Plant Growth and Development, BO/GN 733

1) What exactly do the authors mean when they say "underlying positional information" pg.494; if they are referring to position of chromatin, what is the "position" of chromatin?

2) Criticism:Why was the wer mutant FISH data in figure 1 not discussed? I can only assume that wer is not required for establishing closed conformation.

3) Are there any other known positional signals that could be used to further validate this chromatin phenomenon?

Chris

1) How does Ago1 interact with the siRNA pathway and how would they explain the changes in phenotype with the crosses?

2) Would overexpression of Ago1 affect the Cuc2 mRNA and change the meristem boundary per the discussion?

3) Did the supplimental data show wild type pictures to collaborate their EM data?

1) On page 512, the authors discuss the affects of the clf mutant on the ago1 phenotype. What do they mean when they say "clf does not rescue the loss of meristem function in ago1-10"? I didn't think a mutated gene,even if it is a repressor, could rescue another mutated gene. Is there evidence of a direct interaction of the CLF gene/upstream regulatory region to the AGO proteins or a protein-protein interaction?

10/5/06
? Boss 2006

 1) In Figure 7, the Arabidopsis plants displayed filamentous
structures. Could these parts be petals, siliques, or some other part in its
very early development or just an outgrowth like extra cartilage on a mammal?

 2) What does VFL and FT stand for?

 3) Did they do copy number for the transgene on either the
tobacco or Arabidopsis lines? I think dosage effect would play a part in the
severity of the phenotypes of these plants.

 
In general, I like this article because of the detail it
shows in the SAM and floral development; the pictures are nice too. Of course,
it would be great if the methodology of transforming grape was fully established
at the time these experiments were conducted.

Ramons wrote:
1) On the Mallory paper, I am still not sure how the transformation of
wild types were able to exhibit mutant phenotype. Should the wild type
copy be able to overcome the mutant phenotype?

The wild type would not be expected to overcome the mutant since it is a dominant gain of function allele.  what this means is that epreession of the mutant RNA is found in ectopic places where the normal RNa will be degraded.  So in these tissues you now have action of ARF17 where the there is normally no ARF 17 from the wildtype RNa.  does this make sense?

Ramon's second question:

2)  On the Vazquez paper, why is silencing shown as seperate pathways
when it seems that they can complement eachother? I suppose my question
is, how do you really know that a certain protein is specific to that
pathway, if it's all redudant?

I think you have a valid point here.  one could draw these liner pathways as more of a network of nodes whit several of the nodes having redundant functions.  The network drawing may reflect reality more closely, but ismaybe more difficult to look at. 

Christina asked: Why did they not do a protein blot? (figure 5) just because there is no cleavage


does not mean that there is protein being produced. The miRNA might bind weakly


preventing translation to occur.

Good point at least in theory.  miRNA have been shown to cause translational inhibition of target RNA's even in plants.  Chen et al demonstrated inhibition of AP2 mRNA by a corresponding miRNA (miRNA172?).  so this is possible.  There theory for how these miRNA resistant or immune forms of ARF17 work is that they are constitutively and ectopically overexpressed.  It woul dbe interesting to test that the phenotypes that they see require a functional ARF17 protein product.  Thus if you introduced a second mutation in the coding sequence of the miRNA immune construct, you expect that all of the "overexpression "  phenotypes should go away.  They haven't done this rather important control.

I guess that the evidence for SHR causing the assymetric cell division is that it is missing in the mutant and they seem to be able to induce assymetric cell divisions in the pSHR:SHR:gr construct plants that are not seen in the controls.What genes would be controlled to position the division site?  How does the cell control where to build the new cell wall that will split the two daughter cells? We dont know this in its entirety, but cell division in plants requires the regulated accumulation of cell membrane containing endosomes at the site of future cell division.  Sone how SHR seems to set off a cascade that is able to alter the orientation of the cell division plane.  This is pretty cool.  Of course it cant do it by it self but requires downstream gene expression.  This is supported by the fact that there are no assymetric cell divisions in the dex plus cycloheximide(the proteinsynthesis inhibitor) experiment.

we can discuss more if you like in class.  or post again.

