Gryphon CAST Episode 2 Transcript
Speakers: Michael Lim and Annette Nassuth
Michael Lim
As we progress through the autumn season, the cold weather is forcing farmers to harvest their crops. One such crop is wine grapes, including the grape species use for Riesling, Chardonnay, and Merlot wines. But did you know when it comes to cold tolerance, not all grapes are made equal? Open your ears and mind. And let's chat about that. Welcome to Gryphon CAST, a podcast where we carefully chat about science coming out of the College of Biological Science at the University of Guelph, and how that work can affect lives around the world. I'm your host, Michael Lim. With me today, a special guest, Dr. Annette Nassuth will be chatting about how researchers are uncovering different methods that grape plants to use to handle the cold. And what that might mean for the wine grape industry. Welcome, Dr. Nassuth.
Annette Nassuth
I'm glad to be here.
Michael Lim
So, if you were to meet some random person on the street, or say a new undergrad, how would you describe your research and what you do?
Annette Nassuth
Well, my research looked really at how plants react to the changing environment. In particular, to the lower temperatures just before the frost really sets in and how they prepared to deal with that at a molecular level, mainly.
Michael Lim
So I guess that's really important here in Canada with are extremely long winter climate.
Annette Nassuth
That's right. Yeah. And especially because the wine grapes that I'm working with. They originally came from the Mediterranean region. And of course, that is a quite different climate than we have here. So you can imagine that there are some challenges there.
Michael Lim
So prior to conducting work with these semi exotic plants from the Mediterranean? Did you ever thought you would be doing this kind of work with grape plants or not really.
Annette Nassuth
I came to it to via the research that I was doing on plant viruses. And so I was looking at viruses in various crops. And that also then included the grapes. And just by circumstance, I started working on the cold acclimation of grapes. And that is because of the loss of faculty member, Dr. Brian McC???. He worked on cold tolerance. And he was leaving the university take a position in industry. Because I was already working these grapes, and had experience with extracting things from them, and it's not so easy, with these woody plants. They asked me if I could continue with that kind of research. And I said yes, so that's how I came to it. So it was really a special circumstance, but it's fun.
Michael Lim
I guess sometimes the best thing you can do or I guess hope for is that you do something that is novel, but still leave a little experience in where you're expanding and learning new skills.
Annette Nassuth
That's right. I they are more challenging plants. Usually, most of the research is done on model plants that are a bit easier to work with. But then the challenge is to see what people find in these model plans and how that translates into more real life plants. So to say, the crop plants that people are interested in.
Michael Lim
So, speaking of research on studying crops that people are interested in. You recently published a study titled "Leaves of more cold hardy groups have a higher density, a small sunken stomata". So what encouraged you to study that particular topic within this wider field?
Annette Nassuth
Well, that this actually because of research that was done in these model plants which had shown that there's a particular regulatory protein that activates both the cold acclimation such as adaptation of plants to lower temperatures, and it also activates the development of stomata. In principle, that's weird that one protein does both things. And so we thought then, "well, is that just some weird things from the smaller plants or is that something that's more general" so that's how I came to look at the stomata.
Michael Lim
So, speaking stomata, a large part of this research in this particular study is based around looking at stomata and characterizing them through a variety of factors such as their size, their density and proposition. For our listeners. stomata are tiny little openings in leaves. So imagine "tiny mouths", that they use to exchange carbon dioxide and water vapor with the surrounding environment. Adjusting the number and how far these "mouths" are opening are critical for survival in stressful environments for a variety of plants. Do you have anything to clarify about that description?
Annette Nassuth
Yeah, so what really what's happening when they open and it allows the carbon dioxide to come into the leaf and therefore the cells can take the carbon as building blocks to grow. But at the same time when they open, then also the plant can lose more water through these openings. And that can be detrimental when you don't have the possibility of replenishing that water. And that's why the opening and closing and the number of stomata are so important to regulate for plants. They want to grow as much as possible, but not in such a way that you cannot take up any more water because that's when they will dry out and die. So it's kind of a balancing act.
Michael Lim
I never really took into consideration how important it was to maintain a certain water level, because well in humans weren't really thinking about the water we're breathing out. But clearly in plants, it's really important to maintain that balance between water and gas exchange.
