More Sustainable Feedstock for Ethanol

A six-year Great Lakes Bioenergy Research Center (GLBRC) study on the viability of different bioenergy feedstocks recently demonstrated that perennial cropping systems such as switchgrass, giant miscanthus, poplar, native grasses and prairie can yield as much biomass as corn stover.

The study is significant for addressing one of the biofuel industry’s biggest questions: Can environmentally beneficial crops produce enough biomass to make their conversion to ethanol efficient and economical?

Since 2008, research scientists Gregg Sanford and Gary Oates, based in the lab of CALS agronomy professor Randy Jackson, have worked with colleagues at Michigan State University (MSU) to cultivate more than 80 acres of crops with the potential to become feedstocks for so-called “second-generation” biofuels, that is, biofuels derived from non-food crops or the nonfood portion of plants. They’ve grown these crops at the CALS-based Arlington Agricultural Research Station and at MSU’s Kellogg Biological Station.

“We understand annual systems really well, but little research has been done on the yield of perennial cropping systems as they get established and begin to produce, or after farmland has been converted to a perennial system,” says Oates.

To find out basic information about how well certain crops produce biomass, Sanford and Oates tested the crops across two criteria: diversity of species, and whether a crop grows perennially (continuously, year after year) or annually (needing to be replanted each year).

Highly productive corn stover has thus far been the main feedstock for second-generation biofuels. And yet perennial cropping systems, which are better equipped to build soil quality, reduce runoff, and minimize greenhouse gas release into the atmosphere, confer more environmental benefits.

Corn, when grain is included, proved to be most productive over the first six-year period of the study at the Wisconsin site, but giant miscanthus, switchgrass, poplar and native grasses were not far behind. At the MSU site, where soil is less fertile, miscanthus actually produced the same amount of biomass as corn (grain included) in the experiment, with poplar and switchgrass within range.

“All of this means that, at large scales and on various soils, these crops are competitive with corn, the current dominant feedstock for ethanol,” Sanford says.

Now in the midst of the study’s eighth year, Sanford says the study will continue for the foreseeable future.

“We know that perennial systems can prevent negative impacts such as soil erosion and nitrate leaching, and that they also provide habitat for native species that provide beneficial ecosystem services,” Sanford says. “But there are still a lot of questions we want to answer about soil processes and properties— questions that take many years to answer.”

Researcher Gregg Sanford stands before a plot of giant miscanthus at Arlington.

Photo credit – Matthew Wisniewski

The Future, Unzipped

John Ralph PhD’82 talks with the easy, garrulous rhythms of his native New Zealand, and often seems amiably close to the edge of laughter.

So he was inclined toward amusement last year when he discovered that some portion of the Internet had misunderstood his latest research. Ralph—a CALS biochemist with joint appointments in biochemistry and biological systems engineering—had just unveiled a way to tweak the lignin that helps give plants their backbone. A kind of a natural plastic or binder, lignin gets in the way of some industrial processes, and Ralph’s team had cracked a complicated puzzle of genetics and chemistry to address the problem. They call it zip-lignin, because the modified lignin comes apart—roughly—like a zipper.

One writer at an influential publication called it “self-destructing” lignin. Not a bad turn of phrase—but not exactly accurate, either. For a geeky science story the news spread far, and by the time it had spread across the Internet, a random blogger could be found complaining about the dangers of walking through forests full of detonating trees.

Turning the misunderstanding into a teachable moment, Ralph went image surfing, and his standard KeyNote talk now contains a picture of a man puzzling over the shattered remains of a tree. “Oh noooo!” the caption reads. “I’ll be peacefully walking in a national park and these dang GM trees are going to be exploding all around me!”

That’s obviously a crazy scenario. But if the technology works as Ralph predicts, the potential changes to biofuels and paper production could rewrite the economics of these industries, and in the process lead to an entirely new natural chemical sector.

“When we talk to people in the biofuels industry, what we are hearing is that creating value from lignin could be game-changing,” says Timothy Donohue, a CALS professor of bacteriology and director of the UW–Madison-based Great Lakes Bioenergy Research Center, where Ralph has a lab. “It could be catalytic.”

After cellulose, lignin is the most abundant organic compound on the planet. Lignin surrounds and shapes our entire lives. Most of us have no idea—yet we are the constant beneficiaries of its strength and binding power.

When plants are growing, it’s the stiffening of the cell wall that creates their visible architecture. Carbohydrate polymers—primarily cellulose and hemicelluloses—and a small amount of protein make up a sort of scaffolding for the construction of plant cell walls. And lignin is the glue, surrounding and encasing this fibrous matrix with a durable and water-resistant polymer—almost like plastic. Some liken lignin to the resin in fiberglass.

