2026 Boyer Award Seminar - Paige Henning

Welcome everybody to the uh Boyer Award for post-doal excellence. Um it's my pleasure to uh award it this year to uh Paige Henning. But I'm going to just say a couple quick things about Paul Buer first. Paul Buer was a graduate student here. Got his PhD in 1943. And as far as I know, he was the only one of our graduate students in biochemistry to go on and win a Nobel Prize. Hopefully some of you will uh change that to more. But uh he then donated some of of that uh money from the prize to the department. One of the things that it was used for was to create this um this award in uh Paul Boyer's name. And so it's a real pleasure to be able to give the certificate for the award uh today to Paige and suitable for framing. So there you go. Now, uh, Paige has done some great work, but I'm going to let uh Mike Susman, her mentor, uh, introduce that work. >> This is really I have my uh, backup hearing aids in, so never mind. Anyway, so next slide. Oh, I do. >> Yeah, you do it. >> I am really happy to talk about Paige, introduce her. This is her anttogyny. Fancy word. She taught me her development. Started at Washington State University for her bachelor's and then did her thesis at Washington State. >> Wow. >> Speak into the mic. >> Oh, I should speak into it. That's good. Now you can hear me. Okay. So she did her bachelor's at Washington State and then she did her PhD and her thesis was really cool. She um was part of a group that sequenced the genome of um of uh Tinera Subulada and uh that um out of that came a new peptide hormone that we know nothing about and that she'll talk about. Can you hear me now? Usually my voice is louder than me, >> but not anymore. I'm getting old. So, >> oh, for the recording. Oh, okay. All right. Um then she got a three-year NSF graduate fellowship um to come to my lab and uh study um the SPH peptide hormone receptor protein in both uh subulada and arabidopsis. It's a large peptide hormone family. She I don't want to take her thunder so I'm not going to say anything about it. Um, talking more about Paige, um, she is both meticulous and creative and these are important traits of great scientists are can be very difficult to juggle and um, uh, she's done an amazing job of that and I'm sure we'll continue. Another uh, particular is that she's very brave. Some of you who know me well would argue just coming to my lab demonstrates braveness. But in fact, she did more than that. She actually switched fields. She hadn't done much protein work and uh she switched from developmental biology to protein chemistry in my lab and has a great sense of humor. Has even come up some new bad jokes that I don't know if she'll be sharing them with you. So to summarize um she came to my lab four years ago with that fellowship um her goals were to learn protein chemistry number one two number two was to find the ph receptor and number three was to teach me about flowers and genetics. uh she's made real nice progress on one and two which while not perfect or complete her experiment suggests that the SP peptide receptor is not one of the usual players in other words it's not a protein kynac protein coupled receptor um and indeed like Marcus some of you may know who's uh the oxen receptor and also a poppyph receptor it may be a new unexpected paradigm for plant peptide hormone action. Goal number three, in other words, teaching me has turned out to be more difficult, I think, than she expected. And here she is starting. She we went to lunch together and she she said, "Mike, this here is a flower." Wow. And then she told me she wants to come to my lab to answer the deepest possible molecular level questions that Darwin himself pondered. In other words, what controls the length of male and female sexual organs and flowers? And also, how am I going to get a job? So, I said, I can do that. I'll help you with both of those. And I am so pleased that she joined my lab and I hope she feels the same. And this is another experiment she recently completed. uh a beautiful baby named Junipa. And um so that's it. I just uh with Oh no, there's one more slide. Now without further ado, I'd like to introduce this year's Boya Post-doctoral Award, Dr. Paige Henning. >> Oh, the seminar is just getting started. Don't leave yet. Okay. Hypothetically, this microphone is picking me up because I turned it on. Okay. Hi. As you all heard, my name is Paige Henning. I'm a posttock in the Susman lab. And today I'm going to talk to you about my favorite topic, sex, specifically plant reproduction. That first part of the sentence was to capture your attention. I saw some head lifts. Yeah. The second half was to set your expectations low. And that explanation was to let you know that I am that guy who gets in front of people and I try to make jokes when giving a serious presentation. Okay, great. Happy for that. So, I'm sure when you thought about sex and reproduction, you thought about flowers, right? And there's a reason for that. So, of the approximate 38200,000 plant species, approximately 87% of them are flowering plants. which uh brings up this question. How have angioperms managed to uh dominate the plant world, the plant kingdom, right? Um angioperm, they're the only plant lineage to actually exist on every continent. So you might think Antarctica, surely there's no plants there. You'd be wrong. There are grasses there. So like I said, how have angioperms managed to dominate? The answer is the evolution of the flower and the fruit, which is why we're here today to talk about flowers. because flowers are pretty and I don't care about fruit. Okay, as long as Mike laughs, that's all that matters. Uh, everyone else, you can pity laugh as well. That'd be real great. Um, so if we're going to talk about reproduction in terms of plants, we're also going to have to know words probably. And everyone in here probably isn't a plant biologist, and everyone in here probably doesn't know floral anatomy. So let's speedun this. Okay. So here in red we have the anther. This is where our pollen is produced attached to the anther. We have our filament. Together they make up the stamon. We then have our stigma. This is where our anther or our or pollen falls. It falls onto the stigma and then a pollen tube grows through the style eventually