Annette Nassuth
Yeah, and the problem is really that if you want to avoid this water loss, and you close your stomata, you don't get the carbon in so that you cannot grow. So it's really you have to find the balance and that is what seems to be the case in the grapes that we were looking at and that they are doing in a specific way by having these different sizes.
Michael Lim
So, a really important technique using this paper is scanning electron microscopy or SEM, and used to look at the differences between stomata across different leaves from different wine grape species. Can you briefly describe this technique for our listeners?
Annette Nassuth
Yeah, that is really a very nice technique to use. But the thing is that these cells are very small. The leaf cells at the surface of the leaf, and that's where the stomata are. So if you want to have a look at how many stomata there are, how big they are, etc. You need to enlarge your view. And so you can do that this light microscope which is a regular kind of microscope. But when you do that the focus is not always so good. And actually what people often use is that you take nail polish to put on the leaf. Then let it harden, and then examine it under the microscope. However, when you have it under the microscope too long, your tissue will dry out, etc. And it's difficult to focus. But the problem with nail polish is that it cannot really take all the dips and valleys that are in the leaf surface. And the scanning electron microscope enables you to look at everything and just enlarge it enough that you can see the different cells. But it still gives you a large enough area. And then you would use a more fancy electron microscope, for example, that requires much more preparation but you wouldn't be able to process as many samples. So, the scanning electron microscope is really very exciting to look at, because you can also put the live tissue under it. So, you really can see I see it very nicely.
Michael Lim
So, I'm clearly very inexperienced when it comes to SEM techniques. That was all very insightful to me. I guess I would describe several series is kind of like, if you feel a very, very fine sandpaper, it might feel almost smooth to your fingers but when you zoom in you can see all these tiny little bumps and crevices.
Annette Nassuth
Yeah, that's right. And and the scanning electron microscope that I used was a tabletop scanning electron microscope. So a simple version of you have very fancy scanning electron microscopes that are more complicated. But this is a relatively small one. It's a little box and you can justput in your sample but you have to work quite fast. So usually we take pictures, and then count to cells or measure their cells later from the pictures.
Michael Lim
I mean, it must be incredibly grueling process to even process a few leaves.
Annette Nassuth
Yeah, and you have to do a lot to to get meaningful data. So we really have been counting thousands of cells. That's why I say all these people were so important that they are willing to do that and stay the course.
Michael Lim
Well, I guess so. As a result of your study shows that more cold tolerant species such as say Riesling grapes, have the highest mountain densities, were smaller, and more sunken. For our listeners sunken refers to one of the positions that somata can be. So, they can be either below the leaf surface level or above or at the same level. So sunken somata are thought to be more beneficial as they're thought to open and close faster and require less energy. And the amount of reduced water loss, I guess also plays into that whole gas exchange balance talked about earlier. Is there anything you'd like to add on to that? Yeah, the finding or the idea that the smaller stomata can open and close faster than the larger one was a study that was not done by me but by some physicists so they are much more knowledgeable about that kind of stuff. But that was interesting to read that paper because that's makes that you can understand that when a plant is temporarily under some better circumstances. It can quickly open the small stomata and still get some carbon dioxide. Whereas the large one they are really you have to crack them open and that takes much more time so that that will not happen then. And what you mentioned also is that they are sunken. Everybody might know when you are in a valley, you don't feel the wind as much as when you are the top of the hill. So that's the same thing as in that leaf. And then when you have the wind going by, that will also increase the water loss. So when they are sunken, you have less water loss. So why do you think there are such a large increase in the small sunken somata in some group species like Riesling compared to others, like Merlot getting ["unclear]" other cold tolerant grape species?
Annette Nassuth
Yeah, well, first of all, we actually discovered this whole thing first by looking at wild grapes. They are far more cold tolerant, frost tolerant than the cultivated grapes on the market. For example, it depends on the conditions, but if you treat them and pretreat them first, it improves, they can withstand about minus 40 degrees Celsius. The cultivated grape like Riesling can withstand Merlot something like minus 23 degrees Celsius, and the Merlot is a little bit higher. Okay, in temperature, so can withstand only minus 18, or something like that. Those differences can be important. But we first looked at the wild grape we first concentrated on looking at the number of stomata. We had no idea that there were different sizes. initially we saw the different sizes, of course, but we said "okay, that there are stomata that still have to grow, that are busy getting bigger", and only when we look at really more mature leaves. So leaves that have finished their development. And we look at those, they still had the small stomata. Then we realized it's really their final size, it's not that they are still developing. And that's how it all came about to look at this. And so the wild grape has also higher number of these smaller, sunken stomata.