1) On the Mallory paper, I am still not sure how the transformation of wild types were able to exhibit mutant phenotype. Should the wild type copy be able to overcome the mutant phenotype?

2)  On the Vazquez paper, why is silencing shown as seperate pathways when it seems that they can complement eachother? I suppose my question is, how do you really know that a certain protein is specific to that pathway, if it's all redudant?

9/28/06
? Mallory 2005

1)      In
figure 1, how can we know for sure that the miR160 is THE miRNA that is causing
cleavage these ARF RNAs and not another miRNA?

2)      In
Figure 4 they only compared the mutant heterozygous lines with the wildtype
control. Why did they not compare with heterozygous ARF17 or null mutant
segregants (I assume the homozygous mutants do not survive)?

3)      Why
did they not do a protein blot? (figure 5) just because there is no cleavage
does not mean that there is protein being produced. The miRNA might bind weakly
preventing translation to occur.

Going back to the Levesque et al (2006) paper. I am still not sure how they were able to discern asymmetric cell divisions (fig 2J). I just feel the authors are overextrapolating, or state it as "matter of fact." I agree that SHR has a role in cell division. But I am not sure if we should assume that the expression of SHR can be directly correlated to asymmetric division.

-Ramon

Let's have a comment frenzy!  The blog is kind of boring if we don't actually use it.

I mean, I know I'm mostly just putting off studying for my biochem exam, but....

1) Pertaining to Surge and Destroy:  Does the lack of lethal mutants with single mutations in the PIN proteins suggest that a certain amount of Auxin can be transported without PINs or that there is a compensation during embryogenesis?

2) Pertaining to Surge and Destroy: If the ARF and Aux/IAA proteins are both repressors then does that sugges that certain cells must use other methods to respond positively to auxin? 

3) Since the DR5:GFP construct needs 5 response elements to show a large response then this suggests that auxin response proteins must interact with another set of proteins to provide the necessary controls for the development so how is the auxin gradient sensed?

4) In response to the Jallais article there is debate over the PI3P Kinase activity in the plant community.  Certain individuals state that there is no PI3P Kinase activity in an cells but the roots and pollen production therefore Wortmannin is actually having more than just a PI3P Kinase inhibitor activity so how can they state that the endosomes are different, could they be altering the processing of the PIN instead?

5) Why did the authors not look at different cell types to see if  wortmannin affected the other PIN proteins in a similar fashion?

1) are there seperate sets of ARFs, AUX/IAA proteins, etc. for IAA and IBA etc?  Do the same PIN proteins interact with IAA and IBA? The generalization of "auxin" is confusing...

2) I'm not sure I understand how (theoretically) the presence of ARFs with repression domains could COMPLETELY obscure the expression of DR5::GFP.

3) Why no comments about the effectiveness of immunolocalization to visualize auxin levels?

4) Could the hypothesis of redundant auxin influx pathways be tested via localized activation of a heatshock::IAA::GFP in a aux1 mutant, then seeing where the IAA goes?

5) What is the value/significance of the "morphogen" label?

6) Wouldn't the arabidopsis embryo be a great candidate for digital in-situ?

7) Is it possible that AtSNXs might play a role in PIN redundancy?

1.-How good or how reliable are results that came from DR5 experiments? By the way described it seems there's too much uncertainty in using DR5 as a reporter of IAA activity.

2.- Why would nph4 and nph4-like mutants exhibit deficiency of cell expansion only in the hypocotyl?

3.- Is there evidance that auxin genes are negatively regulated by the gene product? In other words, is it fiar to suggest a negative feedback loop?

-Ramon

9/19/06
- Jaillais 2006

1)      In
figure 2, I don?t see much difference between n-p and q-s.

2)      How
would the IAA- in columella cells be affected by these same gravistimulation experiments?

3)      How
would PLT1 and 2 gene be affected by the treatment of BFA and wortmannin
(visual fluorescent protein fusion also)?