reaching an ovary and fertilizing an egg. Uh here's a picture of a rabbidopsis flower. Um, I've highlighted the anther here. Well, I forgot I have a pointer. Uh, I've highlighted the anther here in red. We can see that it actually has multiple anthers. And there's our little filament in blue. We have multiple filaments. And here we have our stigma. And then we have our style. So, just to recap why you should care about any of this, our male gamtates are produced in the anther with our pollen. Our pollen contains our male gametes. And then within the style somewhere we have our ovary which is where our eggs exist or our female gametes. So I'm specifically interested in destyly right there are so many plant reproductions reproductive systems you got to pick one and I picked dy um it's a form of heterostyl and it's really cool because a species will have individuals that either have es more flowers or elmore flowers. So the S just stands for short style morph. So you can see our style is short in this individual and our anther is placed high in the flower because we have this long filament. And then we have our Lmorph individual and that just stands for you guessed it, long style. Um so yeah, we have our long style and then we have our short filament phenotype. This is comparable to animals. At least that's what I tell people to make it easier for them. So you can either be an Smorph individual or an Lmorph individual. You can't be both. Just like a human can be a male or a female, they can't be both biologically. Um so Dyl is super cool because it's a case of convergent evolution. It's found in at least 28 families and 187 genre with like a ton of different like points of evolution. Like it's been evolved a tons of different times and even within a genre. For example, uh the linen, it's evolved multiple times in linen and is of economic importance hypothetically because jasmine, starfruit, and buckwheat are all die stylus and we do eat starf fruit and we do eat buckwheat and jasmine can be used in perfumes. So you should care at the very least because you enjoy eating. Hypothetically, maybe you don't. There are five general key characteristics of a datus plant. So, as we talked about these anther height dimorphisms where in one morph we have anthers higher in the flower than the other. Um, and then our pistol length dimorphisms. So, we have our stigma lower in one morph than the other. We have differences in our pollen grain. So, hypothetically, even if you're not a plant biologist, you can see that there are clear differences between the pollen grain in figure A. Then in F, we have differences in our stigmatic papial. So hypothetically, even if you're not a plant biologist, that is very obvious in our figures B and E. And it's hypothesized that these dimorphisms evolved to discourage self-pollen from sticking to self-stigma. And then occasionally we have self- incompatibility. So that's an individual's inability to reproduce with itself. So a lot of plants, they can just mate with themselves and they don't need anyone else. um self- incompatibility just means that you can't do that. Um sometimes we also see intramorph incompatibility and that's when an Smorph individual is incapable of reproducing with an Smorph or vice versa. So there are a lot of Dylist species that are studied at the genetic level and I spent my PhD studying Dylan and Pascllaci specifically in turn. So these are pictures of turner seulata. Uh, one thing Mike was right about is that you can't teach him how to pronounce things. I've been telling him as Turner this whole time. That wasn't I don't care that he doesn't know how to pronounce it. That was a joke. I'm sorry, Mike. That was a joke. But at the Okay, I'm moving on. So, here's a picture of Turner Seabulada. Again, it's beautiful. It's a subtropical shrub. Dicy is an ancestral trait, meaning the last common ancestor had Dasty. So any species within the genus that is dyus uh is going to hypothetically have the same genes controlling that. Um it has self and intramorphine compatibility which makes genetics easier. It has an annotated genome which makes it um an exceptional model to study in because we like to have genomes and I I annotated it not to like um and it has 3S genes. So it's a relatively the genetic basis of dicile is relatively simple in turner compared to other dicilus species. So we talked about s genes like for one second on that last slide. Let's dive deeper into the genetics of dying. So yeah I'm sure you guys all came here for a plant talk in biochemistry award. You're welcome. Um so the genetic basis of dicilate in all dilus plants is called the slocus or the self-compatibility locus. Uh today it's always a hemzygous super gene. So super gene is just multiple lossi that are inherited in a mandelian fashion as if they are only one gene. Um today all of the dilot species that have been studied um the super gene is esmorph specific. So this means there's one cop if it is a diploid individual there's one copy of the s locus and then the other chromosome would not have anything there's no alilic region. Um in terms of turnaround specifically we have our female mating type and morphology gene. We have a male mating type gene. So mating type just refers to who you're capable of mating with. Right? So if you're an Smorph individual, your mating type means that you're capable of only breeding with Lmorph and vice versa. Then we have our male morphology gene. So that's involved in establishing those filament dorphisms. So our female gene we found was this uh ail transferase that's involved in brass steroid inactivation. We called it bad after the name of the super family it's from. It's what causes that short style phenotype and establishes female self- incompatibility. We have yuck 6. It's a member of the yucka family which is involved in oxin bio synthesis through the tryptophane dependent pathway. It's the most bioactive pathway for oxin biosynthesis. It establishes that male mating type. And then we have ph1 or which is a member of the sproin homalologue family. It's a family of cysteine rich peptide hormones. Um and it's what contributes to that long filament phenotype. Now I'm going to highlight this because as you may know there are citations for these other two uh genes and that's because I characterized