Michael Lim
So kind of taking the idea and flipping it on its head. Would you expect to see larger stomata in like warm tolerant species? Or is it more of like a stressful environment? Yeah, now let me explain because I was talking about water loss. And maybe you wonder what does that water loss have to do with frost? Well, when you have frost you get ice. That means that a lot of the free water is not free anymore. It's in an ice block. So, there's less free water. So, in fact, the grape is exposed to a drought condition. And so in the same way, actually, we then looked at some papers where people had been looking at it from Spain had been looking at some grape cultivars, and looked at their stomata. But they were looking at different kinds of things. But when we took their data that they published in that paper and analyzed it in our way. Then we found that a cultivar that was more drought tolerant and also had more smaller stomata. And so in both cases, it has the same function. And at least that's what they think that it will help the plant to reduce its water loss while still enabling it to open quickly. When time and conditions are okay so that they still can grow. So, speaking of surviving drought, I read your paper as well and I thought that was really interesting. Would you say that this [unclear] stomata is something that all plants use across all stressful environments to survive various stresses? Or was it more something you need to investigate?
Annette Nassuth
Well, in environments where they would have drought stress. And another circumstance where plants would experience drought stress is when you have salty soil. And that is relevant. You might have seen some reports for example, from Australia when they have their crops and they fertilize a lot, then part of the fertilizer is not taken up by the plant and that stays in the soil. And so these soils become more and more salty. And that's a big big problem actually for the agriculture. So also there you would expose the a plant to more drought condition because the salt attracts water. So it will take it out of the plant and dry out the plant. But the whole idea is so about the small stomata where the drought is more general to other plants that I don't know. But, I bet that other plants that have also this kind of phenomenon, but people like I said the more common way of looking at the stomata would be by taking nail polish imprint, and that wouldn't show necessarily these sunken stomata. Basically people have to look again, then with the scanning electron microscope to see if they have the same scene or not.
Michael Lim
So out of curiosity is the SEM technique a somewhat new or modern technique that many scientists of the past have used. I'm just curious why people have opted more for the analog nail polish style, instead of the more, I guess, advanced SEM you used.
Annette Nassuth
Well, this is a dedicated kind of equipment, it's actually not that expensive, relatively speaking, I think you can buy one for about $10,000 or something like that.
Michael Lim
It's quite a lot of money!
Annette Nassuth
It's quite a lot of money. But compared to an electron microscope, it's cheaper. And we actually use it at the university also in our course. And because it is relatively easy to use, so some students can use it to make a picture of whatever they are working on in that course. But it is a piece of equipment that not everybody used for many different things, so many people won't have it. And that's why they they don't look at it. And because they saw that the nail polish technique was pretty good. And that was easy. So why would you try something else until now, we realized that unfortunately that doesn't catch all the stomata. But when you don't know that, then you think you're fine, right?
Michael Lim
Yeah, I mean, it totally makes sense. And so zooming out, what is your favorite part of this research project? And why?
Annette Nassuth
Now, because I mentioned earlier, this regulatory protein in the model plants are thought to increase the number of stomata and increase frost tolerance. But that, in principle initially didn't make sense. If why, then you would have more stomata, you would think you have more water loss. And you just think straight it is all the same kind of stomata. So that was from the data do that. And they were excited then to see that "Yes, there are more stomata but there are these small sunken stomata, so it's not necessarily leading to more water loss. And that was, of course, satisfying to find the kind of solution to the problem.
Michael Lim
I bet that probably ties into being the most surprising part of your project. We're not expecting to see a lot of stomata, and suddenly you looked at it. And there's an overwhelming amount. You had to explain stomata what the heck is going on here?
