Senior Scientist, Dewpoint
|Type||Kitchen Table Talk|
On November 10, the condensates community and Dewpoint scientists gathered around the Dewpoint kitchen table to hear from Lucia Strader, an expert in plant biology from Duke University. Lucia has been studying Arabidopsis biology since her PhD at Washington State University and her postdoc in the Bartel lab at Rice University. After opening her own lab in 2011 at Washington University in St. Louis, she stumbled upon condensates in plants that were doing some very interesting biology. And like many other condensate scientists out there, Lucia couldn’t resist joining the effort to understand condensates!
Now Lucia is at Duke University and her lab continues to produce deeply impressive work at the intersection of environmentally responsive condensates and plant biology. In her talk, she showed us the fascinating involvement of condensates in plant development. Enjoy her video below as part of our Kitchen Table Talk Series.
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Christiane Iserman (00:00):
So I’m very excited to introduce Lucia Strader. Lucia got her PhD at Washington State University, followed by a Postdoc in the Bartel Lab at Rice University. And most of her training was focused on Arabidopsis biology, which tells you how dedicated she is to plant biology. Lucia started her professor in 2011 at Washington University in St. Louis, but recently transferred to Duke University. And I’ve been deeply impressed by her lab’s work, which is very wide and not just focused on condensation.
Christiane Iserman (00:31):
There are many reasons why Lucia’s work lays close to my personal heart and just is very cool. My own personal background is in environmental responsive phase separation, which has taught me how important this process is for sessile organisms such as plants. Just imagine phase separation is essential for so many processes, but is highly environment dependent. So plants need to have this process optimized throughout the day and year. And Lucia’s lab looks into exactly this. How do plants control their development when the environment keeps changing?
Christiane Iserman (01:02):
And the Strader lab has uncovered condensation of the auxin response factors, which are transcription factors. They’re also called ARFs. And these ARFs can form condensates in the cytoplasm of cells which drives the transcription landscape and plant development, but she’s going to talk all about this in her talk today. So Lucia, the floor is yours.
Lucia Strader (01:20):
Thank you so much, Chrissy. I’m really delighted to be here and as I was telling Jill and Chrissy earlier, I’m actually pretty jealous of all of the fantastic scientists that Dewpoint has recruited to their company because I feel like you guys probably have these amazing conversations all the time that I would love to be a fly on the wall and just listen to so it can inform my own work.
Lucia Strader (01:44):
So as Chrissy mentioned, I’m a plant biologist and my lab is really interested in this one particular plant hormone called auxin. Auxin was first postulated to exist in this really marvelous book called The Power of Movement in Plants, written by Charles and Francis Darwin in the late 1800s. And in this book, they describe a phenomenon that I’m sure most of you have seen in your own gardens, which is if you expose a seedling to directional light, it’ll bend to grow towards that light, as you can see here. However, if you remove the tip of the seedling or if you put a little hat on top of the seedling, this bending response no longer occurs. So clearly, the tip of the seedling is necessary for this directional growth. However, this growth is being driven by asymmetric cell expansion at a site distal from the tip. So in this book, the Darwins postulated that there must be a mobile signal involved and indeed, there is. It is a plant hormone auxin or indole 3-acetic acid…
Lucia Strader (02:45):
So my lab jokes that IAA does not actually stand for indole 3-acetic acid, but rather for involved in almost anything because it’s become apparent over the past century of work that every cue that you can imagine that results in a growth output, auxin is involved. And auxin somehow interprets each of these cues into the correct developmental output, right? So whether it’s a temperature response or a change in light or a developmental program, auxin is involved to drive what happens downstream of that.
Lucia Strader (03:19):
My lab mostly uses the model plant Arabidopsis thaliana to understand how auxin works. And you can imagine over here is an Arabidopsis seed and this is the first 10 days of Arabidopsis growth. So who amongst you the in the audience has ever eaten a peanut? Right? So a peanut is a seed, right? And so you can imagine an Arabidopsis seed is a miniature peanut. The two halves of that peanut will become, they’re the cotyledon, so they’re these seedling leaves. And then that little nubbin inside the peanut, that is actually the embryonic axis. So one end of that nubbin will give rise to all of the above ground parts of that plant and the other end of that little nubbin in the peanut will give rise to all of the below ground parts of the plant.
Lucia Strader (04:11):
Because plants don’t have morphogenic cell movement, all of this development has to be driven by really carefully regulated cell division and cell expansion events. Because once you divide a cell and make a new cell, you can’t really go back from that decision the way you can do so more easily in other systems. And auxin regulates both cell division and cell expansion, so it’s really critical for every aspect of plant morphogenesis.
Lucia Strader (04:38):
In fact, there’s a recurring theme in auxin biology that there are particular places in the plant, such as down here at the root meristem, that’s here. Or here where you have this nearly emerging lateral root, where there are high levels of auxin transcriptional activity, as shown by this blue staining in this reporter line that are really critical for driving these developmental events.
Lucia Strader (05:01):
Okay, so how does this auxin transcriptional activity get regulated? When I started my lab in 2011, what we understood of the process was this. There is this family of transcription factors called the auxin response factors, or ARFs, and their activity is inhibited by interactions with these Aux/IAA repressor proteins.