those during my PhD or I helped characterize them. Uh the PH one however eluded me because there's only so much time and only so many resources um and so this guy kind of got left out. So I made it my posttock mission to characterizing this is my mission. Okay. So let's talk about the S- protein homalologue family um just in general just background knowledge. So like I said it's a family of cysteine rich peptide hormones and there are two members with known functions. So as you might have guessed there's ph1 I'm going to specifically refer to the copy from turnar cellulada. So tsp1 um it's from dilus turna um it's involved in filament dorphisms and how we know that is through this little mutant right here. So here we have our wild type subulada esmorph wild type Elmorph and then our little mutant. Um this mutant is an Smorph individual and you will notice it has short filaments causing its anthers to be at the same level as the stigma. Um when we looked at expression of the 3S gene, so bad yuck and PH in this individual, we found that PH was not expressed. And based off of that, we decided that's hypothetically our filament dorphism gene. This was back in like the paper was published in 2019. So you'd imagine a lot of work has gone come out since then. Um yeah the other member of the S P family with a known function comes from Poppy. So Papai um so it's this PRSS. It's the founding member of the SPH family. Um it just stands for papari stigma s protein. It's involved in self- incompatibility and it's an alilic system which makes it really confusing and difficult to talk about. Basically, an individual will have their little slocus, the poppy individual, and that s locus will contain a copy of PRSS and its receptor PRPS. Um, and you're basically wanting to mate with someone who has a different copy of both of those genes from you, right? Um so hypothetically if you mate with yourself let's say this is us our ph P RSS is secreted from our stigma and then because it's our own receptor we're going to be able our pollen tube is going to perceive our self as PH right and it's going to cause a response within the pollen tube it causes death it causes death of the pollen tube if we breed with someone else who has a different alals for these two proteins. Um, when we secrete our PRSS, our SPH from our stigma, it's not going to be perceived by the pollen tube specific receptor because it does not fit. Hypothetically, that's what this diagram is showing. Um, and that's going to allow for the pollen tube to continue elongating through the stigma into the style and eventually the ovary and then it will fertilize the egg. Uh, so that's like our crash course on self- incompatibility and poppy. It really does not matter beyond that PRSS is the founding member. And another thing that's extremely important to know is that our receptor PRPS is papaveracy specific. So PRPS does not exist outside of papaveracy and it really only exists in uh papaver and then a very closely related genus that I cannot remember off of the top of my head and I didn't write down so I failed you if you were interested in that. But that was the wrong reason to come to this talk if that was your point. Okay. So now we know about our two phes. Let's compare notes. Um so we have our Turner cellulata SP1. We know that's involved in filament elongation. And while I didn't have a slide about this, I did search the Turner cellulata genome and there are 77 family members of inulata. Hypothetically there could be more because our genome is only at the scaffold level. We're not fancy. We're not at the chromosome level. In poppy, our poppy PRSS, our founding member, it's involved in self incompatibility. It does this through program cell death and it does have a receptor that is poppy specific. The one overlap that we see between our two proteins, our two uh peptide hormones is that they're involved in reproduction because self incompatibility and filaments. Yes. So this is a diagram that summarizes everything Franklin Tong's group has found about diet styling or about self incompatibility in Poppy. And it's really just a lot and I'm not going to like go through all of it except for I kind of am. But if you want like a really detailed version, you can see this review which is where I stole this from. So basically what the poppy people know is that when we have perception of the SPH prss by its pollen tube specific receptor PRPS, we see an increase in cytoolic calcium and a decrease in pH um of the cytool and then we see increase in Ross and decrease of ATP all within the pollen tube. So this is in the pollen tube. We have this inhibition of endoccytosis. We have in um inhibition of this space which is a pyrohospitase. This protein here MPK9 it triggers program cell death and is involved in actin alterations. Um this protein here PGAP it is involved in um deacilation of anitols. They think hypothetically this might be the result of aha inactivation. There's actually like a paper this year about mitochondria and like related to this but uh that's not in the diagram so we're not going to talk about that but if you're eager to know about that exists um and they hypothesize that um deacilation of is probably critical for SI response in poppy and they think hypothetically based off of their data but they had nothing to prove this or like support this hypothesis that there might be a GPI anchored protein that's acting as a co-ceptor with PRPS. And I only bring all this up because it's going to be cool in like 45 minutes if you can stay awake. Not 45, that would be excessive, but whatever. So, we've increased what we know about PRSS. Obviously, it's inhibits endoccytosis. It increases ROSS and calcium levels. Decreases ATP, that's a typo, and cytoolic pH. Uh there might be a GPI anchor protein co-ceptor. Um yeah, so we know a lot about poppy. Don't know that much about PH. And now you might be asking what about our favorite plant ever, Rabbidopsis. Well, not much is known about arabidopsis that orphabidopsis stalliana. We know that there are 98 family members and we have the NMR structure for PH15. Um, one thing that we do know based off of this analysis in general is that the best way to determine if a peptide is an isn't actually sequenced because they are very divergent in their sequence, but