Katie Murphy (UC Davis): How Corn Fights Crop Stress

Katie Murphy (UC Davis): How Corn Fights Crop Stress


I’d like you to think about the last meal that you had. I hope it was delicious. Maybe it looked something like this. And I hope that it was filling. But did you think about where that meal actually came from? How did it get to your plate? I think about this a lot, because I spend a lot of time in a cornfield. This is me, doing my graduate research, in my cornfield at the University of California, Davis. I love corn. I love corn so much my students have nicknamed me the Corn Queen. Now, if you didn’t have corn on the cob in your last meal, I can almost guarantee you ate something that was made with a corn byproduct. And if you ate meat, you ate something that has eaten corn. It is the backbone of the American food supply. That’s part of why I love corn so much. It is so important to helping us eat. But I also just love being out here in my field. Corn is fun, because I get to start here. Just a seed, something so tiny in the palm of your hand. And that is where all of your food comes from. I get to put this seed in the ground and then watch it slowly pop up like this. Here is where the biochemical beauty begins. Everything we eat comes from three simple ingredients: sunlight, air, and a little bit of water. Here, plants are able to do a miracle, bringing these three things together to make a plant, something so beautiful and green covering our planet. After these little things pop up, and I give them a lot of love and care, they begin to look like this — corn plants taller than me. Here, I watch them interact with their environment, cope and thrive, continue to grow. And if we’re lucky, we get to see this: a beautiful harvest. Lots and lots of corn that eventually makes it to your plate. Now, while we were all watching this corn grow up, it was also doing something different: reaching down. In my research, I study roots, this whole world underneath our feet that oftentimes goes forgotten. Plants have to anchor themselves into their environment. They can’t move. And these roots connect them with the world beneath us. Now, while I see a lot of healthy plants, I also see a lot of plants die. Now, this nasty ear of corn is caused by a fungal disease. This fungus is called Fusarium. Now, Fusarium not only makes our food unappetizing, it also produces toxins, mycotoxins that are hazardous to both human and livestock health. Now, disease, drought, and other types of stress cause about 10% crop loss of corn in the United States every year. What does 10% of our corn crop look like? It’s a cornfield that covers the entire state of Florida, rendered totally inedible. Now, rescuing crop from disease is so, so important. It allows us to grow more food. Asking questions about what makes a plant sick and what makes it healthy is so exciting to me as a scientist. On your left, you can see a really healthy corn plant. It’s gotten enough water. The one on the right looks not so good. You might recognize this, because this is what your houseplants look like when you leave for vacation and you forget to water them — totally wilted. I ask myself, what differentiates a healthy plant from a sick one? How does a plant deal when it begins to get sick? Not only is this question scientifically interesting, it’s also really important. The reality is today there’s a lot of people in our world who go hungry. And 20 years from now, we expect 2 billion more people on this planet. That is 2 billion more mouths to feed. We have to find a way to grow more food using less resources. Food in the future could become a luxury. We simply don’t have the arable land, the water, and other natural resources to produce enough food. We only get one planet. We all want to eat. And if we can grow food in a more efficient way, we can help preserve a world for our future. Now, as a scientist, I wake up with hope every morning, because I know that my research and that of others can help pursue this dream of growing more food and feeding more people. I’m excited to tell you about that research today. First, we’re gonna talk about stressed-out plants, this fungal disease that I mentioned earlier. Then, we’ll get into a brand new group of molecules, that my research has found, that corn makes in response to this disease. Then, we’ll talk about how to use these molecules to make stronger crops and food for our future. So, just like you, plants get stressed out. Maybe you had an exam, maybe it’s taxes, maybe you can’t remember if you left the oven on this morning and if it’s gonna burn your whole house down and did you even cook… but we get to relax. We’re human. We have ways of dealing with stress. Now, plants get stressed too. Drought stress causes significant loss of crops, because water is essential to plant life. Herbivores, like the caterpillar that you can see here eating the corn that was supposed to be in my barbeque, chew up our crops and destroy them. And the fungal disease Fusarium also causes significant crop losses. When plants get stressed, we lose them to disease. Now, plants do have a way to cope with stress, just like we do. One way they do so is using chemistry. Plants are the greatest chemists that we