Lucia Strader (05:22):
In the presence of auxin, this repressor protein forms a co-receptor for the hormone, along with this TIR1 component of this SCF E3 ubiquitin ligase complex. This auxin driven association allows for the polyubiquitylation of the repressor targeting it for degradation via the 26S proteosome and relieving repression on this transcription factor so you can have output occur.
Lucia Strader (05:49):
When I started my lab in 2011, we were looking at this pathway, and we really thought this seemed too simple to explain all the complexities that auxin has to drive, right? There had to be missing layers of regulation to this system. We decided to interrogate this as a first pass by trying to understand how this interaction between these transcription factors and repressors worked, because although I’m showing this as a really simple type of interaction, what we know is there’s 29 members of this repressor family in Arabidopsis and there are 23 members of this transcription factor family in Arabidopsis, and they don’t all interact equally. And so we first wanted to understand the molecular basis of that.
Lucia Strader (06:29):
To do so, we took a structural biology approach. We solved the structure of this interaction domain from ARF7, one of these transcription factors in this family and what we found is that this folds into a single globular domain called a PB1 domain. And here I’m showing you one chain with all the negatively charged surface residues in red and all the positively charged surface residues in blue. Amongst all of these transcription factor and repressor family members, there’s a conserved lysine, which I’m showing in this interaction chain. And this is the same pair of chains except now I’m showing you conserved aspartic acids on this acidic face interacting with this basic patch.
Lucia Strader (07:10):
For decades, people had drawn these interactions as these pairwise interactions, but what our data suggested was that these are actually interacting in this head to tail fashion, with these miniature bar magnets, with the plus face interacting with the minus face. And because these are miniature bar magnets, for every interaction that occurred, there are two additional interfaces for additional interactions that could occur. So raising the possibility that rather than simple dimers, these transcriptional proteins acted as oligomers or at least had the potential to do so.
Lucia Strader (07:44):
In fact when we express this protein heterologously and purify it, it behaves as a large soluble oligomer in solution. Whereas if we mutate either that conserved lysine or those conserved aspartic acids so that the domain either has a negatively charged face or a positively charged face, these proteins behave as pure monomers in solution because there’s no bar magnet available for it to interact with. This is really important because it means that we can make really simple point mutations in these electrostatic residues on either side of this domain and completely control how these proteins can interact with themselves and with one another.
Lucia Strader (08:24):
So this is all very good. We identified the 3D structure of these interaction domains. We found that they have the potential to oligomerize in solution, but ultimately I don’t really care about that if it’s not important to the biology of the plant. I’m trying to understand how this system works in the plant. So then the question became, do these proteins oligomerize in the plant, and if so, does that drive any aspect of the biology.
Lucia Strader (08:56):
To address this, I recruited a new graduate student to my lab, Samantha Powers, and I asked her to fluorescently label some of the transcription factors in this family and then we can use that to start to interrogate whether they multimerize. What she showed me were these images, so I’m going to orient everyone to the fact that these are Arabidopsis root cells. These are long cells, about 100 microns in length and about 20 microns across. These are not unusual in any way, they have a single nucleus within the cell. And what we saw was this transcription factor was all in these cytoplasmic puncta. We thought that perhaps she had done something wrong. I had her PCR amplify these constructs back out of the plant. We had put these into the mutant backgrounds, they rescued mutant phenotypes, which told us that the localization of these proteins were where they were functioning or they wouldn’t have rescued the mutant phenotypes.
Lucia Strader (09:53):
So it was very confusing to us until we realized that Sam was taking images from this part of the root, right? So Arabidopsis roots and plant roots in general are marvelous developmental models because this is a continuously growing organ from that one nubbin side of the peanut and down here at the root tip is all meristematic tissue and this is where all of the active cell division and cell expansion is happening, and each cell is cemented in place. So along the longitudinal axis of a root, every cell is held within the cell wall, like this, and they’re exactly six hours different in age from the cell above them and the cell below them. So you get a full developmental trajectory at a single time point in a single individual.
Lucia Strader (10:40):
When we look in the same individual down here where all the active growth is occurring, these transcription factors are predominately localized to the nuclei. Indeed, when we looked at the progression at intermediate steps, we go from a place where we see predominately nuclear localization of these ARF transcription factors to a place where we start to see appearance of these cytoplasmic puncta. If we go a little bit further along development, we see mostly puncta, but we can still see some nuclei here. And then we get to a place up here where actin growth has ceased, where we can no longer detect nuclear ARF protein.
Lucia Strader (11:19):
This process is really dynamic. These protein bodies are being formed within the nucleus itself and then they get expelled out of the nucleus relatively rapidly, into the cytoplasm. So these protein bodies are not being formed in the cytoplasm itself, as far as we can tell.
Lucia Strader (11:39):
The formation of these puncta reminded us of our initial question, which was, does PB1 domain multimerization affect protein behavior within the plant? And so we wondered if perhaps that was necessary for formation of these cytoplasm bodies. Indeed, when we mutated the single conserved lysine in the PB1 domain, so this protein is over 1,000 amino acids in length. That one conserved lysine–the same one that we had used in our in vitro studies–in those same cell types, we no longer see formation of these cytoplasmic puncta. Instead we see purely nuclear signal.