rather the number of cyine residues they have and the number of beta sheets. Uh, so secondary structure is more important for determining if it's an or not. What I have found through just downloading RNA seek data and then being a nerd with my computer, you can laugh at that, okay? Is that apparently the phes they're only expressed in reproductive tissue. So this is just RNA seek data um that I've turned into a a heat map for you and I've artificially set the cap of expression level and we can see that all of our SPHs are only expressed in reproductive tissue. um we don't see any expression in vegetative tissue. And if we look specifically at um just like I've summarized it here for you, we can see again that we have very high expression of these in the sphes and the pollen. If I artificially cap it again, we see that we do actually have expression in the um infllorescent apical merist stem and then also seed development. We look at the spec like the specific family members in only reproductive tissue, we can see that we actually do have expression in a bunch of different reproductive tissue. Um yeah so overall this kind of suggests that in arabidopsis uh we're also seeing that the phes are important for reproduction. We don't know how. This is just based off of RNA seek data. Uh for the arabidopsis phes like I said we have that structure for PH15 and there are 98 members based off of the number of members in arabidopsis the number of members in turnaround and then I did a very large fogyny of the family back in like during my PhD sometime so I don't know whenever that was. Um this is true across a bunch of different genres. So the SP family is massive. That's all. That's all. So now let's actually talk about cool experiments I've done and not just things I've downloaded or papers I've read or Yeah. Um so the question is for me at least because I'm interested in destiny filament elongation. And so the concept that I came here with was to do a multi-omic analysis of filament development. Um and when I came here I was very naive in the world of biochemistry and I did not realize how hard biochemistry was. Mike so >> yeah and I still don't like a rapidopsis but I'll use it. Okay. Yeah. Um so the concept here is that I'd take the filaments from the smore um do some RNA seek and then we'd make a co-expression network using the RNA seek. Uh one thing to consider is that really this lab is the only lab that's doing anything right now with turner um a molecular level and so there really aren't that many resources available for turner outside of what I've done. Yeah, what I've done. I'm going to take I'm going to take the claim for that. So um yeah we needed to generate some RNA seek because RNA seek just did not exist for the filaments only the stamon um and then we decided it's time to enter the world of proteomics with dile um and turner. So we did some protein isolation. The idea here is that once we had these isolated uh protein, we'd do a phosphoenrichment so that we could look at phosphorolated peptides and differences in phosphorilation between the S and the Lmor. And then we could take our non-phosphorilated peptides and just look at differences in protein abundance. And this is under the hypothesis, if we remember, SPH1 is hemisygous and Smor specific. So only the Smorph has a genomic copy of SPH1. the Elmorph does not. Um, which is beautiful because we don't have to make knockouts or anything. We can just hypothesize that there are going to be differences due to SPH1 because it just only exists in one genotype, right? Yeah. Yeah. Okay. So then once we have all these differences and this co-expression network, we could combine them to create a final network. We can look within that final network to find clusters that contain SP1 and then we could make a model for filament elongation. And so here I'm just showing you this uh so that we have a picture to look at while I give you numbers. So I found a cluster that had SP1. Uh it consisted of 165 different genes. um of the genes that were expressed so the eight different RNAs that we found um 67% of them had peptides and then 59% of them had phosphorolated peptides which are denoted from by the uh circles versus like squares and yellow versus blue. When we look at go terms because everyone loves go terms. Uh we found that reproductive structure development was one of our top terms. Binding was a top term and membrane bound organels was a top term which is exciting because again we're looking at filaments in the flower. So I was hyped. I'm sure Mike was hyped too even though he hates go terms. >> Okay. >> So next what I did is Sorry. So, I'm so sorry. Okay. So, next what I did is I took those uh proteins that were had phosphorilation events that were specific to one morph or occurred more in one morph than the other. And um I compared it to a database of arabidopsis phosphorilation events. So it shows every single site on a protein that has been previously documented as capable of being phosphorolated. Uh I don't remember the name of that database, but you can read about it here. Um or I guess email me. Um um so I did that and I came up with this list of proteins that have previously identified phosphorilation sites. Um, specifically these five are the ones that have the previously identified phosphorilation sites. We see that this one is interesting because it's only phosphorolated in the Elmorph. So the morph that lacks SPH. Um, the rest of them are phosphorolated only in the Smorf or there are some phosphocosytes that only that occur at higher rates than um in the Elmorph. So if we look at the actual roles of these proteins based off of their arabidopsis homalogue. So I assigned all turner proteins, rabidopsis homalologues so that we could have some idea of what a function might be. Um we see that these three proteins are involved in RNA processing which is pretty cool. Uh we see that this AT5 PTA 12 um is a type 2 anitol polyphosphate five phosphatase um which is cool because that means it's involved in phosphonesides. Whoops. Oh, and then I forgot that I didn't put description here because this is just an ethylene uh protein. So it's just a protein that's involved in ethylene response. Um but that was pretty exciting because if you remember the Poppy people were talking about um phosphonesides