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one of the things I told you about all the hormones and we’ll start right now with finishing off oxen is the fact that oxen and all the hormones are involved in a lot of different things so unlike animal hormones where each hormone only does one or a few things and there’s a bazillion hormones in plants we got a relatively small number of hormones but each of those hormones does a lot of different things and as we mentioned in the last lecture that has to be controlled developmentally what signal transduction pathway is there how those cells respond to the hormone in all these different ways is determined developmentally okay so the one thing that we wanted to talk about is apical dominance so in all plants or all now wait a minute not in Dyke not in monocots but in all dicots at the base of every petiole or lateral branch there is this axillary meristem an axillary bud and basically what this is is a remnant of the shoot apical meristem that is these cells in this bud are capable of meristematic activity but under normal conditions that meristem that activity is arrested it isn’t doing anything and one of the things that people who have known for a long time is if you want a plant to branch more if you want a flowering plant to produce more flowers the thing you do is you pinch off the apical bud and you see what’s happening here the axillary buds have become active and they’re producing new shoes okay so the the the picture here is that there’s some signal that’s starting in the apical meristem that is inhibiting the growth of these axillary buds and when you remove the apical meristem that signal disappears and the axillary buds start growing that all that’s certainly consistent and makes sense and it also makes sense in the context of the things that we’ve been talking about that that signal is oxen right that the shoot apical meristem is one of the main sources of oxen in the plant and the direction that oxen is being transported from the shoot apical meristem is down the axis of the stem and where is it being transported where is that oxen being transported from what in what cells from the shoots to the roots yeah and the prank um but in the parenchyma inside the vascular bundle so the the phloem per income and stuff like that not in the vascular tissue itself okay so let’s think about what that means what you would expect that the response to be at those axillary buds if the shoot apical meristem is there what’s the auxin concentration going to be in the cells that are conducting the oxen down the stem high or low higher right and if you take away that meristem the concentration of auxin would drop right so for a long time it’s been assumed that it is the low oxen that stimulates the growth of these axillary buds makes sense there’s only one problem if you measure the concentration of auxin in those axillary buds in the presence or absence of the shoot apical meristem and the presence of the shoot apical meristem the concentration in the buds in the axillary buds is low when you remove the shoot apical meristem the concentration in those axillary buds is high how do we explain that yes so the increase in auction in these axillary buds after the shoot apical meristem has been removed is a consequence of the activation of those narratives right we know that shoot apical meristems normally produce high oxen so is this the beginning of a signal transduction pathway or the end of a signal transduction pathway okay both yeah right thank you that’s correct it’s both but in terms of auxin signaling from the

original shoot apical meristem once you’ve removed it is the production of oxen and the axillary buds the beginning or the end is that the result of the loss of oxen that was being transported down the chute yes of course so the question is how does the signal get from the loss of oxen going down the axis of the chute to turning an oxen synthesis in the activation of the axillary buds where is the oxen being transported not in the floo floo brinkema okay so where is that auxin concentration most likely to be sensed the change in the auxin concentration moving down the stem where is it most likely to be sensed in those cells right in the phloem parenchyma because that’s where the oxygen is movie or something immediately adjacent to it so there must be a signal transduction pathway that carries the information that’s responding to that low auxin and carrying the information to the axillary buds and telling them okay the shoot apical meristem is gone you guys are now the shoot apical meristem start start producing oxen start producing new shoots okay the details of this are not understood but the long time held picture that it is low oxen in apical buds that causes them to take off is incorrect its low oxen in the chute itself and there’s a signal from the chute to the apical to the axillary buds to start growing what is the difference heading in the right direction but it’s not quite there remember you’re going to respond to increase or decrease in the concentration of options two things have to be there the option in the signal transduction pathway is the option that is going down the chute getting to those apical buds no it’s moving down through the parenchyma right so the oxen isn’t going there the response is in the cells that are seeing the auxin in the parenchymal cells and that must turn on some other signal that goes to the axillary buds it turns it on but as I said we don’t know all the details of that what we do know is it’s not the oxygen is not being sensed in the axillary buds okay right and this look at this is what I mean about thinking about these things because how many times have we talked about the response depends upon two things whether or not the hormone is there and whether or not the signal transduction pathways there if the hormone is not there it can’t respond okay so think through these things from a very general of the way hormones and signal transduction pathway work don’t think of these things in isolation figure out how it works in the context of a general model that’s what’s really important okay one last thing that I must say about oxygen because it’s a pretty cool experiment and that is the role that oxygen plays in wounding responses in fact this picture is showing you two aspects of what oxygens doing so the red is showing cells where oxygen is being produced or where act oxygen is present so we can see in this particular wound that option is being produced all along the surface of the wounds so auxin plays an important response role in the response of wounding in terms of producing new tissues that cover the wound that part has been well known for a long time but the other thing that’s that’s going on here is also pretty interesting here’s a vascular strand that was broken by the wound and what you see is auxin leading to the development of new vascular tissue that is going to connect these two strands to basically work its way

