What’s the BUZZ about? with Dr. Karen Sanguinet

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Resources:
New Phytologist article
Gene required for root hair growth, nitrate foraging found in grasses
Sanguinet lab members
Pacific Northwest National Laboratories (PNNL)
Environmental Molecular Sciences Laboratory (EMSL)

Contact information:
Karen Sanguinet, WSU Department of Crop and Soil Sciences, 509-335-3662, karen.sanguinet@wsu.edu

Episode transcription:

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Drew Lyon: Hello, welcome to the WSU Wheat Beat podcast. I’m your host, Drew Lyon, and I want to thank you for joining me as we explore the world of small grains production and research at Washington State University. In each episode, I speak with researchers from WSU and the USDA-ARS to provide you with insights into the latest research on wheat and barley production.

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My guest today is Dr. Karen Sanguinet. Karen is an associate professor of crop physiology. She joined WSU and the Crops and Soil Sciences department in 2014 after a brief stint as an assistant professor at Iwate University in Japan. Her research program focuses on root development in temperate grasses, as well as trying to understand the molecular mechanisms and traits underlying cold and drought tolerance in both crops and model plants. She teaches an undergraduate crop physiology course on how plants interact with the environment.

Hello, Karen.

Dr. Karen Sanguinet: Hi, Drew. Thanks for having me.

Drew Lyon: It’s good to have you on again. So, your program received $40,000 from the Washington Grain Commission in the current fiscal year.  I’m wondering what these funds allow you to do that would not be possible without the additional support.

Dr. Karen Sanguinet: Thanks, Drew. Yeah, the Grain Commission has been wonderfully supportive of my program over the past nine and a half years, and we’re really grateful for their support. In this fiscal year, this money is going to support a graduate student in my lab, Luigi Peracchi, who’s finishing his Ph.D. this spring. He’s been investigating root development in wheat cultivars and in particular in a landrace that has a very proliferative root system that also produces a tremendous amount of lignin.

And lignin is a polymer that associates with the cell wall and makes xylary elements impermeable to water and helps rigidity of stems. It’s also important for straw breakdown in wheat, and we’re really looking at it in roots. So, there’s been a lot of intensive study. If you think about forage analysis in wheat or grasses or straw, a lot of what they’re looking at is lignin composition and lignin content.

And we’re, in particular, interested in this landrace that produces a lot more lignin, both in the stems and roots. And so, we’re studying how the lignin is deposited, how those root systems respond to drought. And so, Luigi, this year has been able to collect a really beautiful dataset using a technique called RNA sequencing, where we isolate all the RNA from the shoots and all the RNA from the roots of the wheat plants, and then do high throughput sequencing and get a readout of all the genes that are being expressed in the various tissues.

And we did it in this landrace that has more lignin in its roots, and we did it under drought and under normal conditions. So, we can see what changes in the wheat plant–and particularly in the wheat root system under drought. So, we’re trying to identify root specific genes that respond. We’re looking at the lignin biosynthetic pathway and how genes change in their expression.

And this wouldn’t be possible without their generous support.

Drew Lyon: Okay. So, without these funds, you wouldn’t have been able to support the Ph.D. student who is doing this work?

Dr. Karen Sanguinet: Yeah. And we’re also able to work on, you know, lines and breeding material that’s important for wheat in the Inland Pacific Northwest. And that’s also really something that’s special about the support, because on a federal level, they want to see, you know, a diversity panel like a global diversity panel, but we can really focus in on things that are important for our region.

Drew Lyon: Okay. You mentioned RNA, seq. I wonder if you could explain what RNA is for those of us who it’s been a while since we’ve taken the genetics class.

Dr. Karen Sanguinet: Right. So, RNA is a basic building block of our cells. It stands for ribonucleic acid. And hopefully we all know that we’re made up of DNA and RNA and proteins and other molecular components in our cells, but in the nucleus we have all of our DNA. And that DNA is translated and essentially expressed as RNA.

And those RNA, it’s essentially a code and a message that allows proteins to be made in the cell, but it can also act as a regulatory molecule. And so, it gives us a good idea that–the RNAs are unique; the DNA is the same in every cell–and that’s kind of the way that way to think about it.

Drew Lyon: Very good, nice explanation. Thank you.

So, I understand you recently had a paper published in the journal New Phytologist titled “BUZZ: An Essential Gene for Postinitiation Root Hair Growth and a Mediator of Root Architecture in Brachypodium Distachyon.” Can you tell us a little bit about that?

Dr. Karen Sanguinet: Sure, Drew, thanks. Yeah. So, this paper is a culmination of over a decade’s worth of work trying to understand how grass roots grow. And I should preface this by saying that Brachypodium distachyon is a model system for other temperate grasses, so C3 Pooideae grasses in particular, like wheat and barley.