having a role potentially in self-inccompatibility response based off of the role of uh that one protein. So that suggests that pis likely are common between uh ts1 and prss signaling. What I also found interesting in this analysis was the presence of this ATM1. Um so this is a kynise that promotes fertility, meiosis, mitosis. Um and it also prevents double stranded breaks from occurring during crossing over during the enduplication cycle. The CDK G2 also interesting because it promotes endoruplication and growth um cell growth expansion. So the endoruplication cycle is just uh when a cell is going through mitosis, it may prematurely stop after the genome has duplicated um and it enters into the enduplication cycle which just allows the cell to m become much larger than it normally would doubles in size. Uh plants use this cycle as a way to rapidly grow tissue like filaments hypothetically. Um so this then brought up the hypothesis is tsspsph1 regulating the indoor reduplication cycle. So as you might remember I assigned arabidopsis homalologues to all of my turnaround proteins and that allows me to take advantage of tools like string which um mine pubmed for experiments um and like actual support for interactions between proteins. interactions is used loosely because like they might not actually be interacting. It might be that this is a homalogue of this protein and this is a homalogue of this protein and they do something together. Um but anyways it allows me to draw more conclusions from my data set than I might be able to if I only had the subulada names. Um, and so by throwing all of my proteins into a string, I was able to come up with this little network and then a model for hypothetically how um, filaments are elongating in the L versus the Smorph. Um, and so the hypothesis here, we'll go through it on the Lmor. Yellow means that the protein was found in both morphs but not differentially expressed, so same level of abundance. white means that it was only found in the RNA seek data. We didn't have peptides in our uh massback data set. Um and then this purple means that the protein was only found in the Smorph individual. Our dark green means that the phosphocite only existed in the Smorf. And then our light green means that existed at a higher level in the Smorph relative to the L. Okay, now that I've taught you about colors, let's go through this diagram. So because ATM is not phosphorolated in the Elmorph, we could probably assume that it's inactive and if it's inactive, it's SOG one is likely inactive because SOG one is activated by ATM1. If this is the case, then that means the CDKB1 is active um which forms a complex with this psych A34 um psych A34. It might be active as is phosphorolated in both data sets. This complex together actually phosphorolates CDKG2 at a site that causes its degradation. So it gets marked for degradation. Hypothetically since CD CDKG2 is not present in the Elmor but it is in the Smor uh we might hypothesize that this is because it's been degraded in the Elmorph due to the presence of this complex. Um if this is the case then we would s like endo reduplication wouldn't be happening and we would not be elongating our filaments uh resulting in our short filament phenotype. Alternatively we do see phosphorilation of ATM1 specifically in the esmor. Um ATM1 it activates the SOG one. SOG one is a negative regulator of CDKB11. Um that would prevent CDKB1 from forming that complex with psych um and it would prevent degradation of CDKG2 by that complex. CDKG2 actually interacts with this psych LA um and is phosphorolated asset that was not related to degradation. um in the Smorph only we see higher phosphorilation of the psych LA in the Smorf relative to the Lmorph. Uh that might suggest that this complex is actually active. Together this act complex um activates endo reduplication um resulting in filament elongation and then resulting in our long filament phenotype. Hypothetically, this is all bioinformatics and so we can't actually say if this is real or not and needs empirical support, but it at least gives us a starting point. See if this hypothesis had any merit. I uh measured a bunch of things. I'm going to zoom in. I'm using fancy PowerPoint. Um and it really made the uh quality of the picture go down, but I'm sure you can understand what's going on. Um here we have the length of uh cells in the esmor filaments, mature esmor filaments. So this is after the flower has opened and we have the cells of the morphs filaments right here. And we can see that the esmorph filaments are significantly longer than that of the elmor. Imagine there's a star there. There isn't because I um am neglectful. So this might support that hypothesis that endo reduplication promotes rapid growth of the filaments of the esmore. Um that might not be so surprising because if you see we do have very rapid growth of the filaments towards that late stage. So we can see that up to a certain point our filaments are basically the same size between the S and the Lmor. Uh and then right before opening we have this rapid growth of the filaments. um basically over like a night. So I don't know if a night is actually that rapid, but compared to pistol elongation, it certainly seems much more rapid. Okay, so back to our little circle diagram, summary diagram. Uh we can now throw indoor reduplication up there. So hypothetically, SP1 may be promoting filament elongation through endoruplication. So does that mean that both of these uh proteins are involved in stopping mitosis from occurring? Um as you might think like programmed cell death that can be a result of premature stopping of mitosis and telling the cell that the cell needs to die and reduplication premature stopping of mitosis telling us that the cell needs to get big. Okay, so that was cool. Paige, you did a little proteomics. But what about our receptor? What about the meat of this talk and why we all came here today? because I'm sure this is the riveting part. Okay, so like I said, there are 77 members of PH internal cellulada and there's ph1. What's cool is that ph1 evolved from a scaffold that contains at the very least five different phes. It looks like there might be two more, but they've been truncated either artificially due to the fact that this is a scaffold genome or