have. They produce special molecules to help them deal with stress. These molecules can act as a weapon, or as a medicine, for protection and support, to help them thrive despite all the environmental pressures that they face. I study a special group of these molecules that are called terpenes. Terpenes help plants deal with stress. All terpenes come from a smaller molecule, a subunit called isoprene. And multiple isoprenes are put together in different ways to make these larger molecules, terpenes. Now, all terpenes are made with enzymes. I think of enzymes as little builders, like you see here. An enzyme will take one molecule, a certain shape, and change it into another shape. A subsequent enzyme, or builder, can then add on new groups, new elements, new functional groups, to the molecule to change it again. All of these enzymes are encoded in DNA; they come from genes. When I started working on this project studying corn, we knew a little bit about the terpenes that were present in corn. We had one group, a subgroup of terpenes that were called the kauralexins. Now, we didn’t know all of the enzymes that made up this pathway to make the kauralexins. So, we asked ourselves a simple question. What genes control the production of these terpenes we know are present in corn? To answer this, we have an exciting method in my lab: rapid gene characterization via co-expression assays. Now, put simply, this means we’re figuring out what genes do. To start with, we look at gene expression. Now, this is our way of looking at genes that are expressed at the same time and in the same part of a plant. Now, if you wanted to know if two builders were working on the same construction project, how would you figure that out? You would look to see if they showed up at the same construction site at the same time. We’re doing the same with genes. First, we look through the genome to see genes that look like they might encode for enzymes that make terpenes. We know this from previous knowledge of what these genes should look like. Once we had a list of these genes, we looked for genes that are expressed in the same time and in the same location in the plant, just like a builder at the same construction site at the same time. So, here you can see three genes: ZmAN2, ZmKSL2, and ZmKSL4. All three of these genes have the same pattern: green in the control and yellow in the treatment. This was our first indication they may be working together to form a pathway to make terpenes. Next, we cloned these genes. This means we take the sequences from corn and put them into a plasmid. That’s a circular piece of DNA that we can then put into a bacteria. We do them separately — AN2 in blue, KSL2 in red, and KSL4 in the green. Next, we take these plasmids and we put them into E. coli. Yes, that’s the same bacteria that can make you sick, that we can grow in the lab. Now, E. coli will take in these plasmids and treat them as its own, expressing the genes, making the enzymes, and those enzymes can then make terpenes. Now, we do this in different combinations — first, blue and red: AN2, and KSL2; blue and green: AN2 and KSL4; and the third combination: KSL2 and KSL4 — to understand how these genes might be working in a pathway. After we express these genes in E. coli, we can grow it for a while, and then finally extract the molecules to see what terpenes might be made. We analyze this using metabolite profiling. This is done using GC/MS, gas chromatography/mass spectrometry; LC/MS, the liquid version; and NMR, nuclear magnetic resonance to understand the chemical structures that are made from this process. So, we did this with the genes in corn that we thought might be terpene synthases. And while we started with a pathway where we knew some of the molecules that were present in corn, we indeed filled in two of the enzymes. We now understand the genes that underlie kauralexin biosynthesis. But then, something a little bit crazy happened. We made something totally new. When we put some of the genes together, we made a totally new group of molecules that we call the dolabralexins. It’s a bit of a mouthful. Phonetically, doe-lab-raw-lexin. Now, this to us was really surprising. A totally new group of molecules, we hadn’t seen them in corn. Here’s what these molecules look like. These were brand new to corn, and we’d nev… frankly never seen them in nature, anywhere. In particular, epoxydolabranol, this one on the end here, has a unique group. It’s an epoxide, an oxygen in a ring with two carbons. Now, typically, these structures are uncommon in nature because they’re highly unstable. But our molecule appeared to be fairly stable. Was it really being made in corn plants? Or was this an artifact of our E. coli production system? We didn’t know, and we needed to show this, so we started looking. We started in the roots. Now, when I told you that we looked for gene expression — genes that were expressed at the same time and in the same tissue — we actually started looking in root tissue. So, this was our indication that that might be a good place to look for these molecules. And what did we find? Corn is making the dolabralexins. We weren’t nuts. This was not an artifact of our E. coli system. These genes from corn indeed