And the genome of Brachypodium distachyon was sequenced in 2010. This enabled assembly of the wheat and barley genomes subsequently because they’re very similar to one another. And so, we initially were interested in the drivers of root growth and development. And we found this really interesting root hairless mutant that we called BUZZ. And the mutant was identified in 2012 in a screen looking for plants that had different root architectures and root systems.

And not only did it have–so the title is Postinitiation–so essentially the little cells on the epidermis or on the skin of the root that make root hairs–well the root hair is initiated so it made this little localized bulge, but they were arrested, they couldn’t be rescued, they couldn’t grow out. And so, they’re stalled. But not only does it have this root hairless phenotype in this small little bulge in the place of where a normal root hair would be, but the roots grow faster and initiate more lateral roots. So, it’s a root architecture and a root hair mutant both at the same time, which was really a unique phenotype.

And so, we chase the gene. It’s like looking for a needle in the haystack. We had to sequence the whole genome and look through all 30,000 plus genes and try to find which one was different from wildtype and mutant.

And so that took us a while. You know, genetics can take a lot of time. So patience, perseverance, and a lot of hard work by a lot of really talented grad students and postdocs and collaborators on this project culminated in this paper and it’s been really exciting for us to see this work come to fruition.

We’ve seen a lot of interest. I was interviewed for a magazine to talk about it with some of the Canadian wheat growers as well. So, it’s been really fun to talk about our research. It is pretty fundamental research, but it has real life applications.

Drew Lyon: So, I’m sitting here thinking, why or how would a mutant that didn’t grow root hairs, what would be the advantage to something like that? How it would have maintained itself without root hairs?

Dr. Karen Sanguinet: Well, that’s the really intriguing question. There doesn’t seem to be any kind of penalty for not making root hairs. And there are certain crops like blueberries that don’t have root hairs. So, we know root hairs are dispensable. And the old adage in the field and the way we think about root hairs is that they’re essential and that they help water nutrient uptake, they facilitate interactions with the soil, with the soil microorganisms. But really what we found is the plants can do pretty okay without the root hairs.

But there is what we think a compensatory mechanism where they grow more. The unique aspect is most of the time when you abolish root hair development in grasses or other plants, you see a penalty on yield or the plants don’t grow as well.

And that’s why we infer, “Oh, well, you know, root hairs are required for growth and they’re not getting adequate water or nutrients.” But what we see in the BUZZ mutant is that the roots grow faster and that seems to be enough to make sure that the roots are able to acquire [the] water and nutrients they need to grow and to support and sustain growth of the aerial portions of the plant–of the shoot, [the] tissues, the flowers.

So, there’s no yield penalty or real growth penalty necessarily.

Drew Lyon: [You] totally shot down all the things I thought I knew about root hairs. So, thank you, Karen. [laughter]

So, why would a wheat or barley grower be interested in this BUZZ gene?

Dr. Karen Sanguinet: Yeah, so, the really interesting connection we found, and we did this same technique where we did RNA sequencing that I talked about earlier, and we looked at all the genes that change their expression in the mutant versus wildtype. And we were expecting to see usual players like transcription factors that control transcription cascades and maybe plant hormones that control growth. But what we found is that there was upregulation of a lot of the nutrient transporters, in particular overexpression of the nitrate transporter and another gene that regulates the nitrate transport signaling pathway. And so, in the mutant we see increased nitrate transport. And so, we’re working on that.

Drew Lyon: Okay.

Dr. Karen Sanguinet: And so, this connection with nitrate and root hairs–at least understanding the molecular mechanisms–it’s really new research in the field.

Drew Lyon: Okay. That could possibly have repercussions for wheat varieties in the future that might be able to make more efficient use of the nitrogen that’s applied to soils.

Dr. Karen Sanguinet Exactly. So, now we’re working on trying to understand nitrogen-use efficiency in Brachypodium in wheat. And this is kind of a new and renewed focus. You know, inorganic nitrogen fertilizer is a huge cost and we use most of it. We know that root systems are at best 50% efficient in taking up nitrogen. So, if we can tap into mechanisms and genes that are controlled by nitrate and nitrate signaling, if we can upregulate nitrate signaling pathways and routes and tweak expression, that’s huge for the farmers in terms of cost savings and also has a huge environmental impact so we don’t generate a lot of excess nitrogen running into waterways and all the environmental and agroecosystem issues that arise with excess nitrogen in the environment.

Drew Lyon: Okay. Very interesting. Not something you had planned on finding when you started down this route but something that popped up along the way.

Dr. Karen Sanguinet: No. And that’s why science is so much fun. You know, you get on pathways and come to things, you know, that you weren’t expecting.

Drew Lyon: Very interesting. So, what are the next steps and how do you see this research evolving in your group?

Dr. Karen Sanguinet: Yeah, well, now that we’ve established that BUZZ is important in Brachypodium, we’re starting to look in other grasses and delve into barley and hopefully wheat and maize.