around the wound so two things that are that are shown in this picture they’re important auxin is important in the surface wounding response to produce new cells to cover the wound but auxin plate also plays a very important role in differentiation of cells to form vascular tissue this is present under north this happens under normal conditions as well as under wounding conditions is this the new vascular tissue here yes that’s a new vascular system but they’re showing up because there’s oxen present there so those cells are expressing some reporter gene I don’t remember what it is but those cells are lighting up like that because there’s a reporter gene that’s being expressed in those cells that’s responding to the presence of auxin okay so the textbook talks about a half a dozen other physiological responses of auxin i’m not going to talk about them all you can easily read through them and yourself what I want you to do is understand how those physiological responses fit in the context of what you know about auxin metabolism auxin transport and signal transduction and development and gene expression okay that’s your challenge is to make sure you understand all of those things and if you don’t send a question to me by email come see me in the office come see Simon talk with your friends whatever works okay all right any other questions about oxygen before we move on to talk about gibberellins okay with auxins where’s the Marisa Marisa and monocots yet sitting down in the bottom right and all the sums it behaves certainly behaves differently in monocots and to be honest I don’t know what the roles of oxygen plates and that isn’t monetize here’s where my limitations as a plant physiologist really show up because my background is in physics and chemistry and less in the details of I don’t know but I can find out the answer to anybody know who’s taken who’s taken botany who’s taken three four one zero to talk about that in monocots versus dicots okay yeah so dr. Nicholas would be the person to ask II least likely to know that kind of stuff okay so gibberellins jibrell another big class of plant hormones that many of you asked questions about from the readings the difference between how gibberellins work versus auxins work and so we’ll we’ll get to that in just a minute but this is one of the things that i want you to be doing while you’re reading is thinking about what are the similarities and what are the differences okay so Hugh Rowland was experienced were discovered by studying stem elongation very similar to what how auxin was discovered so auxin was discovered by looking at elongation of cells associated with phototropic responses in coleoptiles gibberellins were discovered by a fungus that happens to produce a compound that is a gibberellin that causes rice plants to grow very tall and so tall that they couldn’t hold themselves up they fall over for the fungus that’s good right if the plant falls over then the plant is sitting down next to the moist ground it’s a much better environment for the fungus to grow in than if the plant is a plant is standing upright so as far as the fungus is concerned that’s really useful not so good for the plant but gibberellins were first extracted and purified from the fungal extracts that can cause these plants to their stems to elongate an interesting thing is what many of Mendel’s mutants on in peas that had different stem heights were gibberellin mutants we’ll talk about some of these in just a few minutes okay so that stem elongation stem height is a is a key thing associated with gibberellins one of the things that once gibberellins were were purified that people found out pretty quickly was that if you take plants that normally have a dwarf phenotype that is short internodes short spacing between

