<TITLE: Control of AHP6, a Central Player in Vascular Cell Fate Regulation
ACADEMIC DOMAIN: natural sciences
DISCIPLINE: biology
EVENT TYPE: seminar presentation
FILE ID: USEMP12A
NOTES: continuation of and continued in USEMD270, seminar also includes presentation USEMP12C

RECORDING DURATION: 13 min 54 sec

RECORDING DATE: 11.4.2007

NUMBER OF PARTICIPANTS: 34

NUMBER OF SPEAKERS: 1

S2: NATIVE-SPEAKER STATUS: Portuguese; ACADEMIC ROLE: junior staff; GENDER: female; AGE: 31-50>


<S2> okay , so the the title is here and wh- what my main interest is actually in the control of this string called AHP-6 that m- many of you probably know , something about it at least er but i will give a short introduction into these and to the model that we are working in our group so we are working with arabidopsis root that has a quite a simple anatomy and that's why actually it's considered such a good model , and there's all these in the cross-section there's all these concentric layers and in the middle there's what is called the (stylet) that by definition it's comprised by the erm , a layer of pericycle cells in green and the vascular bundle inside , and the vascular bundle is self-sufficing (root) just formed by a xylem axis and the xylem it's er by definition it's now composed by what is called the protoxylem (part) and in the middle you have the metaxylem and this is quite important because i will be really talking about th- about these two protoxylem poles the two also can be , well i will show you the english (xx) but it's the also the secondary walls that you can see that protoxylem has these angular rings so it's quite distant from the other cells okay apart from this xylem axis it also has the two phloem poles and in the middle there is some er two important (xx) cells that will later in development go right to the secondary development and will be back to cambium and so on , okay so i will focus in these genes so AHP-6 this ring it's it was shown by <NAME> and <NAME> that is really important in the for protoxylem specification so in wild type you always have these two protoxylem poles and in the AHP-6 many many times at least in one of the sides it's missing this you can see with confoc- confocal pictures this is quite clear so here you just in in wild type you would see always the two in the AHP-6 mutant you just have one or you can see actually here the breaks are you seeing it right mhm . so the protoxylem it's this thing that look like rings here and the metaxylem it's what is in the middle in the AHP-6 mutant there there is always some kind of breaks and you don't have the protoxylem anymore this never happens in wild type and in wild type it should be like this having two sides this protoxylem's parts , mhm . when we see t- what is the AHP-6 type expression actually it's quite interesting because it's really specific to these two protoxylem poles and always with to the two pericycle cells adjacent to these two protoxylem or so actually this chain of course it's doing something here and what we when we kno- w- erm see what is the molecular nature of the AHP-6 actually it's er little (xx) transfer protein so in the cytokinin the signalling pathway you have the receptors and then you have er all this kind of the phosphor transfer proteins that will transfer its phosphor to the response regulators in the (xx) AHP-6 actually it's in it's missing because er it's still in this conserved (xx) in (xx) to phosphor an- and so on so actually what (xx) show is that this phosphor transfer doesn't happen and what is missing is that this cytokinin pathway it's block so actually AHP-6 is down-regulating the cytokinin signalling and it's down-regulating si- er cytokinin signalling specifically in these two protoxylem er er poles because we also know that two protoxylem to (be formed) actually it's cytokinin signalling it's inhibiting the protoxylem plate because when we er treat wild type er roots with cytokinins , the protoxylem just completely disappears when we compare this with the wall mutants so the wall er mutant it's er it e- it's a mutation in the one of the receptors of cytokinins so there is no cytokinin signalling at all in this mutant and what you see why the cytokinin signalling is missing is that all vascular bundle it's just with protoxylem <P:07> on the other hand er cytokinin actually also may th- also regulates the the special domain of the AHP-6 expression what happens is that when you don't have the er cytokinin signalling the AHP-6 its expression it's rather it gets increased like you can see it in the u- mhm (xx) and when you have alpha regulation of cytokinin signalling like it happens in the AHP-6 mutant also the er expression of the AHP-6 actually gets a bit not (xx) so just the conclusions of this @@ is that cytokinin signal (which) protoxylem specification , also that the AHP-6 it's enabling po- protoxylem formation in in these two two poles by inhi- inhibiting cytokinin signalling and cytokinin signalling actually (xx) repress er it puts the the the AHP-6 expression in into these two two