made these molecules in the roots of corn plants. Now, the graph that you see here is a quantification of these molecules. The three columns you see are three different varieties of corn: B73, Golden Queen, and Mo17. You can see that B73 only makes really low amounts of these metabolites in its roots. B73 also happens to be the variety of corn that most researchers use and grow in their labs. Perhaps this is why no one had found dolabralexins before. We were all using a variety of corn that only made very low levels, and only under certain conditions. And frankly, underneath our feet, where a lot of us were not looking. This was exciting. We now know that dolabralexins are made in corn roots. This brings us to our next important question. What are dolabralexins doing for corn plants? We already know that terpenes are important for how plants deal with stress, so were corn plants making these in response to stress? Were they protection against some of these stressors? We were really excited to answer this question. And we had a tool already in our toolbox that we didn’t know was previously useful to us. This is a mutant plant. Mutant plants have a mutation in their genes, here, in the one particular gene that you can see here with the red X. A mutation is caused naturally or by scientists in a lab, and that gene no longer works. It cannot produce the enzyme. This means that any downstream products — both the kauralexins and the dolabralexins — are not made in this mutant plant. Now, I like to think of this experiment when I think about a car. Now, I know how to drive a car, but I know nothing about what’s under the hood. Say I pop the hood open on a car, and I go in and I cut any line that I see… just one. I’m gonna close the hood, get into the car, and take it for a test drive. I’m driving along, and then I go to make a right turn. And while I turn the steering wheel, the car keeps going straight. What does this tell me about the line that I had cut? Whatever I cut must be controlling the steering of the car, because when it’s broken, I can no longer steer. Now, analyzing a mutant plant works the same way. I am physically going to be breaking a gene. It no longer works. We’re gonna take the plants for a test drive. Whatever goes wrong tells us what that gene normally does right. And so, our collaborators had already taken these mutant plants for a test drive, at the time not knowing that these mutant plants didn’t make dolabralexins, because they didn’t know that dolabralexins existed. And first, what they did was saw how this mutant responded to fungal disease. Now, on the top you can see some corn seeds that are a little bit fuzzy. This is a normal plant. It has a working copy of the gene. And we’ve appl… they have applied fungal stress to these seeds. On the bottom, you can see the mutant plant. An extreme amount of fungus is growing on here. It’s extremely fuzzy. You can see it with your own eyes. You don’t even need to quantify it to know that this is important. The mutant cannot deal with fungal stress. This is an indication that what that gene normally does with those terpenes — the kauralexins and the dolabralexins — downstream from this mutation must normally protect this plant from fungal stress. This was our first indication that dolabralexins were important for how plants deal with stress. I decided to keep test-driving this mutant a little bit further. And so, I planted the mutant and a wild type, or normal, plant next door. And I looked at what might be happening in the roots. Now, just like we have microbes in our gut that keep us healthy, plants have microbes in the soil surrounding their roots, again, part of this underground system that we often don’t think about. And just like the microbes in your gut help you stay healthy and digest your food, microbes in the soil give plants access to new nutrients, protection from drought and disease. It’s a very exciting and ongoing area of research across the globe. So, I wanted to understand, are the microbes surrounding plant roots affected by terpenes in the roots? And the answer was, yes. I looked at these microbial communities by looking at some genetics and understood that, indeed, the microbial communities surrounding the roots of the mutant were different than that of the normal plant. This to me was one of the most exciting times in my graduate student career, because I was able to do this experiment not knowing what was gonna happen and, finally, I saw that result knowing, yes, these microbiomes are distinct. Terpenes are influencing the microbes that surround plant roots. Now, at this point, I was working with a mutant that lacked both the kauralexins and the dolabralexins. I wanted to understand specifically how dolabralexins, our new group of molecules, were related to the plant stress response. Here, I went to healthy plants. Are dolabralexins produced in normal plants under stress? Are corn plants making these to protect themselves, to fight off drought and disease? I started with drought stress. Here’s a very unhappy-looking corn plant. In order to simulate drought stress, we apply copper sulfate to the soil of growing plants. As you can see, they’re very unhappy. And then we looked into the roots to