Anna Dobritsa - podcast === [00:00:00] Anna Dobritsa: Different plant species have exine patterns that can look very different. Some pollen grains can have pretty smooth exine .Others can have some sort of spikes growing on the surface. Others may have like ward like structures. So it's very interesting. [00:00:23] David Staley: This one here looks almost like the exterior of a cantaloupe. [00:00:26] Anna Dobritsa: Uhhuh. It does, yes. Right. So they're very fascinating in that sense. [00:01:07] David Staley: I am pleased to be joined today in the ASCTech Studios by Anna Dobritsa, associate Professor of Molecular Genetics, the Ohio State University College of the Arts and Sciences. Her areas of expertise include pollen development, cell biology, and plant molecular genetics. Dr. Dobritsa, welcome to Voices. [00:01:26] Anna Dobritsa: Thank you very much. I'm really happy to be here. [00:01:29] David Staley: You study pollen grains. And I think I wanna start there because when I think of pollen, I think of my allergies and so is that what you study? Do you study allergens? [00:01:40] Anna Dobritsa: Not really. So people are very familiar with allergies that are caused by pollen, but that's not what plants are using pollen for, right? So pollen, came on the scene much earlier than humans came. Right. And it is actually a male reproductive structure in plants. So pollen is a collection of individual pollen grains that are produced by male organs and plants and. Each tiny little pollen grain is actually composed of three cells. One of them occupies the bulk of the pollen grain. So imagine a big cell. And inside the cell there are two small cells, and those cells are sperm cells of the plants. And so when pollen flies on the wind or when it is transferred by pollinators, by insects, for example. This little sperm cells are traveling inside the larger cell and the goal is to deliver them to the female parts of another plant. Right? And once pollen lands on the female parts of the other plant, it actually, the sperm cells cannot travel anymore and they don't have any structures that allow them to move around. So what happens is that this big cell is going to grow a large tube, and this tube is going to grow through the female structures and bring the sperm cells towards the egg cells, which are residing deeply inside the floral tissues, flower tissues. [00:03:29] David Staley: So when you say, Sperm and eggs and male organs. [00:03:33] Anna Dobritsa: Mm-hmm. [00:03:33] David Staley: Do you mean that like an analogy or are we talking like what we have in animals? [00:03:38] Anna Dobritsa: No, absolutely same thing. A little bit more complicated because plants actually have to have two fertilization events. So two events of meetings between sperms and female cells. One of the cells is going to be an egg cell. And so if you combine a sperm cell and an egg cell, then an embryo is going to be produced a plant embryo, and the second sperm cell is going to combine with a second cell called central cell. And this is going to create a group of cells inside the seed that are going to nourish this developing plant embryo. So, in a sense, you can consider it maybe almost like placenta. So the second thing, right. [00:04:28] David Staley: So and I, I understood you to say that this can only happen if there's a pollinator, like a bee or some other sort of insect. Is there any that's the only way this can happen or [00:04:37] Anna Dobritsa: No, not really. So some plants are self pollinated, so basically they produce pollen and then just drop it on their own female parts. In other cases, many plants, like corn for example, produces tons of pollen and it travels by wind. So pollinators are very important for some plants, but all the plants came up with other strategies of how to ensure successful pollination. [00:05:10] David Staley: So within these pollen grains, I know that you and, and your labs study specifically what are called exene. [00:05:16] Anna Dobritsa: Mm-hmm. [00:05:17] David Staley: Tell us a little more about what exine refers to. [00:05:20] Anna Dobritsa: So exine is a special cell wall that is going to cover each of the pollen grains, and basically plant cells are always covered by some sort of cell walls. This is sort of a very big difference from the animal cells. So they tend to produce a fairly rigid structure so that plant cells cannot move on their own, and they're just surrounded by this cell walls, and most of them are made out of the material called cellulose that you probably heard about. [00:05:56] David Staley: Mm-hmm. [00:05:57] Anna Dobritsa: But not exim and not pollen. So pollen covers itself with very unique and pretty fascinating cell wall called exine. And this cell wall is made out of a very unusual material. It's also cellulose is a biopolymer, but this material is also another type of a biopolymer. [00:06:20] David Staley: When you say biopolymer, that means... [00:06:21] Anna Dobritsa: it means that there are chemicals that are usually pretty small. In the case of cellulose, it has a glucose, for example, that are making long chains, right? They're