the leaves and you apply gibberellins to them to things generally happen one they grow tall and the other thing is that plants that normally grow short when they tall they when they grow tall they also flower right so gibberellins are doing two things here one they’re causing the plant to bolt to increase those internode lengths a lot but they’re also initiating a transition from vegetative growth which is in most plants is an indeterminate sort of growth to reproductive growth produce flowers and then that meristem is gone right so it’s changing from indeterminate to determinate growth in many dwarf plants certainly not all but in many dwarf plants gibberellins can replace the normal environmental signals that trigger flowering and so this led people to think that gibberellin plays an important role in plants in transferring that environmental signal which is perceived in the leaves to the meristem to initiate flowering and it turns out that’s completely wrong gia berlin plays no role in most plants in signaling the plant to flower so this is a good example of where exogenous application of hormones to the plant gave a response but that response has no physiological significance as we see when we talk about control of flowering the signal that goes from the leaves to the flock to the meristems – to tell the meristem to produce a flower he’s not gibberellin it’s some it’s a messenger RNA in fact so it’s a good example of how you can be steered in the wrong direction by the non physiological response to exogenous application of a hormone okay and another important thing about the response of plants to hormones if you take a normal corn plant normally has long intro notes and you apply gibberellin to it there’s no response but if you take a dwarf corn plant and give it gibberellin so there is a response so that tells you something important when does it tell you when you read this what did you think Anna yeah so that’s one thing right it must have something to do with the amount of jibril and since there and that with the signal transduction pathway that’s responding to it good what else does it tell you about the responses to gibberellins I mean that’s right so in other words there is a maximum in the response to gibberellins in the normal plants there’s already sufficient gibberellin presence to induce this assembling that you see any word does not have that effect right so it basically says the response at Lisa this saturable right those are both important characteristics of understanding these particular movements and their role in signal transduction right right away it tells us that we should expect two very broad classes of mutants in any type of hormone mutants that are involved in production of the hormone and mutants that are involved in response to the hormone why didn’t we talk about any mutants in Oxon production that we talked about oxygen response mutants why didn’t we talk any about any mutants in Oxon production it didn’t say this in the chapter we’ll know more about this next week but can you take a guess what do we call it an oxen deficient a plant that can’t make oxen dead yeah it’s lethal oxen is absolutely required for plants to grow right an oxen mutant is embryo lethal can’t even make an embryo okay so that’s why we don’t worry about oxen synthesis mutants because you can’t do anything with it you can’t study them gibberellins obviously you can so gibberellins you can take away Jabril and sand the plants grow it just alters the morphology of the plant that’s a good question that’s a good question so is it encoded in the DNA is the

maximum height that a plant can grow to encoded in the DNA say that again it depends so that’s not a very definitive answer is it depends on what let’s go it let’s at least carry it that far depends on what okay so for example if we think about annuals versus friends but so what determines architecture okay so to large extent how tall a corn plant will grow in the presence of saturating gibberellins is somehow to determine in the DNA so I’m complicated but it’s there right it’s also there how it responds to the gibberellic acid is part of the DNA but it’s the presence of saturating gibberellic acid something else has to be determining how much the plant grows it’s like photosynthesis right even if we give the plant plenty of light and plenty of co2 there’s still a limit to how fast it can go that’s determined by the characteristic of some enzyme in this case it’s got to be something a lot more complicated than that right yeah yeah so most plants that grow up right like this the limits of how tall they can grow are mechanical right what’s the what’s the strength of the plant versus the things that would tend to tip it over but for ideas is supported yeah maybe I can grow you know hundreds or thousands of the arts long I don’t I have no idea but still if that’s the case if it still has to be somehow those characteristics have to be encoded in the DNA okay so gibberellins are involved in a number of other things one of the things that we’ll see next week no week after next gibberellin a number of its functions are antagonistic with another hormone of Cizek acid ABA so the relative amounts of gibberellins and upsizing acid play important roles for example in seed and bud dormancy these are controlled by the ratio of gibberellic acid to ABA the higher the ABA the more likely that are stay dormant or the seed won’t germinate the higher the jibra acid relative to ABA the more likely this the bud will break or the seed will germinate okay so chapter talks somewhat about the role that gibberellic acid has in breaking seed dormancy and also talks about the role that that once the seed germinates particularly for cereal seeds the role that gibberellic acid has and we’ll talk more detail about that in just a minute it’s also will some may say something briefly about anther development so gibberellins play an important role in antler development if you screw up jibril and synthesis or response in flowers you get you get poorly formed anthers and pollen dozen development doesn’t develop it and as we saw in this picture here gibberellins can play a role from a non physiological perspective in causing certain types of particularly dwarf plants to flower as I said that’s not what controls it naturally but gibberellins can replace external environmental signals in causing plants to flower so if you want your cabbage plants to flower in short days you spray some gibberellins on it and they and they flower normally cabbage plants require long days to flower okay so it can be used so for example in an agronomic perspective just to stimulate flowering in some types of plants but don’t read that as meaning Jia Berlin’s control flowering because