Lucia Strader (12:18):
Clearly PB1 domain oligomerization is necessary for formation of these protein bodies. However, when we tag just the PB1 domain itself, we no longer see formation of these protein bodies, but when we add back in to our construct the long, intrinsically disordered region of the ARF protein, we once again see formation of these. So since I’m talking to the condensates.com group, it’s obvious that these protein bodies are consistent with the definition for a biomolecular condensate. These are non-membrane bound concentrations of protein that are somehow relying on both the combination of this oligomerization domain and this intrinsically disordered region for their formation.
Lucia Strader (13:02):
And when we look closely at individual condensates, we can use an approach called number and brightness analysis, which looks at the correlated diffusion of proteins in and out of pixels to understand oligomeric stage of proteins within this condensate. When we did this, what we found is that in an individual protein condensate, which I’m showing you here, in the very center of it are extremely high order oligomers. So these are 10-mers to 20-mers, to even things that were very difficult to tell just how high we were getting. As we move from the center and go towards the periphery, the oligomeric state of these proteins starts to go down so that at the very periphery of this protein body, we see only monomeric and dimeric protein.
Lucia Strader (13:53):
When we do FRAP assays on these condensates, we also see that in the protein bodies where we had these higher order oligomers in the center, we see very little diffusion of protein within the center and relatively rapid diffusion of protein along the periphery. And so that’s consistent with the oligomeric states that we’re finding. When we look in the nuclei, we don’t see proteins of these higher order oligomeric states. All of the protein is either monomeric or dimeric in the nuclei themselves.
Lucia Strader (14:29):
But then we’re left with the question of how can the combination of an intrinsically disordered region and this oligomerization domain facilitate formation of these protein condensates? And when when we looked closely at the sequence of our middle region, or this intrinsically disordered region of our transcription factor, what we find is that there’s a prion-like domain that is highly enriched for glutamines as you can see here. So this is consistent with other IDRs that prefer to self-interact rather than interact in a solution. And when we look at younger condensates that have not yet gone through an aging process that we see in the plant root, these exhibit very much liquid-like behavior where if they fuse, they form into a single body that has a minimized surface area.
Lucia Strader (15:20):
Right now, the model that we’re working under is that ARF condensation is being driven by PB1 domain associations, which are driving up the local concentration of the protein so that it can overcome the biophysical threshold necessary for phase separation to occur. Since there’s a lot of experts in the room, I will say that if we highly overexpress just this IDR by itself in the plant, like, way outside of what’s physiologically relevant, we can also see condensation happening, but we need to have a very high level of protein driven behind a very strong promoter to get that to occur. [It would again be 00:16:01] consistent with our model that the role of the PB1 domain association is likely to concentrate this protein. Obviously, also adding another site for multivalency to occur along this protein as well.
Lucia Strader (16:13):
When we look at the 23 members of the auxin response factors in plants and then you analyze intrinsically disordered regions of each of these, really only some of them are predicted to exhibit this behavior. These exist in three ancient clades that have existed for at least 500 million years and it’s been known for a long time that all of the proteins in this clade, of which there are five in Arabidopsis, all have transcriptional activator activity. We also find, when we examine them in my lab, that they all undergo condensation. These are also involved in recruitment of the SWI/SNF chromatin remodelers that is taking place somewhere in this intrinsically disordered region.
Lucia Strader (17:05):
Conversely, proteins that are present in the second ancient clade, the Class B, tend to have very serine-rich middle regions and they’re known to be involved in transcription repressor of target genes. Both Class A and Class B ARFs have nearly identical DNA binding domains and PB1 domains. They can compete for the same binding sites and it’s really, the transcriptional activity has been encoded in this IDR for each of these different clades of ARF proteins.
Lucia Strader (17:35):
When we look at the protein localization of proteins in that repressor class of ARFs and we look across the same developmental trajectory that we’ve examined for ARF7 and ARF19, we see that these proteins are nuclear in every cell type that we examined. We don’t see these cytoplasmic condensates forming in any of these family members that we’ve looked at.
Lucia Strader (17:59):
In fact, we can take the middle region of ARF19, which is an activator ARF and replace it with the IDR of ARF2, which is one of these repressor ARFs, and we no longer see formation of cytoplasmic condensates. This was done in protoplast cells so you can isolate individual cells out of a plant and transiently transfect them with a construct of interest. However, if you look closely, we do see condensate formation it’s just those are retained in the nucleus and they’re much smaller than what we typically see with the wild-type ARF19 protein.
Lucia Strader (18:34):
We can also replace, historically we’ve called all the activator ARFs activators based on this glutamine-richness to their middle region, and repressor ARFs we classify this on serine-richness, but it turns out that doesn’t really dictate protein behavior. We can mutate every glutamine in this middle region to a serine and still see formation of these cytoplasmic ARF condensates, although if you look closely, we do see differences in some of the properties of these protein bodies when we make this glutamine to serine substitution, as you might expect.