combined with each other into a long chain. And in the case of Spar Pollin, there are also some building blocks out of which this component is made and it is, by the way, so it's called Spar pollen because it's only found in spores and in pollen. [00:06:49] David Staley: Okay. [00:06:49] Anna Dobritsa: And people have known about it for a very long time, at least since 1930s. And since then they've known that it is a material that is very unusual, very rigid, very robust. It's very difficult to destroy it by chemical means. You can, for example, boil the structures in concentrated sulfuric acid and they're still not really destroyed by that. Pollen can also be a _polling scene_ can be found in fossils like a hundred million of years ago it was deposited and it still can be found recognized. So the material is pretty interesting and however, because it is so chemically inert and so robust, it's actually been very difficult to really understand what the material is made of: what are its building blocks. And recently because of some advances in chemical studies, as well as in some genetic experiments, people started kind of getting some idea what the building blocks are. For example, fatty acids are seem to be an important chemicals that will go into making spiro pollin. Right? So that, that's one interesting thing about ion . [00:08:15] David Staley: Mm-hmm. [00:08:16] Anna Dobritsa: Another interesting thing is if you've ever seen pictures of the pollen grains, you can see that they're beautiful. And so if you've never done it, I would strongly suggest that yes, you can just Google pollen images microscope and you will see tons and tons of images. [00:08:38] David Staley: I did exactly that and I'm looking at some right now. And they are, they are, they are strikingly beautiful and very distinct from each other. [00:08:46] Anna Dobritsa: They're distinct and different plant species have exine patterns that can look very different. So some pollen grains can have pretty smooth exine .Others can have some sort of spikes growing on, on the surface. So spora pollen assembles into those spikes. Others may have like ward like structures. So it's, it's very interesting. [00:09:13] David Staley: This one here looks almost like the exterior of a cantaloupe. [00:09:15] Anna Dobritsa: Uhhuh. It does, yes. Right. So they're very fascinating in that sense. So why there are so many different patterns? So there are literally thousands upon thousands of different patterns of exhibits and different species and well, presumably, yeah, it is important for plants to be able to make those patterns and assemble spora, pollin into these patterns. In one species pattern would be different from the other species. [00:09:51] David Staley: You say presumably, [00:09:53] Anna Dobritsa: Presumably [00:09:54] David Staley: that implies maybe we don't understand what these patterns mean. [00:09:58] Anna Dobritsa: Right. We don't understand it very well, so the usual hypothesis that people are putting forward is that it must be some sort of adaptation that allows pollen grains to be either attaching itself better to the pollinators bodies, right? Or maybe in the case of the wind pollinated plants, maybe they can travel better by wind, so maybe it improves their a aerodynamic properties. And there are some general correlations. For example, wind pollinated species tend to have a eczema that is much smoother and is devoid of a lot of fancy ornamentations While Insect pollinated plants tend to have eczema that is kind of more intricately developing. But nobody have done any experiments to really, really understand it. And those experiments are not easy to do. [00:11:00] David Staley: Why not? [00:11:01] Anna Dobritsa: Well The best ones would probably involve mutants in which you would change an exene pattern in the same species from one pattern into another pattern. And in my lab, we are working with a plant species called Opsis, which is a weeded plant that is the best studied plant, a model organism for plant biology. And we have tons of mutants in which ex in patterns change. So, in principle, we could have studied this question, but the problem is that ADOS is actually a self pollinated plant that is grown in the lab. So probably there is not much. It can pollinate pretty well even when it's in patterns are disrupted or sometimes even when exine is missing altogether. But in nature it might be different. And so it would be great to be able to change exine patterns in some insect pollinated plants and convert them from one type to another, and then actually measure what effect it has on pollination and on ability of pollen to fertilize. So I guess that that's one thing. So pollen interacts with insects, for example, but it also interacts with female structures. [00:12:18] David Staley: Mm-hmm. [00:12:19] Anna Dobritsa: So another possibility is that maybe the female organs that have to capture pollen maybe they're also sensitive to different patterns and people should definitely look into it. [00:12:33] David Staley: Like Velcro or something like that. [00:12:34] Anna Dobritsa: Like