that is not the case okay so let’s think about sort of the chemistry of gibberellins and they’re complicated compounds they’re diterpene so they’re either 2020 carbon compounds of the dye terpenes it can lose this carbon to form this interesting epoxy side bridge here to form a nineteen carbon one so all of gibberellins are either 20 or 19 carbons they’re derived from the normal terpene biosynthesis pathway and plastids and they all have this basic 4 ring structure 1 2 3 and then there’s this fourth ring here so they’re relatively complicated in their structure but they’re also all relatively similar in their structure that is they all have this basic 4 ring structure and what gets modified is hydroxyl groups or double bonds and things like that so in terms of the general shape of the molecule they’re relatively similar to each other ok yep my guess is they do because it because it’s relatively easy to you know you apply jibril and then you do a microarray and see what you know what genes are turned on but I don’t know what the answer to that is I don’t know what what what’s actually causing that okay so if we thought talking about Jibril and biosynthesis the details of this are not important but one of the things that you should be doing is thinking about how this diversity this huge range so I can’t remember what the I forgot to look it up I was gonna go to the Jibril and website and look to see how many gibberellins have been identified now last time I looked I think it was a hundred and forty six so goes from G a 1 to G a 140 something okay the interesting thing is that only a few of them let about a half a dozen are bioactive what do we mean when we say bioactive that some JS are bioactive and some of them are textbook uses that term all the time okay so couple things let’s just look at the general structure of of gibberellins here is this molecule likely to be membrane permeable or membrane impermeable so you said what it’s pretty big so it’s gonna be impermeable it’s it’s it’s relatively big yeah I mean it’s the size of you know it’s larger than the largest amino acid but it’s not the size of a protein either right what other characteristic is important besides size polarity is that polar or nonpolar no it’s pretty nonpolar yeah it looks you know Scylla aromatic sorts of stuff and things like that they’re pretty nonpolar so gibberellins are membrane permeable okay did you see anything in the chapter about gibberellin transporters across the plasma membrane no that’s because there aren’t a least as far as we know they just moved through so where was the Jibril and receptor that had talked about was it on the plasma membrane where’s that G GID one cytoplasmic right right so that’s remember that’s the thing that distinguishes membrane permeable from impermeable signals remember brain impermeable ones the receptors on the plasma membrane membrane permeable ones that can be anywhere inside the cell this one happened to be in the cytoplasm okay so I lost track of what we’re talking about here Oh bioactivity yes so what what do we mean by bioactive the textbook used that term a lot what do we mean by bioactive these guys are bioactive they can function these guys are not what does that tell you right away about our picture that we put on the board at the beginning of last lecture about the things that control the distribution of of hormones at any given spot in the

plant so what would make them bioactive well what what can what changes this to this it’s an enzyme right right so whether the cell has that enzyme or not right so if this is what’s being transported if this is what’s moving around in the plant does this have any effect so which cells will respond to the gibberellins the ones that can convert this to this or you can stop the response by converting an active to an inactive one so this is one of the things that you should be seeing when you read the chapter that the book does not spell this out for you and I don’t know why in this sense I think it’s a very poorly written book because the thing that distinguishes you tell me what distinguishes what controls the local concentration of gibberellin from what controls the local concentration of auxin yeah transport is what what matters for auxin and active versus inactive what enzymes can activate inactive forms or deactivate active forms is what’s important in gibberellins and it’s critically important that you understand that right I wish the book would just spell that out but at the same time you should know that’s the kind of thing I want you to be looking for understand it’s not all these gory details of signal transduction pathways see the big picture okay so the interconversion of these is the one of the most important things and gibberellins in terms of determining where responses happen obviously they have to have the signal transduction pathway there as well but in gibberellins inter conversion of active and inactive forms is much more important than it is an oxidants basically auxin is auxin and it’s transported everywhere all right it’s not transported everywhere its transported in specific places but it’s always transported as oxen okay let’s see so the story I just told you you see this figure in the textbook remember this figure these are mutants along the path of jibril and biosynthesis so here’s a mutant that’s in the very first camed committed step in in jibril and biosynthesis of converting journal geranyl pyrophosphate this is a this is the starting point for all the C 20 die terpenes so GA 1 is right up here at the beginning it’s a mutant in this CPS enzyme that produces the first committed precursor in the pathway ok so let’s look at this picture mutations along the path and think about what that means in the context of the statement that these are bio active and these are not what do you see in the response of these mutants as you go along this pathway okay so somewhere down the farther are you along the pathway if you have gibberellins that are basically farther along the pathway right what about the stem height response that you see here is it do you see no inter notes no internodes no Internode’s no inter nodes then you get down here to the to the correct enzymes and you see the right height or these guys bigger than this guy they’re bigger right so in other words what that means and this is important in an experimental sense but not is important in a physiological sense that’s not that these are inactive they’re less active okay and if you as you look through the description of mutants in the biosynthetic pathway it should be clear that it’s not so much that these precursors or these degradation products are totally inactive they’re just significantly less active than the ones that we call bioactive okay that’s why I asked that question because bioactive doesn’t mean no activity it just means significantly less say that again isn’t that the term that they used in the textbook to talk about the the that the GA one and GA four were bioactive and GA