poles so that is a kind of feedback you would (xx) so but actually my my main main question here it's what actually is upstream of the AHP-6 er expression so always it's a shift it's such a specific erm expression of the gene in these two two poles now of course we know that cytokinin signalling is inhibiting this but we don't know nothing about the molecular nature about it so it's kind of , one of the points what is the molecular nature of the metho- er er regulation by cytokinin signalling and of course what are the positive (up signal) factors so . i have two approaches to to this to answer these two questions the first one and the main one i'm developing these er for the genetic script for the identification of AHP-6 expression and parallel to this i'm erm , kind of analysing what are the regulatory elements within the AHP-6 promoter and i will talk this because it's first about this promoter analysis because it's really simple @@ and i don't (xx) (this) so much so basically i just amplified different parts of the AHP-6 promoter so to what the AHP-6 promoter so the promoter GFP construct actually it's 1.6 er KB that er drives this specific er expression and i'm amplifying less and less erm , er versions of of of this promoter to see actually to i- try to <SIC> identificy </SIC> what is the minimal part that actually still drives this GFP expression and if these different models are the same or not (xx) to respond to cytokinin signalling , but and these i'm just still cloning things so nothing (xx) , but these th- the screen for the altered of the AHP-6 expression , so what i did is i had these lined up actually i think , so i had this AHP-6 GFP er promoter fusion that it's quite specific like i said to these two protoxylem er poles and to the two pericycle adjacent cells and with this line , i mutag- mutagenized this line , grew it er in pools in NM-1-1 i screened in the M two four both or loss of the AHP-6 GFP or for the (amendment) or for for everything that it (xx) being the rational of this is that if i get things that that reduce the GFP expression i will get genes promoting the AHP-6 expression and the other way around so if i think that (xx) AHP-6 expression i will get genes that inhibit AHP-6 yeah , this is @and@ this it how it looks in the greenhouse it was so every (pod) it's a different pool so yes mutagenesis was quite successful i got ze- zer- 0.5 per cent of albino plants in the end pool this is supporting th- what the literature says that it's a successful mutagenesis and i have this number of pools collected 800 and so i i i maintained that the pool size quite small that in case if i get erm sterile mutants or i could still go to the pools and re-screen them then and try to find the (xx) , yeah , okay so the AHP-6 expression it's actually how it looks like in the stereomicroscope you really have to amplify a lot to see something and so you see really these two poles in a nice way of course when the root is turned you just see one and i'm getting i'm getting two classes of mutants or i'm picking these two or four classes of mutants so no GFP at all things with faint GFP enhanced GFP and everything that looks strange with outer pattern i'm also thing- i- picking that , so this is to exemplify so i worked without any GFP @@ faint GFP you still s- er s- still see something fluorescence there but it's very very faint , enhanced GFP and strain GFP estimated (xx) okay this one that has then er i'm i'm actually starting really to work with two of these mutants one of them it was this enhanced GFP so the AHP-6 expression it's in all the vascular bundle if you see it in er cross sections it gets into the procambial cells to the pericycle cells as well , many times just in the middle but it's supposed to be the metaxylem it's not there but these are still with erm @@ really characterising the phenotype of this mutant erm maybe you have come across so this is really preliminary , of course the o- one of the things i have to see with this this method is actually if it's a (xx) transmutation because it actually can be a (xx) mutation (xx) , so it's worth (thinking) , another one that i find very interesting is that it was one of the ones that was picked out in strain GFP and interestingly enough many of the times the AHP-6 expression it's just in one of the poles , sometimes it appears also in in the second pole but it's always very (white) and this can be very interesting to know what (are the) layers and mechanisms and so on and and the root also doesn't look normal so the root growth it's er i- they are shorter and maybe some of the roots it's completely messed up so , yeah but again i'm still also characterising phenotypes and making all all the crosses to start mapping these these mutants so actually this leads me to my @@ future plans that is the characterisation of the phenotypes of the mutants that i'm working with and of course (xx) and of course these are the shorter future p- plans they are quite clear and that's it thanks </S2>
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