Annette Nassuth
Well, and especially because we first didn't realize that the smaller stomata was the final size. We first looked at younger leaves, and we saw it. Okay, they are still developing or something like that but we can still count them. But then we realized "No, that's the final size. Look at all the leaves that have finished developing". And then we saw "Okay, yes, we get more but they stay small".
Michael Lim
Exactly. So, if you go back in time and change one thing about your study. What would it be? And why?
Annette Nassuth
Well, it would have have been great if we had realized earlier that the small stomata was the final size, because we did a lot of counting and we didn't measure the size in the beginning because we didn't think it was important. So that was a lot of work to do. And then we had to basically redo it again and look at the older leaves too and then measure the sizes to get to the final picture. But you know, that's how research goes you have to be open for whatever you get and realize that there are different explanations for things and try to find out what the possibilities are. So this is exciting to go through that process.
Michael Lim
Yeah, I bet. It's not very often that you find a finding that goes completely against what you expected based on previous work and literature. And you go "What the heck is going on here?" Yeah, that's gonna be really relieving to like, ah, of course.
Annette Nassuth
Yeah, exactly, exactly it. Thank you. I do feel satisfied that you, at least you know, I don't know for 100% sure but it correlates. So we think that is the case, but 100% proof is difficult to get in science.
Michael Lim
Just a reasonable amount of confidence, right? Yeah. So um, I think we both agree in general this study kind of highlights that there are new techniques such as using SEM to study stomata and this should be used for a variety of other species. But what are you hoping that the general public takes away from this research?
Annette Nassuth
You can take a step back and use it for the practical application to grow grapes in a vineyard. It usually takes about three years before you get a yield. And it's always that there are other cultivars that people think they make good wine and they want to have it. So, they want to grow it here. And it would be nice if you know in advance if they are able to withstand the temperatures here and to survive. And so, if you could now look at the stomata of the plants when they still in pots and look at that and compare them with the cultivars that you know. You can say, "Okay, it's more like Riesling or say it's more like the Merlot that doesn't do so well here". In that way, you could already preselect a little bit. Although that's not the whole story why they would be called tolerant, which you have a higher chance if you choose them a new cultivar that might be able to grow here well and before you plant it out. Because you can imagine how much work that is you have to plant the whole field and grow things. Then wait two or three years and then you figure out "Okay, was not such a good choice, that's not nice". So, I hope that it can be used like that. Now besides that, like I said, the SEM is relatively simple, might still need a dedicated person to use. But one thing that I think would be exciting if it will be possible to have a kind of community project. Because the most of the time with this kind of research is really the counting and regards to look at to take pictures from the leaves, yes, that takes time. But the most of the time comes afterwards when you have the pictures, and you have to count. So it might be fun to try to have a kind of community project that people get the pictures and then do the counting. And can see something like that, so that at the same time people see the pictures they will understand more about a plant and how it looks like - just a little bit bigger than then they normally see. And they might then appreciate plants a whole lot more than they do now maybe. But you know, that's I'm always dreaming about these kinds of things. I don't know if it's possible to do. And you have to have, of course, people that are interested in doing it. Or you have to have a teacher that is interested in doing something like that. But you have to be motivated.
Michael Lim
I think it's a great idea. It'd be really fun. I mean, there's no age where you can't be a researcher.
Annette Nassuth
Exactly, exactly. And for me about what they show me the exciting thing about research is to be ready for the unexpected and to be open thinking. So I think this type of thing might open the eyes of some people that they might have heard of stomata in high school but I find these things are so abstract when you haven't seen it yourself. When you see it yourself you get excited you see it "Oh yeah, you know, here are these are open and then these are closed and etc. So, I think it could be something.
Michael Lim
Yeah, for sure. I think so too. It'd be a lot more fun than say like, you know we have a drawing of leaf and you're like yeah, okay, cool. But we're actually seeing the surface of the leaf is more engaging and impactful. Well, that's all the questions for me. I want to ask you some that are coming from our social media now? So our first question is, "Are cold hardy grape species native to Canada, or were they specifically bred to be able to be grown here?"