20 and GA nine were not bioactive no bioactive means significantly more did I say it backwards okay it’s dyslexic brain thank you very much yeah okay so that’s important another thing that we can do with gibberellins is pretty much the same thing we talked about in terms of auxin use a gibberellin dependent promoter to look at where gibberellins are being synthesized or transported in plants so this is again a Guus promoter so it’s turning that artificial substrate blue so we can look in five days seedling three-week-old seedlings and in flowers and in embryos and see that gibberellin is being synthesized in not everywhere but in particularly in in seeds and in embryos quite a bit of gibberellin in mature plants that seems to be restricted mostly to the vascular tissue and there’s Gia Berlin since being that are being expressed during flower development okay so this gives us some idea under both different developmental states in in different morphological positions within the plant where gia Berlin’s are being synthesized the blue wherever you see blue that’s a good question I don’t know what’s the difference between these guys and these guys I don’t know but it’s the blue regions here in here that are important in terms of where the jibril is being synthesized yep that’s the blue sorry yep okay so this this gives us another powerful tool to think about where gibberellins are either being synthesized or where they’re being transported okay and obviously the physiology that we’re going to talk about or that you’re gonna read about are related to these places where the gibberellins are being synthesized under different developmental conditions okay the last thing is that we can if we look at a number of the different mutations that Mendel was studying in terms of stem height and peas three of those mutate three of those mutants that he was studying are related to jibril and biosynthesis and interestingly so there are two mutations the mutation that is the NAM mutation which is way back here is up here causes the ultra dwarf the le mutant is further down it produces slightly taller plants here’s the normal plant and then that the SLN slender mutant is actually a mutant in not jibril and synthesis but jibril and degradation so in this particular plant increasing the concentration of bioactive gibberellins actually increases the height of the plant so it tells us in peas compared to corn the effect of the gibberellin and stem length is not saturated under normal conditions right so if you did this same mutation in corn we would expect the mutant wouldn’t be any taller because adding more G exogenous GA to the corn didn’t make it grow any taller but in the case of Mendel’s slender mutant it does make a difference okay so we want to talk to skip this for a second we want to talk a little bit about the signal transduction pathways associated with gibberellins and the receptor that we know about for gibberellins is GID one it is a cytoplasmic protein that binds gibberellins quite specifically and clearly there must be it must be able to distinguish in terms of its binding affinity between the bioactive gibberellins and the let’s say less bioactive your berlin’s right it’s got to bind these preferentially less than these other well that’s that’s what determines why these guys are more more create a larger response than either of these two right it’s the affinity to the to the receptor that’s important yes are any of these steps reversible

mmm so could you go from here to here here to here not that I know of so when you want to inactivate a bioactive form there is a way to go or when you want to activate an inactive form there is a way to go but there they’re involving two different substrates I’m not aware that these reactions are reversible I mean this this is a gross oversimplification of the jibril and biosynthetic pathway the gibberellin biosynthetic pathway is sort of like it’s four parallel pathways where and where there’s four different intermediates along four different paths that conversion from one to the next is catalyzed by the same enzyme so this one may have a hydroxyl at carbon 17 and this one may lack a hydroxyl but the conversions going on doesn’t involve that hydroxyl and for something else involves something else so for example the enzyme that’s involved in converting GA 9 to GA 4 and the enzyme that’s involved in converting GA 20 to GA 1 is the same enzyme these guys only differ in the presence of a hydroxyl group right so the the biosynthetic pathway of these are are kind of weird they’re they’re very different than the sorts of linear pathways that we’ve been talking about before the book doesn’t go into this it used to actually two volumes ago it had a very nice diagram of the complicated pathway and I think they must have figured that this confused students but it is a very messy pathway in terms of synthesis and one of you asked why why do you have all these different gibberellins well that’s a good question you know we’ve got one oxen and oxen can do a bazillion different things why do we need all these hundreds of different forms of gibberellins when only a few of them are bioactive and why do you need is there necessary necessity to have the difference between GA 4 and GA 1 it’s a good question I don’t think either from an evolutionary perspective or from a biochemical perspective we know the answer to that okay so we were talking about the jibril and receptor and this is the only one that we know about in it was discovered in rice because there’s a single jibril and receptor in rice and rabbit abscess there are three of them and obviously that makes studying a rabid abscess mutants in jibril and signal transduction pathways receptors and anyway a little bit more complicated and here’s the figure from the textbook that’s showing there’s wild-type and here’s the three different mutants are gid one a B and C right so single mutants in either any one of these basically no difference from wild-type so what does that tell us about each of these receptors what does that tell us about each of these receptors Eugen how about you what does that tell us the fact that each of these single mutants in either a B or C looks the same as wild-type what does that mean Patrick they’ll you can do this they can replace each other basically right if you knock one out the other two must work just fine because they all look phenotypically the same as wild-type how about if we look at the double mutants are they identical as far as the double mutants are concerned okay what’s what are the ones that are important to have a and C right if you have either a or C it looks fine but if you get rid of both a and C then the plant is short so that tells you B isn’t equivalent to a and C right this is the sort of thing that you should be able to determine if I gave you data like this you should be able to figure out what’s going on are they equivalent or not and if they’re not equivalent what is the characteristics of that non equivalents all right yes yep so these that’s what the typical notation means these are these represent loss of function of those particular genes correct okay like do a and B or B and C complement each