Lucia Strader (19:13):
We’ve clearly seen that ARF localization, and its condensation, and its nucleocytoplasmic partitioning is dependent on the cell type and developmental state along this root axis, and we wanted to understand what role this condensation might play in the biology of the plant. And thinking about this reminded me of one of the very first experiments I’d done as a brand new post doc in Bonnie Bartel’s lab where I’d taken this really classic auxin transcription reporter called DR5::GUS, you can see it here on the left. You can see that auxin responsive transcription is happening down here at the root tip, and if you treat this with auxin, even really high levels of auxin, you can get an induction of auxin transcriptional activity in many cell types, but you really don’t see much happening in these upper root cells.
Lucia Strader (20:03):
And when I first did this experiment as a new post doc, I went into Bonnie’s office and I said, Bonnie, I just did this experiment and I did something wrong because clearly my auxin is working, but I’m not seeing a response everywhere the way I would have expected, what did I do? And she’s like, “That’s okay, Lucia. Everyone sees this, that’s why everyone crops down their DR5 data just down to the root tip and they ignore everything else that’s happening in the rest of the plant.” And so that’s what I did, but it always bothered me because we had other evidence in the field that auxin is being perceived in these other cells, there just wasn’t any transcriptional output that was coming from that auxin perception.
Lucia Strader (20:41):
So I wondered if perhaps this ARF condensation was a regulator of this tissue-specific competence to respond to auxin. When we give this reporter line an extra copy of the transcription factor, the one that is forming these condensates in these upper root cells, it looks very similar to wild type in the transcriptional activity and where that’s located.
Lucia Strader (21:08):
However, if we give the plant a copy of ARF19 that cannot form condensates, that is purely nuclear in every cell type in which we examined, we see now that every cell in the root is now responsive to this hormone. So a single point mutation in this one transcription factor that disrupts condensation is sufficient to turn every cell in the plant into an auxin responsive cell. This tells us that ARF condensation is a huge regulator of the competence of individual places in the plant to be able to respond to this hormone.
Lucia Strader (21:44):
When we do RNA-Seq analysis on plants carrying this non-condensating version of ARF19, we go from having about 15% of all genes in Arabidopsis being regulated by auxin to having about 30% of all genes in Arabidopsis regulated by auxin. So I want to clarify this data a little bit, because we don’t think that when we create this non-condensating version of ARF19 it’s finding new targets. I think that because we’re doing these RNA-Seq experiments on entire seedlings, by making every cell in that seedling responsive to auxin, we’re probably unmasking auxin targets that were only in a small niche of cells and now more cells are transcribing that target in response to auxin.
Lucia Strader (22:33):
And it’s not just that there are more genes that are auxin responsive, but when we take our RNA-Seq data and we make self-organizing heat map, what we see is that non-condensating version of ARF19 that carries that lysine mutant, when we treat it with auxin, there’s a huge shift in the amplitude of auxin responses that occurs in the seedlings. Not only is condensation affecting which cells can respond to auxin, but the amplitude of that response as well.
Lucia Strader (23:03):
These poor plants, we give them this non-condensating version of ARF19, they are not very happy with us. So they are very small. They look like they may have been auxin treated. They have small seedling leaves. They have short roots. And they take nearly a full year to complete their life cycle, which is in contrast to wild type Arabidopsis plants that typically take about six weeks. So no one in my lab really likes having to grow these because it takes so long to get their seeds. This is not a good thing to do to these plants.
Lucia Strader (23:40):
All of our data leads us to a model where ARF condensation and it’s nucleocytoplasmic partitioning acts as a mechanism to attenuate auxin responses. You can imagine if these transcription factors are being held in these protein bodies out in the cytoplasm, they can’t be doing their transcriptional activity even when these repressor proteins are removed by addition of auxin.
Lucia Strader (24:07):
We know that this is happening in a cell type specific manner, and it gets back to our big question. The big over-arching question is, how can you have this one molecule that’s seemingly involved in everything, but it’s doing the right thing in the right place at the right time? We think that ARF condensation regulates tissue-specific auxin responses so you don’t have inappropriate auxin responses in response to different cues and you’re only getting the right growth output in the right place in response to these various cues with this hormone.
Lucia Strader (24:38):
So if this is the case, then this must be a regulated process. And so this is one of the directions my lab has been moving in, trying to understand what regulates ARF condensate formation. So there’s some confusing things about this. I’m sure all of you have, since I told you that these form in the nucleus, like you can see here, and then they’re expelled out into the cytoplasm, you probably think that shouldn’t happen. I think it shouldn’t happen. These protein bodies should not be able to just squeeze through a nucleopore and get back out in to cytoplasm, we’re definitely trying to figure that out. The fact that they’re very diffusible and liquid-like at this stage of condensation probably helps with this process.
Lucia Strader (25:28):
The other thing that’s really confusing to us is if this protein body is being formed in the nucleus, which has a fair amount of ARF protein in it, once the condensate gets out of the nucleus, it’s into the cytoplasm, it should be back in a dilute solution of this transcription factor and it should remix within the solution if it’s following the rules of simple two-component phase separation. That’s not what we see happening, so clearly there’s additional layers of regulation on top of this that are keeping these protein bodies intact once they’re back out into the cytoplasm in a dilute solution of this transcription factor. These are some of the questions we’re asking ourselves and trying to figure out.