Velcro. Right, right. Something like that. [00:12:37] David Staley: Well, you said that* *no one is, no one is asking, or no one's working on this. You say in part because it's difficult. Is it perceived as an important problem? I mean, is this a question worth asking? [00:12:46] Anna Dobritsa: I think it's definitely a question worth asking. Yes. Yeah. [00:12:51] David Staley: When you were describing this, I almost thought like snowflakes. Is that an apt metaphor here for the different kinds of forms we're looking at? [00:12:58] Anna Dobritsa: Well, to an extent. So with snowflakes, people usually say that they all look completely different. [00:13:05] David Staley: Right? Right. We don't repeat Right. Which I don't think is true. Right. [00:13:07] Anna Dobritsa: First of all, within the same species patterns tend to be identical. Also, patterns between closely related species tend to be much more similar to each other than if you compare plant species that have been evolving for a very long time separately, right, like separated long time and evolution. [00:13:33] David Staley: So I know part of what you're interested in is the study of those surfaces that lack enes protection. [00:13:41] Anna Dobritsa: Mm-hmm. [00:13:41] David Staley: Right? These are pollen apertures. Do I have that right? [00:13:45] Anna Dobritsa: That's right, that's right. [00:13:45] David Staley: Tell us, tell us what pollen apertures are. [00:13:47] Anna Dobritsa: Okay, so in most species, not the entire pollen surface is covered with exine. Usually pollen grains leave specific sites on their surface that are going to be devoid of exine formation. So somehow they can label this areas of their surface in a way that makes it clear to the molecular machinery that deposits exine, that they should just let this regions alone. They shouldn't deposit exine on those regions. And those sites are becoming, as pollen develops, they are turning into pollen apertures and the usual function. So there are several functions for pollen apertures that, people think. So one of the most important ones is that in many species, they serve as the site of exit for the pollen tube. So I mentioned the pollen tube. Mm-hmm. Inside of which the sperm cells are giving to travel to the female eggs and apertures are the least robust sites on the pollen surface. So it's the easiest way for the pollen tube out of the pollen grain. Some other functions of apertures is that they help pollen to kind of collapse when they are released to the dry air from the male organs where they're kept in a watery environment. So they're released to the, to the dry air, they start losing water. And in order to prevent complete desiccation of the cells, exine essentially closes. Hmm. And this closure happens, or this closing happens at the aperture sites. And then when pollen, lands on the female organs, it often actually, it needs to get the water back in order to kickstart this process of growing pollen tubes. And so it can sort of absorb the water from the female organs and it swells and the swelling is also happening. Apertures are helping pollen grain not to burst during the swelling. Right. And so we are not so much interested in studying the function of this structures, but we are interested in how they're forming and how the cells that are going to become pollen grains know which regions of the pollen surface to leave without ion deposition. Because in different plant species, just like with exine patterns, there are also different aperture patterns. [00:16:35] David Staley: Really? [00:16:35] Anna Dobritsa: So some of them can have, for example so our favorite species, _ados_ has three apertures. That a place like if you imagine pollen as a globe, they will be placed like three meridians. Mm-hmm. That are. Placed at equal distances from each other in some other species. Species like lines of longitude. Right, exactly. And in some other species there could be six meridians and other species, they would not look like lines at all. But as little circles. And there could be two of the circles, one circle or sometimes. Dozens of those circles. So they can differ in their shape; they can differ in their position. They can differ in their numbers. And so we are interested how the cells know where to place them, how can they count how many to produce? Because in _opsis_ it's actually very, very stable. Essentially all normal pollen grains will have exactly three apertures. They will be long and narrow in place like three _Acquiescent Mary dance, _right. Well, how do they do it? Well it turned out that, and there are special proteins that are going to be deposited at those sites. And we found several of those proteins. There are some proteins whose function we are still trying to understand that help this proteins that are going to end up at aperture sites to end up there. So somehow they're directing them to those positions. And this somehow serves as a signal for the proteins that are involved in exam deposition that, well, guys, you don't like leave the sites alone, right? Don't deposit exam at those positions. [00:18:26] David Staley: Did I understand