other yeah so is that an alternative way of so you could say either needs a or C but can you this one here is B functional or B not functional in this one not functional so that really the only functional gene here is C right so that means with C alone it basically behaves like wild type so in the absence of a and B C works just fine in the absence of B and C a works just fine but in the absence of a and C B doesn’t work just fine so I think I’m in interaction between the a and B or C and B cannot be in camp cannot be deduced from this data this one here oh this one here this this is a this is a mutant in the G a biosynthetic pathway its way that’s way back very early in the biosynthetic pathway so these are comparing a mutant in the synthesis versus mutants in the response and so we’re seeing that the triple mutant actually has less growth than the than the one that can’t make this can make very early GA s but not not the later ones okay so this is a good good example of how mutant analysis can be used to sort out when there are multiple genes that encode a certain component of the signal transduction since signal transduction pathway and we’ll see this it’s common in a rabid OPS’s why are why is having multiple forms of a gene common in a rabid OPS’s we talked about this way back at the beginning of the semester and you probably don’t remember the answer to it no it’s not specific to a rabid OPS’s yeah it’s related to ploidy right so although we consider a rabid abscess to be a diploid it contains in its genome remnants of two polyploidy events so if you look back in the rabid abscess the you know sort of look at fit the history of rabid abscess to some of its neighbors some of its closely related species you can see remnants of two polyploidy events where in rice for example the last polyploidy event is much further back in the plants history so that means rabid OPS’s compared to rice is more likely to have multiple forms of genes when you have a polyploidy event you double everything and over time evolution either changes the function of the duplicate genes or gets rid of the duplicate genes so the longer the evolution has gone on since the polyploid event the fewer duplicate genes you’re going to have a rabid abscess has two relatively recent ones and so it’s more likely that you’re going to have larger gene families in rabid abscess because of those polyploidy events okay so when you compare when you see three and rabbit opposites only one gene in rice that’s that’s the that’s the most common explanation for that okay so let’s think about I want to step back from thinking about the specifics of gibberellin signal transduction pathways to sort of bring something that was talked about in the gibberellin chapter in the context of question five from the exam so remember question five from the exam that was the positive and negative regulators that most of you didn’t do very well on so I want to go over positive and negative regulators in the most general sense and then bring them back in the context of the gibberellins so let’s think about where we have a positive effect er something that that turns on a response that is normally off so let’s think about it like this here’s

a component of a signal transduction pathway and the normal state of that component in the absence of anything else is that that that that step is off because remember every step in a signal transduction pathway has to have two states off and on right so there’s another state over here at the on state and there is a process that couples these something in the previous step converts this from the off state to the on state okay right and so on and so on and so on we’re just gonna so we’re just gonna look at one step and what’s thinking about what’s happening in before and what’s happening ever so in the case of positive regulators what’s the normal state of the step above in the absence of the signal right so if there’s no signal this is also off right so that means in this case there’s no response the response is turned off every subsequent step in the signal transduction pathway and the response is off right so this is the case when there’s no signal when the signal comes in what happens it’s turned from off to on because this guy has been turned on right and now we have responds describe for me the phenotypes the possible phenotypes of mutants in this the protein that controls this step of the signal transduction pathway what are the two possible phenotypes how about you Jonathan what are possible two possible phenotypes for mutations let’s just say this is a protein we’re not going to specify what it is it’s a protein that functions in the signal transduction pathway what are the two possible phenotypes for mutations in this protein okay so let’s let’s not think about it that way so you could imagine a mutation where the protein is not even made let’s consider only those mutations where the protein is made but it does it no longer functions in its normal way what could be the characteristics of those mutants there’s obviously more than one characteristic from the way I’m asking the question right it’s important to to read the professor’s questions and understand and Sysco that’s basically it always on are always off so we can imagine a mutation where even when this has turned the previous step is turned on this can’t be converted to on that would be always off no response or we can imagine the scenario and even when this protein is off the Mutai is in causes the protein to be in this state always on okay got that now let’s think about the same characteristics in a pathway that involves a negative region a negative regulator let’s make sure I get this right okay so in the case of a negative regulator we have the same sort of thing except for it’s sort of backwards that the normal state in the case of a negative regulator is that am I getting this right no I got it backwards sorry I want to mess you up took me a while to figure out how to draw this but the normal state of a negative regulator oh jeez I still got it backwards I’m sorry let me hold my paper right here I don’t want to confuse