Annette Nassuth
The cultivars that are being used to make wine they all have been imported from for the Mediterranean or more bred for the quality of their wine. The wild grape that I was talking about that is like very cold hardy I mentioned was minus 40 that it can withstand and that one grows all over Canada. In Ontario, Vitis riparia is the only wild grape that is growing wild so if anybody goes out and sees it, they know immediately what it is but it can go very far to the north also in Manitoba, for example. But unfortunately it doesn't give nice wine. We refer to that as funky. But you have to realize that also at a certain moment, you get to an area where there might be a lot of snow cover. So actually, the plant itself might not even be exposed that much to very low temperatures because it's protected by those snow. So, you have to be careful how you interpret things, but it can grow very well but like I said, the problem is that the wine doesn't taste very well.
Michael Lim
So that kind of connects to our next question. Do cold hardy grapes species produce the same quality of wine is regularly species, if not, what is different about them?
Annette Nassuth
So yeah, but what has happened is that people have crossed a wild grape with the cultivated grape and then of course you don't get only this cold hardiness trait from that wild grape you get also the funky taste a little bit. So, what they have done, they crossed that and then that new plant that they got have crossed again over and over with the cultivated grape to try to select for keeping the cold hardiness but getting better taste. And so some of those plants are being grown as well. But it depends on which group of drinkers you belong to that you want to have to pure wine grape or that you go for this hybrid wine.
Michael Lim
So we can't really touch upon this already with selectively breeding for cold tolerance. But are you aware of any genetically modified species out there they've been engineered for a particular trait.
Annette Nassuth
In many cases it's not that easy to make a transgenic plant. In many cases people have used or made transgenic plants to test the concept. So for example, with these proteins that I talked about that are important for the cold tolerance, they can introduce that into a plant that's not called tolerant and then test if it now has a higher cold tolerance or not. For the commercials or making a transgenic plants [unclear actually the most interest there is to make grapes virus resistant. And that is because grapes are really from from a disease point of view, if you are work on diseases you like them, but if you don't work on disease, you dislike it because they they can have many many different diseases. And viruses can spread through the whole vineyard. So that is not something that people like so the most effort that is going on is to make virus resistant plants and those are in the process of being developed. And you have to realize that with grape the plants are grafted. In a vineyard they are grafted so you have a root stock, That's the bottom part with the roots. And then on top of that, you put the desired grape. So one of the ideas that is out there is to mate the trangenic part to bottom part. So that the top part where you have the grapes that is used to make the wine is from a part that is not transgenic. [Oh, okay]. So in that way, you know that you can have something acceptable for consumers to take those plants. So, there are different ideas that are out there that people are working on.
Michael Lim
Okay, it's really interesting, because a previous study I worked on, so I know that there's a concern about viruses in the soil. So if it contaminates your field suddenly had to burn and sacrifice.
Annette Nassuth
Yes, exactly. Many viruses are for example transmitted by nematodes, little worms that are in the soil? Yeah. So if you can can make a plant to adjacent to those, then you don't get the virus and the plant will be healthy.
Michael Lim
Very interesting. Unfortunately, we're coming to the end of our chat today. But do you have any final comments to make make your work? And if our listeners only take away one thing from our chat today? What do you hope it is?
Annette Nassuth
I think what I hope is that people can take away is to keep your eyes open. I mean, for my work, okay, that's about a stomata. It's fun. But I can imagine for people that are not into this, it might be less fun. But for the takeaway is really to keep your eyes open. Be open for any possibility, so that you recognize it, and then you get it. That's always the difficult part. If you only have a preconceived idea in your mind and it doesn't turn out that way. They might be disappointed. But in fact, it might actually be quite exciting to find out what's happening in reality.
Michael Lim
And with that, we've come to the end of today's podcast. A big thanks again to our guest, Dr. Annette Nassuth. [Thanks for the invitation]. You're very welcome to come back and join us again. Gryphon CAST is brought to you by your host me, Michael Lim. With editing assistance from Ian Smith. If you're hungry to learn more about different science topics, please check out SCRIBE research highlights at scribe S-C-R-I-B-E research highlights on the University of Guelph website at uoguelph.ca or you can follow us on social media at UofG CBS. You can find us on Instagram, Twitter, and Facebook. Music in the podcast comes from uppbea.iot with details in the show notes. For next time, stay curious.