you earlier to say that that you are able to manipulate these patterns? [00:18:32] Anna Dobritsa: We can do it to some extent. [00:18:35] David Staley: Why, why do it? [00:18:36] Anna Dobritsa: well right now we are just trying to do it because we can and because we want to know how how this patterns are controlled. But there are in principle some, maybe some good reasons to do it in the future for agronomical reasons. So, for example, sometimes it is useful not to have fertile pollen. So usually you want to have plants that are fertile, but it also could be important for creation of hybrids. For example, pretty much all the corn that is grown in the US is a hybrid corn, right? And that means that you take two lines of corn and you cross them together. So you're putting pollen from one line onto pistols of and another a line, and then their offspring is actually going to be physiologically superior to the parents. Plants will be taller, they will produce ears that are longer, there will be more nutrients and so on and so forth. So there are a lot of reasons for why you may want to mate pollen plants of different lines. But in order to do it, you actually need to have a female line that doesn't produce pollen so that it won't be self pollinated, right? And so that could be achieved, for example, by creating pollen grains with no apertures because in corn, as we found, if no apertures are formed, then pollen tubes are completely unable to emerge from the pollen grain. And so this pollen becomes completely infertile. And so are the plants essentially. [00:20:19] David Staley: And so you've been successful at manipulating? [00:20:21] Anna Dobritsa: To some extent. Yes. [00:20:23] David Staley: How do you do it? What's the process? [00:20:24] Anna Dobritsa: We are. Looking for genes involved in the process of aperture formation in _Opsis_. And we have found about eight genes so far that are important for this. And we can then look and see whether these genes are so similar genes are present in some species that are much more agriculturally important than_ a opsis. _And in many cases we can, and I think actually in the case of all these _age genes_, there are similar genes in other species. And then there are already mutants available, like in the case of one of the genes that we study. We just went to the list of the available mutants in corn and looked to see if there is one available for our favorite gene. And there was. In other cases you can use CRISPR to inactivate these genes. In some other cases, you may be interested in not inactivating the gene function, but you may be interested in over expressing, producing much more protein and much more RNA and protein from this gene. And that can also be done by genetic modifications. So you are essentially adding an extra copy of this gene or maybe you're trying to regulate it in a way that it will be producing much more protein. [00:21:54] David Staley: I'm struck by the scale at which you're working. Presumably you're using electron microscopes. What sort of technology [00:22:01] Anna Dobritsa: right, [00:22:02] David Staley: Do you use to work at this scale? [00:22:04] Anna Dobritsa: Right, so we do use electron microscopes occasionally. We also found that sort of a smaller microscope that is still very complicated and sophisticated called confocal microscope, which is not an electron, but light microscopes. It uses light in order to allow you to visualize whatever you're trying to visualize, whatever you're trying to see. And so we are using confocal microscope primarily in order to look at this patterns and it works usually pretty well for us. But yeah, microscopy is my favorite part of this work. [00:22:44] David Staley: How come? [00:22:46] Anna Dobritsa: It's just fascinating, right? Just looking at the structures and seeing what changes, for example, in the mutants or where our proteins localize. So when we, for example, found this aperture factors, aperture determining proteins, and found out that some of them can go specifically to the regions on the cell surface, where apertures will eventually form and assemble there in this three long lines that predict positions of the three equidistant apertures, that was fascinating, right? And we saw it with the help of this confocal Microscope. [00:23:27] David Staley: Well, and there's great beauty too, as I've, uh mm-hmm. As I've indicated. [00:23:30] Anna Dobritsa: Yeah. [00:23:30] David Staley: I'm curious to know how you ended up as a molecular geneticist as opposed to, I don't know, a chemist or an historian or something like that. What was your path to becoming a molecular geneticist? [00:23:41] Anna Dobritsa: Right. Well, I think, as a child, I actually wanted to be a linguist. [00:23:46] David Staley: No kidding? [00:23:48] Anna Dobritsa: But it also happens that both of my parents were microbiologists. So I do come from a family of scientists and I also grew up in Russia in one of this places that are called academic cities. So it is a very small place. There are several of them in Russia. Ours was about two hours away from Moscow. And it had only something like 