Lucia Strader (26:13):
As Chrissy mentioned, my lab takes a really diverse range of approaches when we’re trying to attack a problem, and one of my favorite ways–when I don’t understand a process–one of my favorite ways to try to understand it is to just let the plants tell us what’s important. One of the ways you can do this is through a forward genetic screen. So what we did was we EMS-mutagenized our reporter line and then we said, okay. My poor student that first did this screen, his name is David, he named all of his mutants David’s High, which I really hope was due to the elevated levels of the protein and the high numbers of condensates and not on his substance status at the time he was doing the screen because I asked him to sit on a dissecting scope for hours and hours at a time and look at all the individuals that had been mutagenized to look for increased levels of condensation.
Lucia Strader (27:12):
Since we moved to Duke, my lab’s invested in a high-throughput screening platform where we can ask the opposite, to look for reduced condensation, but that’s a really difficult screen to do by eye under a scope. And so one of the first things that David isolated was David’s High 8, or DH8, and you can clearly see that compared to wild type, which we’re now using an over expression construct that accumulates a lot more protein than the native promoter, and we do see some condensates forming in the root tip in this line, because we’ve really over expressed the protein. We can see this DH8 line has way more condensates and larger condensates than wild type.
Lucia Strader (27:54):
So when we crossed that back to native promoter lines, we still see the same thing. We see increased numbers of condensates in these roots compared to wild type. And in addition, when we look at all different parts of the plant, this particular mutant hyper-accumulates protein in each of these tissues in addition to having increased numbers of condensates. I’m not going to show you all the biochemistry, but we’ve identified the causative mutation in this line, it’s defective in a previously uncharacterized F-box that incorporates into an SCF E3 ubiquitin ligase complex and it directly interacts with ARF proteins. So we believe that the ARF proteins are direct targets of this SCF complex that’s mutated in this original isolate from our screen.
Lucia Strader (28:45):
What we know is that this SCF-AFF1 complex regulates ARF condensation, we see hyper condensation in our mutants. It regulates ARF accumulation, we see increased levels of protein accumulation in our mutants and what we were surprised to see is that it also somehow is affecting ARF nucleocytoplasmic partitioning. So when we look at roots of wild type compared to the mutant, what we see is that in wild type in the root tip, we see predominantly nuclear localization of this transcription factor and the nuclei are depleted of this transcription factor in this mutant.
Lucia Strader (29:26):
We don’t entirely understand what’s going on right now. When we’ve done Westerns of ARF19 protein in wild type and mutant, we see that not only is there a depletion of polyubiquitylated ARF protein in the mutant, but also a depletion of monoubiquitylated ARF in this mutant, suggesting the possibility that SCF-AFF1 might be either adding a different ubiquitin moiety onto the ARF to expose a nuclear localization signal to allow it to go in or that there’s some sort of ubiquitin relay system that needs to be worked out for this particular transcription factor family.
Lucia Strader (30:10):
Okay, so these mutants that are defective in this process where it was very confusing to us at first because they have these phenotypes of elongated leaves that curl under and this was confusing to us because before we understood the nucleocytoplasmic partitioning phenotype in this mutant we thought that these should be hypersensitive to auxin, and this particular phenotype of long leaves that curl under are what we typically see in mutants that are resistant to this hormone. So we couldn’t understand for a while why, if you had more of this transcription factor, you would end up with less output from this transcription factor. When we directly assess by looking at targets of it and finding that in mutants defective in this F-box, we have less induction of these downstream targets. We think that simply because somehow in this mutant, we have increased condensation but we also have depletion of the nuclear fraction of these transcription factors.
Lucia Strader (31:10):
So we’ve been working on building this updated auxin signaling model in which ARF protein accumulation, condensation, and it’s nucleocytoplasmic partitioning can all be used as ways to modulate sensitivity to this hormone in a developmentally and environmentally relevant context. We’re still working on several aspects of it.
Lucia Strader (31:40):
We’re trying to understand the dynamics of ARF condensation. At the moment we’re creating photo convertible fluorescent tags so we can look at situations in which we see disappearance of these cytoplasmic condensates and reappearance of nuclear ARF protein to see if that’s actually protein from the condensate being redissolved and going back into the nucleus. I’m not comfortable saying that with a single fluorophore unless we can really track the source of these proteins. Our screen is revealing additional layers of ARF condensation regulation. We haven’t fully characterized all of the mutants that have come out of the screen, but we have lots of things that are coming out. We think that phosphorylation is playing a role in this regulation and lots of other modules for other pathways to regulate whether the ARF proteins are in the diffuse nuclear phase or if they’re in cytoplasmic condensates.
Lucia Strader (32:37):
In addition, we’re working with Alex Holehouse and Dolf Weijers to try to understand the evolution of condensation amongst these proteins. So the ARFs are a fantastic system because they’ve been conserved for over 500 million years and they’ve retained their same function over that same time period. So we have functional data from species such as the hornworts and the mosses that last shared a common ancestor with Arabidopsis about 500 million years ago, and these protein’s function is absolutely conserved. I can tell you that unlike the Arabidopsis ARFs where we only see condensation in these class As, in the mosses and liverworts we see condensation both in class A and in class B ARFs. And in that system, these never get expelled into the cytoplasm. This is only happening in angiosperms that are examined. We think there’s something really interesting going on there.
