcium channels? (i.e. have any other downstream events been identified?)

Ans.: Changes in calcium concentration definitely affect microfilament assembly and disassembly, and this dynamic process is critical for cytoplasmic streaming. A recent review that covers this topic is: de Ruijter N C A, and Emons A M C .1999. Actin-binding proteins in plant cells. Plant Biology (Stuttgart). 1: 26-35, and an original article that discuss this phenomenon is: Yokota E and Shimmen T. 1999. The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity. Planta 209: 264-266.

Q.:What is the connection between the polar ratio and the widening/narrowing of the stream to maintain equal cytoplasmic volume on each side of the cell? Is this widening/narrowing just a RESULT of the unequal streaming rates? Or is it also regulated by gravity?

Ans.: I believe one could argue either way from the results. I.e., the real effect of gravity may be to narrow the stream on one side, which results in a faster flow on that side.

Questions on Blue Light Photoreceptor

Q.: CRY I is the photoreceptor for photomorphogenesis, right? But on p. 1700 of the article you gave out on 03.07 entitled 'Arabidopsis NPH1: The flavoprotein with the properties of photoreceptor', on the last paragraph on the right side, it says 'involvement of cryptochrome in phototropism'. I don't understand that.

Ans.: Yes, CRY 1 is a photoreceptor for photomorphogenesis. The same paragraph you refer to on p. 1700 presents strong evidence against the hypothesis that cryptochrome is involved in phototropism. This point may be a bit controversial, but for the purposes of this class, we will accept the arguments of Christie et al. (1998) given in the handout and designate NPH1 as a most likely photoreceptor for phototropism.

Q.: I do not understand the graphs on page 1699 of the article by Christie et al. (1998). Can you explain them?

Ans.: The Figure 1 on p. 1699 shows several blots. The blots are: A. SDS-PAGE gels showing bands stained with a dye that binds to proteins (Coomassie blue). The take-home lesson here is that the gene for NPH1 can be expressed in insect cells, that most of the expressed protein (which has a molecular weight near 120 kD) is insoluble, but some of it is soluble. B. This is an immunoblot. It is stained with an antibody that specifically recognizes the NPH-1 protein, whether it is expressed in wild-type plants (lane marked WT), or is expressed in the soluble fraction of proteins extracted from insect cells that are expressing NPH1 (lane marked BacNPT1). The middle lane is a control showing that the antibody does not recognize ANY proteins expressed in a mutant that is lacking NPH1. C. This is an autoradiogram showing that the NPH1 protein expressed in insect cells (BacNPH1) can autophosphorylate itself when it is irradiated with blue light. The lanes show that this light dependent autophosphorylation occurs in wild type plants (lanes marked WT), and in the purified BacNPH1 expressed in insect cells (lanes marked BacNPH1), but NOT in the nph1-5 null mutant or in extracts taken from insect cells that are not transformed with the NPH-1 gene.

Q.: NPH I is the phototropism receptor, right? It binds to flavin (a chromophore) - the binding makes it a photoreceptor?

Ans.: NPH1 is a photoreceptor for phototropism, but it may not be THE only one. Yes, it binds to a flavin chromophore, and this forms a holoprotein that is a photoreceptor for phototropism.

Q.: NPH 1 binds to NPH 3. What exactly does NPH 3 do? Does it just relay signals?

Ans.: All we know about NPH3 is that it physically associates with NPH1 and that without it the light absorption by NPH1 cannot be transduced into a phototropism growth response. So it is a necessary component of the signal transduction chain that converts a blue light signal into phototropism.

Question on Annexin

Q.: Does Annexin induce increased rates of exocytosis in cells? What is the relationship of Ca++ with annexin?

Ans.: Yes, the point of the article we covered in class was that exogenously added annexin can induce increased exocytosis in cells. The relationship of Ca2+ with annexin is that in the absence of Ca2+ annexin has no documented function. Ca2+ activates annexin, which is a Ca2+-binding protein, and the binding of Ca2+ to annexin is necessary for its function in promoting exocytosis.

Questions on Auxin

Q.: How does auxin get asymmetrically distributed in roots so that it causes a differential growth response to gravity? Does polar transport do this? How?

Ans.: This material was covered in Dr. Estelle's lecture, which I did not attend. Based on my knowledge, and on what was covered in my lectures, I can say that polar transport of auxin is critical for root gravitropism. I can also say with confidence (and I think that Dr. Estelle will agree) that HOW auxin gets asymmetrically distributed in roots to cause differential growth has not yet been conclusively demonstrated.

Q.: How does GA interact with IAA to promote growth? One article (5 classic horomones) says 'the relationship between auxin and GA awaits resolution, but this article is kind of old.

Ans.: What we indicated in class is that GA prolongs the response period that newly made cells can respond to auxin, but that without auxin, GA would not be able to promote much growth.

Q.: One article says that auxin 'cause reorientation of auxin.....thereby promo[ting] elongation of cells that have stopped growing' (five classic horomone handout) and another says (arabpdopsis AUX1 gene: a permease-like regulator...) 'IAA reglates gravity induced root curvature by acting as an inhibitor of cell elongation. I don't understand the difference - can you explain?

Ans.: At relatively low concentrations, auxin promotes root growth; at higher concentrations it inhibits root growth.

Question on Methyl Jasmonate

Q.:Why does incubating with sagebrush allow for production of proteinase I and II in tomato plants? do the sagebrush plants produce a signal that is used by the tomato that the tomato can't produce itself? Is the substance produced by sage methyl jasmonate?

Ans.: The sagebrush naturally produces methyl jasmonate, and this volatile substance can move from the sagebrush to the tomato plant and induce increased proteinase inhibitor in that plant, even though it is not injured. Ordinarily the level of proteinase inhibitor in the tomato plant would be very low in the absence of an injury stimulus.