This has actually enabled us to clone a gene in maize. It’s a long-standing root hairless mutant in maize that’s never been cloned. We knew that there was a mutant, we knew where it mapped, but we didn’t know what the gene was. And it turns out we were able to identify through orthology or similarity between the gene we identified in BUZZ in Brachypodium and clone a gene in maize that’s essential for root hair growth as well.

So, we know this gene is conserved in grasses. We also did research in Arabidopsis, which is a Brassica species. It’s also a model plant, showing that gene function is also conserved in Arabidopsis. And so, this is readily translatable to both, you know, dicotyledonous crops as well as monocotyledonous crops in cereals and grasses. And those are our next steps.

We’re also investigating this link and really probing the interaction between BUZZ and nitrogen-use efficiency and trying to get to the molecular mechanism. What is the protein doing? What are the other proteins that are interacting with BUZZ to do its job or carry out its function, its cellular function? And that’s been really fun as well.

Drew Lyon: It’s interesting to me that you found this mutant through just a general screening and that’s led to all these things down the road that…

Dr. Karen Sanguinet: Yeah, hopefully it’ll keep me busy for the rest of my career. That’s the hope. That’s the hope, Drew. [laughter]

Drew Lyon: It sounds like it just might. It looks like there’s a lot of things evolving from this work.

So, I also understand that you received an Environmental Molecular Science Laboratory Exploratory Award, which basically allows you to conduct research in collaboration with scientists at the Pacific Northwest National Labs. Can you tell us a little bit about this research and what’s underway there?

Dr. Karen Sanguinet: Yeah, thanks for this question. This project and exploratory grant dovetails in this kind of the next steps in the BUZZ project. What we were able to do with this grant is start to get to some of the molecular mechanisms and look at, on a very fine scale, specifically nitrogen in the BUZZ mutant and where it’s moving and how it’s moving at a very fine scale.

We can actually image using N15 nitrogen. So, radiolabeled, it’s an isotope of nitrogen and we can look in the cells as scientists through methods–I won’t get into the technical methods, but essentially we can see individual, not necessarily isotopes, but we can see movement of these isotopes between and within cells at a very fine scale, at a nanometer scale. We can see it moving in through the root, through the cell layers of the root, and see where the nitrogen is in relation to the cell and the cell layers of the root. It’s pretty fantastic.

The other thing we’re doing is looking specifically in the epidermis that makes root hairs and we’re looking at all the genes and all the proteins, not just in the root itself, but in that cell layer that makes root hairs.

And we’re looking at all the genes that are differentially expressed, all the RNAs that are up or down in BUZZ–in the BUZZ mutant versus wildtype. And we’re also looking at all the proteins that change in abundance in the epidermis.

And this is only possible at PNNL with their scientists. They’ve developed these really special techniques for looking at the proteins at this very fine scale in very small quantities. We essentially make very small sections of the root and we use a laser and cut out the epidermis, which is the outer cell layer of the root. And then we ask what are all RNAs or all the genes being expressed in that cell layer? What are all the proteins that we can find and identify?

Drew, how many proteins do you think we can identify in like a tiny 30 micron section of a root? Can you guess a number?

Drew Lyon: Proteins? There’s a lot of them. I’m going to throw out 150.

Dr. Karen Sanguinet: [laughter]

Drew Lyon: I sense I’m a little bit off.

Dr. Karen Sanguinet: Well, actually, the scientists, they use a technique called mass spectrometry and you identify proteins based on mass, and they are able to identify over 4500 proteins that were present in these sections, which is pretty remarkable. We were expecting–we were like, “if we can get 100, we’ll be happy.” But they’ve refined their techniques and, you know, they just have been incredible collaborators.

It’s been incredible to work with them. I feel so fortunate to be in the Pacific Northwest and be able to have Pacific Northwest National Labs close by and support of their scientists and support of EMSL and the Grain Commission. So, it’s kind of, you know, it’s the perfect marriage, so to speak, of different entities supporting us. And I hope that what we find is useful for growers and useful for the community as well.

Drew Lyon: Very interesting information, Karen, I learned a lot today. Thanks for coming in and sharing this work that I think will carry you for at least quite a ways in your career and hopefully with some real positive impact on the agricultural industry down the road.

Dr. Karen Sanguinet: That’s the hope. That’s why we do it. Thanks, Drew.

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Thanks for joining us and listening to the WSU Wheat Beat podcast. If you like what you hear don’t forget to subscribe and leave a review on iTunes or your favorite podcast app. If you have questions or topics you’d like to hear on future episodes, please email me at drew.lyon — that’s lyon@wsu.edu — (drew.lyon@wsu.edu). You can find us online at smallgrains.wsu.edu and on Facebook and Twitter [X] @WSUSmallGrains. The WSU Wheat Beat podcast is a production of CAHNRS Communications and the College of Agricultural, Human, and Natural Resource Sciences at Washington State University.

I’m Drew Lyon, we’ll see you next time.

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The views, thoughts, and opinions expressed by guests of this podcast are their own and does not imply Washington State University’s endorsement.