Lucia Strader (33:45):
In addition, my lab has found that when you combine these types of Janus-type oligomerization domains and intrinsically disordered regions in Arabidopsis, all of the naturally occurring systems with those features form condensates in the cytoplasm and they all get expelled into the nucleus. Interestingly, for each of these systems, it’s a different environmental cue that drives whether they’re diffuse and in the nucleus or in cytoplasmic condensates. So some of it’s a nutrient cue, for some it’s an environmental stressor that regulates that process. But it’s the same biophysical paradigm that’s operating in each of these.
Lucia Strader (34:27):
We’ve also been using ARFs and their oligomerization to understand the non-stoichiometric assemblies of the ARF condensates and how that might be of interest in understanding the biophysics of these processes.
Lucia Strader (34:41):
And finally, we are also finding that the ARF proteins, their accumulation is regulated by different environmental events. So for example, at 22 degrees, which is a nice happy temperature for Arabidopsis compared to 28 degrees, which is a warm day but not necessarily heat stress, we see a huge difference in the accumulation of this protein. And not only do we see more protein, but we actually see that we have more diffuse ARF proteins in the nucleus at the elevated temperatures, along with increased auxin responses, suggesting that there may be a shift along this temperature axis and where this cSAT is occurring for this particular protein. [inaudible 00:35:30] actually a regulator of auxin responsiveness in plant growth at different times of day.
Lucia Strader (35:35):
We’re continuing to uncover these mechanisms that regulate auxin response under really distinct contexts, and that’s really critical because if you have this one molecule that’s integrating all of these cues into very distinct growth outputs, and you have an organism that is born with this really simplified body plan, like our peanut, and then for all plants, all development is being driven post-embryonically. So when it needs to flower, where it needs to make additional organs, is all really integrating these environmental cues to make sure that the final organism and the final form of that organism is really tuned to the environment in which it finds itself.
Lucia Strader (36:19):
So I’d like to take just a moment to be a little bit more philosophical about protein condensation in plants. So I’ve really focused on what my lab’s interested in, which is these auxin response factors that are driving cell division in response to these different cues. And we’re seeing not only developmental regulation of condensation, but also some temperature and other environmental cues that are regulating condensation. And this is a recurring theme in plants. Plants don’t regulate their own body temperature. They’re highly responsive to light. It’s been known for a long time that many of the light signaling components form photo bodies depending on which wavelength of light they’re perceiving. So it’d be like PHYB proteins or CRY proteins. In addition there are proteins that undergo condensation, again, in the cytoplasm, based on oxidative stress and pathogen response conditions.
Lucia Strader (37:20):
And then two of my favorite proteins are ELF3, which is a prion-like domain containing a transcriptional repressor, and when the temperature becomes elevated, this repressor forms condensates that remain in the nucleus, but they’re no longer able to repress their target genes. So similar to the ARFs, the condensate form is the inactive form of this protein. And more recently, there’s a protein that’s known to be involved in vernalization, so this is the process where a plant to be exposed to cold, a period of cold temperature before it can transition to flowering. This is why we have all these beautiful flowers in the spring. The plants are measuring how long they’ve been exposed to cold through this FRIGIDA protein. And for FRIGIDA, you need an extended period of cold and when that happens, this protein forms, again, nuclear condensates, and it’s no longer driving expression of repressors of flowering.
Lucia Strader (38:21):
I think it’s really cool to think about how you can have an organism that’s really sensitive to all of the things that are happening in its environment. It needs those cues to know when to do what it needs to do, and it’s a recurring thing that we’re seeing that protein condensation is a driver of that. And at least in these three examples of the ARF proteins, ELF3 and FRIGIDA, the condensate forms of each of these transcription factors is actually the inactive form of these transcription factors. So I’m curious to see whether this continues to be a trend for plant proteins.
Lucia Strader (38:57):
With that, I would really like to thank all of our collaborators, so the members of my lab, David Korasick and Sam Powers did a lot of the early ARF work. Hongwei Jing is working on this SCF-AFF1 protein. And then this would not have been possible without all of my marvelous collaborators who are much more ingrained into the IDR field and condensate field than I am. With that, I am happy to take any questions you guys might have.
Christiane Iserman (39:30):
Okay, so I think we have.
Jill Bouchard (39:32):
We’re clapping over here, just so you know.
Lucia Strader (39:35):
Jill Bouchard (39:37):
Sorry to interrupt. Chrissy, go ahead.
Christiane Iserman (39:39):
So awesome talk, Lucia, very fascinating. So there’s a first question from the chat right now from Sunita, do you want to ask the question yourself? I can also read this out.
Christiane Iserman (39:56):
Okay, then I’ll read it out. So, if type B IDRs do not form condensates what would be the purpose of that diversification from type As, if type A causes auxin signaling to be more regulated?
Lucia Strader (40:14):
I’m reading the rest of this question, too. Because I’m, yeah. I think this is a really fascinating model. The class A ARFs, we now know, can directly recruit the transcriptional machinery. My lab has recently identified the activation domains embedded in those IDRs. The class B ARFs actually recruit chromatin remodelers that alter the methylation status of the histones around the target. And the prevailing dogma in the field right now is that since these have the same target genes that it’s the ratio of this class A and class B ARF that dictates whether there’s going to be the transcriptional output happening or whether that region is going to get repressed by the residence time of those class B ARFs.