18,000 people, but I don't know, 50%, well maybe not 50, but a large portion of them were PhDs and there were seven research institutes. So I guess that was, sort of on my mind. And then when I was 15 years old, that was kind of in high school. And at that time, Perestroika was happening in Russia. [00:24:42] David Staley: So late 1980s, right? [00:24:44] Anna Dobritsa: And there were borders* *opening. And so it became possible to, for people to start going to other countries. And at that time, children were considered to be ambassadors in the thawing relationships between the US and Russia. And I managed and I really wanted to go and see what US was about. And so I managed to get into the summer camp in the US, and it was a biologist summer camp and probably didn't know that much of biology at that time, but I could speak English. That's why. That's why I ended up going there and. I ended up liking it. And so when I came back in the end of the summer, I told my parents that, well, it was just before my last year of the high school. And I said, well, I think I would actually like to apply to the biology department and not to the linguistics department. And so within a year, I kind of turned around and started focusing on chemistry, biology and went to Moscow University and ended up studying molecular biology. Molecular genetics. Right. Yeah. [00:26:01] David Staley: Looking at your field, what's been the biggest paradigmatic change in the last, I don't know, 30, 40 years? That the way we do molecular genetics today is very different from the way they were doing it, you know, in the 1980s. [00:26:15] Anna Dobritsa: Right. Well, there's. Tons of tons of discoveries and obviously molecular biology, molecular genetics are moving so fast, but technology development probably drove a lot of it. And the way we are able to sequence genomes and_ transcript tube _the way we are able to do studies of_. Proteomes and Metabolomes. Right. All this Ss. _But I would say that yeah, really ability to, to read genomes and transcriptome completely transformed. [00:26:56] David Staley: We tend to associate that maybe with reading the human genome, but it, right, it's as important for plants. [00:27:00] Anna Dobritsa: It's for everything. Mm-hmm. Like for all the species. And there are already thousands of species that well, maybe hundreds, maybe I should take it back, but there are, there is a very large number of species whose genomes have been sequenced and understood to, to some degree, both plants, animals. Microorganisms and so on and so forth.Hmm. [00:27:22] David Staley: Tell us what's next for your research. [00:27:25] Anna Dobritsa: We have been building sort of a collection of mutants and collection of genes, understanding of genes and inventory, I would say, that are important for *ex information *and aperture formation. We still don't really understand how all of it fits together and how these genes are working together. And recently for example, we discovered a group of genes which decode specific proteins that seem to be very important for aperture formation and they seem to be very ancient. So they go all the way through flowering plants to fir trees all the way down to algae. You can find these types of proteins. So they must be doing something and important. And in addition to a couple of genes, from this family that are important for aperture formation, there are a few more that don't seem to do anything with aperture, so they probably are doing, have, having some other function. But we have no idea what exactly they're doing. Like what do they do in algae? What do they *do in Opsis? We* still don't really understand what these proteins are doing biochemically, so that, that's the next thing for us. [00:28:37] David Staley: Any theories? Hypothesis, maybe I should say hypothesis. [00:28:41] Anna Dobritsa: Yes. Although they're not really being confirmed at the moment. So these genes are actually also or similar genes are found in animals. And in animals it is a little bit better known what they're doing. And so we kind of assume that they're probably going to have similar roles and plants. So far we are not really seeing it. So maybe they are doing something different than plants. [00:29:08] David Staley: So what does that mean then, for your research, if you're not seeing what's being hypothesized? Does this mean what you have to ask new questions, new hypotheses. You give up? What's, what's a scientist to do here? [00:29:19] Anna Dobritsa: Yeah, that, that's a great question. Well, usually you try to come up with some other hypothesis, or sometimes you can try a brute force approach, but kind of think about maybe a different technological way of how to approach this question. Like if you are not finding something that you have hypothesized, maybe sometimes you can just try looking for all the proteins that are interacting with your protein of interest and see what kind of proteins are they, and maybe this will allow you to then come up with a new hypothesis. [00:29:55] David Staley: Anna Dobritsa, thank you. [00:29:58] Anna Dobritsa: It was great to be here. Thanks.