see if dolabralexins were being made under this stress. The answer was a resounding yes. Here you can see the accumulation of one of these dolabralexins, epoxydolabranol, under stress. The control makes almost no dolabralexins. This might be why we never noticed them before. We weren’t looking at the right conditions. Under stress conditions, epoxydolabranol is increasingly made. Plants are making this to protect themselves from this stress. All the other dolabralexins in that pathway were also increased. Next, I wanted to look at biotic stress, so we looked first to fungi. Now, while the fungi I showed you earlier was growing on kernels, it can also infect the roots of plants, where we previously had found dolabralexins. Now, why might roots want to protect the plant from fungi? Fungi can move from the roots up through the plant and into the ears, where they affect our food supply. Even before that, they can cause plants to die. So, I can grow fungi — this fuzzy thing on the plate, here — and apply it to the roots of healthy plants, and see how those roots respond. Are they making more dolabralexins? Again, the answer was yes. Here you can see in the control condition — no fungi was applied — only small amounts of epoxydolabranol. With two different types of fungus, we were able to induce production of these metabolites, again, a protecting mechanism against this biotic stress. Next, I asked myself, why might plants be making this molecule? How are they protecting corn plants from this fungus? Fortunately, we can grow our fungus on plates, like you see here, with my undergraduate researcher, Karen. We can grow fungus first on a plate and then move it to liquid media. Now, I told you earlier that I make these molecules in E. coli, in addition to finding them in corn. Using E. coli, I can make and purify large amounts of dolabralexins, and I can make them separately. This allows me to apply them one by one to see how they affect the growth of fungi in liquid media. The result was so fascinating. What you see here is a growth curve. Time across the bottom, and, in black, a fungus that has had no dolabralexins applied. A smooth curve. Over time, this fungus grows as it’s eating up the media. When I apply dolabralexins — here, we used epoxydolabranol in two concentrations, the blue, and higher in red — we see that growth curve is reduced. Yes, dolabralexins inhibit fungal growth in this in vitro system. They’re antifungal. Now, at this point I was curious. What about these new molecules might be providing this fungal protection? And so, we tested this, because I could purify them separately. Interestingly, dolabradiene, a precursor in the pathway that we also find in corn plants, has no effect on fungal growth. Epoxydolabranol, with this very interesting epoxide group I showed you earlier… it is… has an effect on fungal growth. These oxygen moieties are so important to this… to the function of this molecule. Now, this is how I used to think of a cornfield — a beautiful place out in the sunshine. What has this research shown me and others? There’s a whole world beneath our feet. We need to begin looking underground at the roots and the molecules that are there. I’ve shown you that dolabralexins are critical for how corn deals with stress: drought stress, fungal stress, and even how to inhibit the growth of these fungi. Now, importantly, how can we use this knowledge? How can we take this research to make our lives better? First, we could think about medicine, because these molecules are being used by corn as a medicine to fight off stress. Could we use them as a human medicine? We don’t know yet, but it’s something worth exploring. We’ve had lots of examples of success of terpenes from plants being used as human medicines. One of these examples is Taxol. Taxol is a chemotherapeutic used to treat cancer. Originally, it came from the bark of the Pacific yew tree, but we could not get enough of this molecule from the bark. After understanding the genes that control its production and the structure of these molecules, scientists are able to produce it in a lab. Another excellent example is artemisinin, the current best treatment we have for malaria. This comes from an ancient Chinese medicine. Again, after understanding how plants make this and the structure of these molecules, scientists have been able to make this into an effective drug. Important, in addition to human health, is our ability to eat. Now that we have this knowledge of these really naturally produced plant molecules, we can begin to translate it to make stronger crops. We can breed corn to have higher levels of dolabralexins, take our knowledge of these enzymes, these molecular builders, in order to make corn that is more resistant to disease. We can move this into other crops. This molecule, as I told you, is totally unique, and we can help other plants become stronger. This is essential. It’s our whole mission. We need to be able to grow more food. We need to do it with less resources. Making plants that can thrive on their own reduces our use of pesticides, reduces our freshwater inputs, and saves some of that crop, producing more food for our future. Thank you.

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