you with this a normal state of a negative regulator is that’s on and it’s converting an active form to an inactive form that’s the negative regulation the fact that this is on is converting something from the on to the off state so again in the absence of the signal with a negative regulator there’s still no response okay so normal way the negative regulator works is to turn the next step off that’s why it’s called negative regulation right and when the signal arrives the negative regulator is turned off its inactivated right that’s how we talked about it happening in plants so in the case when the regulator is off now the conversion of the on state so the off state is inhibited and you get the response okay so in the case of a negative regulator when you turn the negative regulator off it no longer turns off the next step right and we talked about several different ways that this could be accomplished by degradation of an inhibitor by changing the cellular localization by changing the phosphorylation state a number of different possibilities there but relevant to Part C of question B what are the phenotypes of mutations in the negative regulator they’re the same right they’re either going to be stuck on or stuck off right so it’s important to recognize that in the case of positive and negative regulators the outcome of the signal is the same you’re turning it from no response to response it’s being done in different ways but we can have mutations that affect the normal state in the state in the presence of the signal in different ways in positive and negative responses so in the textbook let’s see if I can label these correctly so in this if you have it stuck and mutation so it’s stuck in the off state of a positive regulator I can’t remember which ones they remember in the textbook had talked about for three different cases of mutations so three of those four mapped to these guys I don’t know why they didn’t include the fourth one probably because they haven’t found any mutants that fit that description but this is this is an example again of how I want you to look at the specific information that is given in one of any one of these chapters in the context of how signal transduction pathways any signal transduction path is a very general model right if this doesn’t make sense to you if you don’t see how these map to the three different types of mutations that they talked about in the chapter come talk with me about it because those are the things that I think are important right it’s not the gory details of everything involved in jibril and signal transduction yes yep these are the regulator’s but that was Part B of question five in other words let’s ask the question how is anything upstream of the positive or negative regulator differ in these pathways does it have two different no so the same signal in one cell could affect a negative regulator in one signal transduction pathway in fact a positive regulator in the different signal transduction pathway the easiest way to think about it is what’s one of the most common ways that a component of the signal transduction pathways turns out wrong phosphorylation right so could phosphorylation from this one I’m sorry turn this one off I’m in this one not off absolutely right so there’s no requirement that there be differences anywhere upstream from it’s including the signal these are just two different