actually just they're just actually truncated. So what we did is we actually uh made recominant protein for a bunch of different PH family members from Turner. So we have SP1 from Turner Seabulada. Um SP1 from Turner Joeli. Joeli is another D stylist member of Turner. Um we have tsp2, four, three, and five. So those are all um part of that area of the genome that SP1 duplicated from. Um we have SP 25 and 29 that we synthesize and this is because they're differentially expressed in the anther, which is cool. Um SP7 is actually expressed in the filament as well. Um which is why we decided to synthesize that. And then we have SP 20 and 21 which are the closest homalologes to a rabidopsis 15. So we had a bunch of different recombinant SPHs. And this was when Mike was like you got to start working with a rabidopsis. And I was like but why? Okay. And then I did. I just did because sometimes you just do what your boss says. You don't question. Okay. So that's what I did. I have my recominant phes. Uh so I did some root assays and so that's what I did. Basically what I did is I took uh rabidopsis seedlings. I threw them in liquid media either control uh containing a control which was just buffer or the ph within its buffer and we found that a bunch of phes actually reduce uh root elongation in arabidopsis seedlings. Uh we also did this with arabidopsis phes. So, Arabidopsis SP22, 23, and 90. And we saw a similar thing where some SPHs are actually reducing root elongation. Um, some do not, and some kind of have an intermediate phenotype. At this point, I decided to hone in on SP4. SP4 had a very severe severe phenotype. Um, and it's closely related to SP1, which was my protein of interest. Unfortunately, at this point, we did not make enough PH1 for me to really do anything with it. So, I had to just say RP to SP1 and do my job. Okay. And so before we get like wild and like do a bunch of different things with the PH, I needed to make sure that it was actually a result of the PH itself and not the recominant protein because this PH was attached to an FC tag. Um, so here we just see that there is a dose dependent effect with SPH4. Here we see that the tag does not actually reduce root elongation. It's just PH4 that's getting the job done. And here we're just showing that structure is important for PH function. So I digested PH4 and then I threw it in the um with some roots and we saw nothing happened. Um, next I was like looking at actual vegetative tissue would be cool. So I set up these massive plots and we're just going to zoom in on something that I submitted and it is low resolution. So I'm sorry. Um, okay. So I set up these plots and I treated each plant with PH. So I literally took my pipette and I just pipetted PH onto each plant because I'm a plant biologist. Yay. I don't know. But that seemed like the best way to go about this without using a ton of PH. Um, and we see 5 days after I treated these plants with PH, we don't really see that much of a difference. But once these plants got out to 33 days, we actually saw that the wild type plants or the control plants, sorry, the control plants continued to grow and they look really healthy. And our SP treated plants really, it's just like this one guy survived. the other ones, they're just dead. They're gone. Um, don't know if it's because they got like they drowned and like fell under dirt or I'm a bad plant biologist. I don't know. Um, but that was cool because it suggests that SPH4 kind of represses vegetative growth, not only of the root, but also of the chute. Um, yeah, no one brought up the patent and I thought at this point I'd be like this is the basis of the patent, but I guess like Mike failed me as a boss today. Okay. Um, cool. So now that we have this like data that showed that FPH can actually do something4 specifically. Um I did some coip with arabidopsis seedlings and that like I actually did it three times because I'm neurotic. So I had N of one here and then both of these had N of three. So yeah, I then took these three data sets and I filtered all of the like proteins that pulled down with SP4 from arabidopsis. I removed anything that I considered a housekeeping gene. So like ribosome related things gone out of there. Um and then I came up with a filtered list of candidada interactors. Once I had this filtered list of candidate interactors, I went to the Arabidopsis Biological Resource Center. That's a typo. Um because I'm lazy and I'm not going to make a bunch of knockouts myself when someone's done the work for me. Um, and so I ordered a bunch of seeds from Ohio State's Arabidopsis Biological Resource Center and I treated them with SPH4. Um, yeah, and I censored things because it's the basis of this patent that I submitted to Warp and I knew that this was going to be online and I didn't want Warpf to come murder me. That's a joke. Warf, please laugh, guys. We're being held hostage. Okay, anyways. Um, God, I'm sorry. Okay. Uh, so what was cool is you'll notice that we have these censored proteins that I've called AP1, AP2, AP4. Um, they all belong to the same family, the P family. Um, two of them were insensitive to SP treatment. So that likely suggests that they are somehow involved in PH signaling. either they are directly interacting which would be the strongest hypothesis considering these were identified through coip right or they're interacting somewhere downstream. This family is cool because it's involved in mitosis. It's involved in transport and it binds uh phosphones anestides sorry what was also cool is that we see this protein AS5 is also insensitive to PH treatment. Um, SP5 is cool or S5 is cool because it's a GPI anchor protein. Um, which you might remember the Poppy people think there's a GPI anchor protein involved in SPH signaling just to make sure that this was not just like some fluke from the line that I had. I ordered additional knockout lines and we continue to see insensitive phenotypes. Um, so like let's go back and summarize. So we've shown that PIPs are important hypothetically through our Frankenstein arabidopsis turner system um for SPH signaling. We've shown that mitosis hypothetically is important since the P family is involved in mitosis and we've shown that there is actually likely a GPIA GPI ink protein that is important for SP uh signaling. So