Lucia Strader (41:06):
So the class Bs don’t undergo condensation. If you increase the class As, you’re going to outcompete them for those sites. But if you have them in condensates, obviously those condensated type As are not going to be outcompeting the type Bs that are still in the nucleus and can still target those sites.
Lucia Strader (41:31):
What we don’t know is, we know that for the class A ARFs, the condensates when we break it, we know that the condensated form of class A ARFs are not transcriptionally active. The condensates might have additional roles. They could be sequestering other proteins in those condensates that we don’t yet understand. But at least they’re not transcriptionally active if they’re in the condensate form. We don’t know if that’s the case for class B. So class B ARFs could be active, even when they’re in a condensate. We don’t have that data yet.
Christiane Iserman (42:06):
Okay, thank you. I have another question here from Sunita that I’m going build up another question from me actually, on top of. So besides development, can environmental conditions alter the condensate behavior of ARFs?
Lucia Strader (42:22):
We’re still exploring this. There are multiple other plant hormones that have crosstalk with ARFs and we haven’t understood the point of that cross talk. We now have evidence that when you treat with these other hormones, you see disappearance of condensated ARFs and you see reappearance of nuclear ARFs. Like I said, I’m a little uncomfortable making strong statements about what’s happening until we use photo convertible tags to understand the source of these nuclear ARFs. Temperature’s definitely shifting the cSAT for these proteins so that you have more auxin output at elevated temperatures. There’s some evidence that salt stress also affects ARF condensation in the lab.
Phi Luong (43:12):
Hi, I have a question. In your forward genetic screen where you found a dominant mutation, that’s pretty amazing, right? What’s up with the F-box proteins because presumably the other alleles of the same gene are okay, right? So do you think these F-box have a common denominator in regulating other types of condensates, like particularly phytochromes?
Lucia Strader (43:39):
Our F-box mutants are not dominant, they’re recessive alleles.
Phi Luong (43:46):
Oh, I see
Lucia Strader (43:46):
But compared to, so Arabidopsis has about 1,000 E3 ubiquitin ligases. So 1,000 out of the 30,000 genes in this species are dedicated to E3s, which is quite striking, right? So I would say that probably nearly every protein you can imagine in the plant is regulated by the proteosome at some level. I’m positive that, I don’t know the specific examples, because they’re not my areas, but I’m positive other examples in this particular diagram that I have up right now are also being regulated by the proteosome. In fact, NPR1 can act both, this guy over here, can act both as a transcriptional regulator, but it, itself, is actually an E3. So it has this dual role in both of these ways.
Phi Luong (44:46):
In the EMS forward genetic screen how do you get a recessive allele, I guess?
Lucia Strader (44:53):
Yeah, so we always screen the M2 generation. So we EMS mutagenize and then we allow, Arabidopsis are self-fertile organisms, so they will self-fertilize. So every- [crosstalk 00:45:06] Yeah, it makes life easier when you have a self-fertilizing. And the other benefit is our lab strain of wild type only has about 200 SNPs compared to the reference genome, so it makes identifying mutations really really easy when you’re working with these incredibly inbred, self-fertile organisms.
Christiane Iserman (45:29):
I have another question concerning the temperature dependence of condensation. So you showed that ARF has these Q-rich regions. Could you see any trends from species that grow at different temperatures, similar to what Amy Gladfelter saw for fungi?
Lucia Strader (45:48):
We haven’t done a good job of that for the ARFs, but for ELF3 in particular. So ELF3 is a component of the evening complex, it’s part of a very complicated circadian clock found in plants. But it also regulates flowering in plants as well. The ELF3 work is out of Phil Wigge’s lab, and they did this really marvelous job at looking at… You can imagine integrating the right temperature cue into flowering is kind of important. If you’re up in Sweden, you might not want to flower based on day length, you want to flower based on temperature. Versus, maybe, if you’re down along the Mediterranean, you would integrate those cues slightly differently. ELF3 is a big part of that.
Lucia Strader (46:40):
ELF3 actually is another prion-like domain and the poly-glutamine richness is completely correlated with the temperature regime that that plant is found in, and it’s critical for correct flowering time for plants. So there’s been a lot of selective pressure on getting the right poly-Q richness, very similar to what Amy has found in her fungal system.
Christiane Iserman (47:06):
Cool, thank you. We have another question: Are you aware of any active plants prion-like domains forming active condensates?
Lucia Strader (47:18):
You say active condensates?
Christiane Iserman (47:20):
This is something where, basically, the condensation doesn’t inactivate the function of the protein, if I understand this question from Philip, here?
Lucia Strader (47:33):
Not that I’m aware of, but I guess there’s prion-like domain proteins that are participating in stress granules, which, I’ll let you guys debate what’s happening with activity of proteins in stress granules. But transcription factors with prion-like domains in Arabidopsis. I’m not aware of any where the active form of the prion-like domain containing transcription factors are the active form.