ways of labeling a step of the signal transduction pathway this may be except that where these types of transcription or maybe seven in the middle of the pathway doesn’t doesn’t yeah I make that segment and lecture I believe but I didn’t see why one is faster than the other but I asked you to tell me your opinion but more important I told you to justify it and there’s a couple of people who said the one in the other was faster and gave a reasonable explanation for it I don’t know I’m not sure I gotta go back look at the answers I don’t remember what they were but certainly just looking at something like this I don’t see a reason why one would be faster than well the signal transduction pathway in which a component of the pathway to has to be synthesized is not a signal transduction pathway it’s all got me there or even nothing happens right so again I’m next to the question five D my expected answer was there’s no apparent reason why they should be different because you’re basically doing the same things interactions are exactly the same it’s just the one conformation is life so no difference in the speed of those okay so I spent the time on this because I’m emphasizing how much I want you to him I want you to think about general models of signal transduction and how they fit into the specifics of what are talked about with any of the hormones that we talked about because remember most of what we know about signal transduction pathways for the specific hormones that we’re going to discuss are derived from mutational analysis right so this is important understanding how this works tells you how you can go from the phenotype of a specific mutant to what’s actually happening in the signal transduction pathway in the protein that that mutant affects okay so if you don’t understand this come talk with me about it it’s important okay let’s move on then to talk about just one component of the gibberellins another component of the signal transduction pathway and gibberellins that’s important and this is this protein called della della turns out to be a very common component of most gibberellin signal transduction pathways in fact there are dela like proteins in other signal transduction pathways but what you should see from this picture is the role that della plays in controlling at least in some aspects of controlling jibril and dependent signal transduction pathways is not much different than the role that that aux /ia a protein played in auxins that is when jibril and binds to its receptor that binding causes dela to by to bind to the receptor which in this whole complex X is an e3 ubiquitin ligase that sticks ubiquitin x’ on to this della grass complex and it gets degraded okay so this is turning off removing a component of the signal transduction pathway does this information here alone tell us whether dela is a positive or negative regulator let’s let’s have a show of hands who thinks that who thinks it proves that it’s a positive regulator who thinks it proves that it’s a negative regulator who thinks it it doesn’t prove anything and who doesn’t know ok it doesn’t prove anything because you don’t know whether this binds to something else and turns it off or it binds to something else and turns it on all you know is it’s being degraded yeah from that perspective the fact that it’s being degraded tells you once it’s degraded it can’t do anything so whatever it does it does only in its presence but does it tell you what it does when it’s there not not that

directly No yep that’s right good you would expect it to be a negative regular that’s for sure but it doesn’t doesn’t prove it and the reason it doesn’t the reason it doesn’t prove it there’s an example given in the book I’m not going to talk about it but it talks about the interaction between gibberellins and light and hypo caudal elongation right and you should look at that and make sure that you understand what the role that dello is playing there is different than the role that dello plays when we talk about cereals and cereal seed germination so I want to spend just a minute talking about this because one of the things that that gibberellins is known to play an important role in and one that’s been studied quite a bit because it’s an easy system to work with it’s the role that gibberellins play in the germination of cereal seeds we call them cereal seeds because they provide us with cereals they provide us with flour from rice or corn or millet or those sorts of things so basically the important is that there’s a big endosperm reserve in there that we can use you know to make flour out over if we wanted to what does the seed use it for yeah it’s basically energy and carbon for growth of the embryo right so one of the things that has to happen when a seed germinates is the endosperm has to be mobilized the endosperm is basically polymers its starch or its proteins can the embryo take up starch or proteins no the starch has to be broken down the monosaccharides the proteins have to be broken down to amino acids before they can be taken up by the growing cells of the of the embryo so one of the things that has to happen here is that the hydrolytic enzymes have to be produced to break down the polymers in the endosperm so they become mobile and so they become available for the for the embryo to grow and the layer of cells in the in the seed that produced the hydrolytic enzymes the L your own so the L your own layer is the group of cells that is responding to the gibberellins that are produced by the embryo when it germinates and are producing the hydrolytic enzymes that break down the endosperm so the question is let’s tease apart the signal transduction pathway in the L your own cells that respond to increased gibberellic acid by producing alpha amylase and enzymes that break down proteins and not just producing them but they excrete them I’d say export them from the cell so here’s the figure from your textbook that shows the pathway for starting with your Berlin coming to the cell and at the end here the cell exporting by exocytosis alpha amylase that then goes out into the endosperm and breaks down the starches okay so the signal transduction pathway we’ve already seen this part of it that the G Berlocq acid just moves through the through the membrane no transporters because it’s membrane permeable gets into the nucleus and binds to Gi Gi d1 and when it binds to GID one that causes the degradation of the dela protein the dela protein is a negative regulator of gene transcription but it’s not the negative regulator of the alpha amylase gene it’s a negative regulator of transcription factors so in the context that we talked about before of early in late genes alpha amp the gene that encodes alpha amylase is a late gene it takes several hours be from the time that these cells are exposed to two gibberellins to when alpha amylase gene starts to be transcribed and the reason is that the transcription factors that control the expression of alpha amylase are the we jeans those are the ones those are the genes whose expression is turned on directly by the gibberellins so the dela protein is a repressor of the transcription factors these are in fallen this class of G a my B proteins the my B proteins are a large family of

transcription factors in plants the GE a my B proteins are gibberellic acid dependent transcription factors okay so the presence of G Berlin releases the negative control of the the G a my B gene that produces the transcription factor which is then used to turn on the transcription of jibril and dependent genes so alpha amylase is and proteases and things like that James say that again uh yeah thank you okay so we’ll stop there and we’ll pick this up again at the beginning of the next lecture

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