it's likely actually the co-ceptor hypothetically. Um we also showed that um SPHs are able to suppress vegetative growth. So if we look at that P family, yeah, okay. Uh this is just an out group. It's not actually a member of this specific subf family, but is part of the super family. Um so it's just there for that reason. We see that P1 is actually highly expressed throughout the plant um compared to the other members. So potentially it's not that surprising that P1 was screaming at us in our data set. Um what's cool is P4 that protein that was interacting with SPH um well that came down in the co but didn't show it in sensitive phenotype is actually highly expressed in reproductive tissue. So perhaps we're not seeing a vegetative phenotype solely because uh P4 plays a bigger role in the flower, specifically the pollen tube, than um the vegetative tissue. Who knows? Um I do know that based off of other research, uh P4 is redundant with other members of the P family. And so hypothetically it just might be that we have to have uh more knockouts like uh the actual P4 P5 P3 knockout that is used in other studies. Okay. So we did our knockout root say that was cool and we found this candidate interactor P. So then we made re combinant P using CHO cells. All of this has been in CHO cells so far. So then we decided we need to show that there's actual binding between these two proteins um hypothetically using uh well yeah this is the arabidopsis. Okay so the first thing I did is I made a really ugly heat map for you. Um so I took the recombinant SP4 and recombinant SP1 2 and three. Um, I threw them in a tube with either recominant P1 or recominant P2 and I did a co-IP assay um to see if uh P1 would pull down more like p would actually pull down with these different phes and if P2 would pull down with these different phes. SP4 was set as the um standard. So it equals one. Everything is ratioed to it. Um, based off of this, you can see that SP1 surprisingly interacts much stronger, hypothetically, with P1 than the other SPHs, even SP4. Um, SP2 didn't really do anything at all. SP3, it does pull down with P1, but to a much lower extent than four. Um, yeah, what was cool is that with P2, um, we actually don't see any of the PH is pulling down with it. um which may suggest that P1 is more degenerate than P2 and it's um who it's willing to perceive. I guess we in a collaboration with uh Arizona State but he moved to somewhere in Texas. Michael Marty Texas now Michael Marty. >> Yeah. In collaboration with Michael Marty but in Texas uh we did some non-denaturing um mass spec. So again, we had our AP1. Uh we went with P1 because we were able to make a large amount of P1 in show cells. The other P's um we did not we weren't able to make like a ton of it. Um yeah, so this this is why we were fixating just on resources alone. Okay, done talking about why we're fixating. Okay, so one thing to note with the P family is at the very beginning of the P family, they have a disordered region. Um, and so it makes all of our graphs pretty messy like this. Um, so yeah, you'll just have to accept this, I guess. Um, I accepted it, but I'm not a Yeah. Okay. So, we see that when we combine P1 with PH, we actually end up getting a uh peak that we don't see in our PH sample and we don't really see it in our uh P1 sample alone. So, that kind of suggests that they are interacting. Um but that's really the weakest of our data I would say because of this like how messy it is. Um and then Ben from our lab um Minkoff that's his last name. Um he did some coalent labeling for me. Um so basically what he did is again he took the two proteins he put them in a tube and he did some coalent labeling mass spec. Um so this is when you are able to identify regions of the protein that are um accessible to the soluble uh solution there. Um and we found that when we add PH2 P1 we have changes in our coalent labeling which is pretty cool because they're occurring in that disordered region. This is a structure that generated using alpha fold. Um and we can see that it's in that disordered region. So that was cool. We have a bunch of in vitro essays. Um, but what would be cooler is if we did the same thing but now in turn sebulada. Um, and so Ben, he also helped me with this. Uh, we had some Turner stamon lysate. So we did whole stamon because it takes a lot of work to get lysate from filaments alone because they're so tiny and you need so much and I can only have so many bushes growing in a growth chamber. So we went with whole stammon because you have a lot more tissue to work with and it's a lot easier to get the amount of protein needed for coalent labeling. Um so yeah we did some coalent labeling um and we did some phosphoproteomics at the same time and I'm going to just show you this fogyny real fast of the turner p family and the arabidopsis p family so that you can understand what's cool and significant about this. So as you might remember P4 is highly expressed in reproductive tissue in arabidopsis and turner we actually have three P4 homalologues um which suggests that maybe that like re uh it expanded in turner right and potentially maybe that's because it's important for dying maybe not who knows um yeah I only bring this up because TP242 we found uh significant differences in coalent labeling as well specifically in that disordered region. So we can see again here's an alpha fold predicted structure of uh P42 and turner and we see labeling in that disordered region. So that was pretty cool but then it was like what if we actually do something in vivo and not just in vitro. Um so then we took turner sebulada intact stamon and we had them in solution and we did some covealent labeling again and what was cool is we found labeling in that same region again. We actually found much more labeling. This is still like all statistically significant. I'm only showing you the statistically significant changes. Um so yeah that was exciting because it actually suggests that yes in its native system PH4 is actually interacting with a member of the P family and this P P homologue P42 might be uh important to reproduction just like how the arabidopsis uh for is only expressed really in reproductive tissue. Yeah. So that was cool. Um, my final bit of data, it comes from an RNA seek data set from