Christiane Iserman (48:08):
Okay, thanks. I’m seeing another question here from Sunita, actually. Have you looked at other tissues besides roots?
Lucia Strader (48:22):
Oh yeah. So in other tissues… The roots are so lovely because you get every stage of development in one time point so you don’t have to think too hard about when you do the experiment, but when you look at other parts of the plant like the young leaves. For example, when this leaf is developing, if we look at ARF proteins in a really young leaf, it’s all nuclear and as the growth starts to cease in that leaf, that’s when we see formation of cytoplasmic condensates. And in a fully developed leaf, we no longer see that.
Lucia Strader (48:53):
However, in both leaf tissue and in root tissue, if we wound. So plants are great because even when they’re fully differentiated, those cells retain some ability to revert back into a stem cell state. So if we wound a plant and cause that reversion to happen, we actually see, once again, appearance of nuclear ARFs in those cell types and then we start the whole process over again from that wound site.
Christiane Iserman (49:23):
Interesting. So I think that’s the questions from the chat. Any other questions from the audience.
Jill Bouchard (49:29):
I’ve got one. I found that the oligomerization in the actual condensate structures fascinating. Have you guys done any work to see why it’s dimers and monomers at the interface? Is it an interface issue, or is it? What do you know?
Lucia Strader (49:47):
What do I know? So what happens that we didn’t describe well in our first paper because we didn’t really know this when we first published this work. When we look at really young condensates that are just formed, we actually see a low occurrence of these higher order oligomers. And so we know that these PB1 domain oligomerization events are necessary to drive formation of these, but those are not stable enough to show up as long chain oligomers in the condensate, in very young condensates.
Lucia Strader (50:27):
And then, similar to many other systems, the ARF condensates undergo an aging process where they become less diffusible in the center and that’s when we start seeing these ultra high order oligomers forming. My favorite hypothesis at the moment is it’s probably that as these protein bodies start to get older, a lot of those associations start to become more stable and then you get more and more packing in the center, and that’s just not happening at the periphery, and that’s probably where, we probably are seeing some diffusion with solution with those peripheral proteins.
Lucia Strader (51:10):
And obviously, we see diffusion with FRAP assays within the protein body at the periphery as well. But I am not enough of a biophysicist to really give you a great answer to that question. I can just tell you kind of what we’ve observed when we’ve looked at these things.
Jill Bouchard (51:28):
That’s very interesting. Anyone else on the table have anything? You’re welcome the unmute.
Christiane Iserman (51:36):
Otherwise, we also have something else here from Kent.
Jill Bouchard (51:38):
Oh yeah. Go for it.
Christiane Iserman (51:40):
Auxin will induce lateral roots, number of which increase with auxin concentration. Do you see any relationship between the condensates level or locations with auxin sensitivity? From Kent, the question.
Lucia Strader (51:53):
That’s a great question. What happens, so for those of you who aren’t plant biologists, as a root develops, there are particular places within that root. I’m going to point to one here or here where some of the cells that are differentiated into a type of cell called a pericycle cell, basically undergo a de-differentiation process and form a new meristem from those cells and that results in this new lateral organ being formed.
Lucia Strader (52:27):
Auxin is critical for this process, in fact the ARF7 and ARF19 transcription factors are critical for this process and what we see is that if we look at sites where this event is going to happen, we see disappearance of these cytoplasmic condensates and we see reappearance of nuclear ARFs in those cells at a very young stage that’s consistent with what we know for the roles of these proteins. And so, and then it’s actually pretty cool because you see this little niche of young meristematic cells in this newly formed lateral root with all nuclear ARF protein, with all of these older cells around it with cytoplasmic ARF protein in it. Does that answer your question or do you have something else that you wanted to ask about that?
Jill Bouchard (53:20):
Kent, you’re welcome to jump on if you have more.
Christiane Iserman (53:27):
That seems like a great answer to me. So we’re out of time, I think there will be time for one last, very urgent question, if there’s anything.
Jill Bouchard (53:37):
I don’t think so, so I guess we’re at a good winding down point.
Christiane Iserman (53:41):
It’s in a very timely manner, so that’s terrific. Thank you so much, Lucia, this was awesome and very exciting to zoom into the world of plants for a little bit.
Jill Bouchard (53:53):
Yeah, lots of applause coming from the world, thank you so much.
Lucia Strader (53:57):
Thank you, guys.
Jill Bouchard (54:00):
All right. [applause] Well, we’ll wrap it up and thanks again, Lucia, for joining us. We were super happy to learn about condensation in plants today and thank you for teaching us about peanuts. I will never look at one the same again, I promise.
Lucia Strader (54:15):
I mean, you’re killing a baby every time you eat a peanut. It’s just a baby plant.
Christiane Iserman (54:20):
Lucia, do you eat peanuts?
Lucia Strader (54:22):
I do eat peanuts, yeah.
Christiane Iserman (54:23):
Lucia Strader (54:24):
I feel no guilt over it.
Jill Bouchard (54:27):
They all have feelings. Anyways, I think our next kitchen table talk will be in 2022, so hope you all can join us again and thanks again for coming today.
Lucia Strader (54:39):
Thanks Jill.Thanks, Chrissy.
Christiane Iserman (54:41):
Jill Bouchard (54:42):