my PhD that nobody did anything with because I was too naive with computers back then. Um, and so I did nothing with it. So, as you might remember, we have this short homicile mutant which has short filaments. Um, and we had RNA seek data for the short homicile that was not published. I also had RNA seek data for the statement of the Lmorf and the statement of the Smorf. This was all stamon data. Um but what was cool is I did a co-expression analysis and what we found is that PSP1 the dileph and um P2 P2.2 two, they're both um the expression of both of these proteins positively correlate with wild type esmor, which potentially means or at least supports the potential interaction of PH with these um this protein family, this P protein family. I want to say the name so bad, it's just hard. Okay. Um yeah, and like filament elongation. So like an actual roll and dice sty hypothetically that we'd actually have to have like transgenics to like prove this. Um so just to highlight the cool things that I saw or like this data suggests suggest that we found that uh GPI anchor protein that the poppy people suggest might exist and we did ex uh support their hypothesis that pips are important for um hypothetically PH uh signaling somehow. So yeah, through this data we supported a potential role of phosphones and PH signaling. We identified a potential co-ceptor that AS5 um which is a GPI AP anchor protein that is probably required for SPH signaling. And then we identified a strong candidate receptor but we require additional stronger support. And yeah, I've been out of the lab for like three months because I had a beautiful baby like Mike showed you. Uh yeah. Okay. And I like to end with this quote. Most people don't know that Charles Darwin was a botnist in his later years and his favorite thing that he studied was Dyl Primrose. Um and you don't have to take my word because he has this quote in his autobiography. And that's cool because Charles Darwin, he made a lot of comments about how he hated himself. I hate myself. Charles Darwin, he loved Dylat, very cool. Um, and with that, I'll stop torturing you with my bad jokes and I'll take any questions. Y >> um >> the flowers now questions. Oh, one thing do want to add the um PH. >> Yeah, >> the SPH uh and the P protein expression in uh CHO cells was done for us by uh Fritz Shamberg and um >> Oh, yep. uh David uh Rancor uh PhD student from Rich's lab and master's lab uh as part of their company Litic Solutions. I'm very thankful for that. Um okay, so now uh any questions? >> Oh, good. >> Wow. Uh that was close. I'll start here. What do you think is the >> What do you think is the the key piece of evidence you need to lock in your receptor? >> Think I have to believe it myself. I that's I think that's my strongest point as a scientist is that I refuse to believe anything because I refuse to think I'm competent enough to get something right. Um, so I think there's a cool data set that Ben generated that I haven't gotten to go through. And Ben's been doing some more experiments while I've been out of the lab. And potentially those experiments will show me that I can be confident in this as an actual thing. I think that really if to prove it to myself, I would need transgenic lines of either where I'm probably like Turner. I' I've wanted turner but it takes two years to generate transgenic turnaround. Uh from experience someone wants to like optimize that for me that'd be cool but um yeah so I think transgenics. >> Okay. Am I allowed to add to it? >> Yeah. What would prove it to you Mike? >> Mike has the confidence. I I don't. >> So I No, I think you said it perfectly well. the uh um mass spec, the non- denaturing mass spec. Uh that was very noisy purple line as you probably saw. Uh we need to repeat that. Uh we've also got some nice SEC data uh which is um also known to naturing interactions. We're also doing some KM KDs. So I think once we get a better idea of the complex uh we have pretty good idea but like I said that that needs to be short up. We'd like to do cryogam on it. Fortunately, it's just under the uh it's about 140 KD. So, um I don't know. Tim tell Tim tells me it's too small. What do you say? >> Huh? >> Thank you. Well, then we're going to be over to your lab tomorrow. >> Rick, >> your alpha predicted a lot of unstructured region in a couple of domains. Okay. So, one of them was the E32 binding domain. >> Yeah. >> And so, you got the other one. Have you mapped to see where this peptide is binding? And also, I did say how long? >> Oh, it's approximately 140 amino acids, but then you have to subtract like I think it's 17 for the tag itself. >> 17. >> Uh, that's the tag itself. the the peptide itself is >> SP is 17. The receptor is uh 60 or 70. What you see here is 554 amino acid long. >> Yes. >> Um um so Rick, you're asking um this is the phosphonosetide binding region that's known. This is the unstructured region that was predicted to have a liant, but they didn't know what it was. We believe now we know what that liant is ph. >> Is that in your alpha fold at all? >> In the alpha fold. Yeah, >> either like these. >> This is the unstructured. >> This is the phosphonetide stuff. >> This you can see it's up here too. >> That's all this any Oh, do you need this? So, oh yeah, actually I'm sorry I should have done this >> really cool talk. Um, so you're studying a a peptide hormone that's primarily involved in um flower morphology in turn. Um why do you think it's uh causing those uh growth defects and arabidopsis in the root and vegetative tissue? Well, somehow you have to stop vegetative tissue from growing so you can start reproducing, right? And so, hypothetically, there's always the possibility that it's involved in stopping vegetative growth so that we can begin actual reproductive growth, right? We only know the function of two of the family members and the poppy one clearly is an exceptional case of neofunctionalization given that its receptor is papa specific. Um, functionalization. >> Yeah. matrix. >> Oh god. Turns out >> so yeah why only has a sequence this sequence is not in any other >> yeah unfortunately >> really amazing and it's probably calcium it's a very small trans uh did I answer the question sufficiently Okay, >> cool. >> Oh, any other questions? Well, again, let's thank Paige.