Without lignin, the pine cannot soar into the sky, and the woody herb soon succumbs to rot. Found primarily in land plants, a form of lignin has been identified in seaweed, suggesting deep evolutionary origins as much as a billion years ago.

“Lignin is a funny thing,” says Ralph, who was first introduced to lignin chemistry as a young student during a holiday internship at New Zealand’s Forest Research Institute. “People who get into it for a little bit end up staying there the rest of their lives.”

The fascination is born, in part, from its unique chemistry. Enzymes, proteins that catalyze reactions, orchestrate the assembly of complex cell wall carbohydrates from building blocks like xylose and glucose. The types of enzymes present in cells therefore determine the composition of the wall.

Lignin is more enigmatic, says Ralph. Although its parts (called monomers) are assembled using enzymes, the polymerization of these parts into lignin does not require enzymes but instead relies on just the chemistry of the monomers and their radical coupling reactions. “It’s combinatorial, and so you make a polymer in which no two molecules are the same, perhaps anywhere in the whole plant,” says Ralph.

This flexible construction is at the heart of lignin’s toughness, but it’s also a major obstacle for the production of paper and biofuels. Both industries need the high-value carbohydrates, especially the cellulose fraction. And both have to peel away the lignin to get to the treasure inside. A combination of heat, pressure, and caustic soda is standard procedure for liberating cellulose to make paper; bleach removes the remaining lignin. In the biofuels industry, a heat and acid or alkaline treatment is often used to crack the lignin so that it is easier to produce the required simple sugars from cellulose. Leftover lignin is typically burned.

The economic cost of these treatments alone is significant, and lignin pretreatment is at the heart of many of the more egregious environmental costs of paper. On the biofuels side, lowering treatment costs to liberate carbohydrates from lignin could change the very economics of biofuels. In these large-scale, industrial processes, saving a percentage point or two is often worthwhile, but the Holy Grail is a quantum jump.

“Because it’s made this way”—Ralph jams his hands together, crazy-wise, fingers twisted together into a dramatic representation of lignin polymerization—“there is no chemistry or biology that takes it apart in an exquisite way,” he says. “We actually stepped back and thought: How would we like to design lignin? If we could introduce easily cleavable bonds into the backbone, we could break it like a hot knife through butter. How much can you actually mess with this chemistry before the tree falls down?”

Ralph’s team had their eureka moment more than 15 years ago, and have been trying to bring it to life ever since.

With a background in forage production and ruminant nutrition, John Grabber, an agronomist at the USDA–Agricultural Research Services’ Dairy Forage Research Center in Madison, got pulled into lignin chemistry through the barn door. On his family’s dairy farm he grew up with lignin stuck to his boots, though he never knew it. But during graduate school he became interested in how plants are digested by cows. Cell walls are potentially a great source of digestible carbohydrates—most plants contain anywhere from 30 to 90 percent of their mass in their cell walls—but it is entangled with lignin. “You quickly find out that lignin is the main barrier to feed digestion,” he says.

Grabber began working on a model system to understand plant lignification—for corn in particular—in 1989. After meeting at a conference, Grabber joined Ralph and plant physiologist Ronald Hatfield at the Dairy Forage Research Center back in 1992. There were many projects ongoing, but Grabber remained interested in trying to fully understand the structural characteristics of the lignin: how it’s made and how to modify it. In his model system they could make any kind of lignin they wanted to study, and see how the changes affected utilization.

Ralph and Hatfield advocated for the work, helping to find funding and offering their expertise. “If I had worked for somebody else I probably wouldn’t be doing this work,” says Grabber. “John and Ron gave me freedom and support to do it.”

Around the same time, Fachuang Lu joined Ralph’s lab seeking a Ph.D. His journey into lignin chemistry was not, at first, his idea. A native of mainland China, he’d enjoyed a successful undergraduate career in Beijing studying chemical engineering, then found himself assigned by the college to a master’s program in lignin chemistry. Lignin is an ingredient in the slurry of chemicals used in oil drilling, and that was his specialty. In 1989 Lu left Beijing for a teaching position at Guangxi University, but three years later he decided to continue his education. Though he’d never met Ralph, he was fascinated by the chemistry and applied to study in his lab.

As Ralph, Grabber, Hatfield and Lu continued to tinker with lignin chemistry, momentum began to build in the lab. Though lignin created a snowflake universe of different molecules, there were rules of assembly. A complex chemical pathway enabled lignin construction, with a mechanism that remained constant across different families of plants, but with many potential building blocks.

Ralph and his colleagues were the first to detail what was happening to lignin as the controlling genes of the biosynthetic pathway were turned on and off, a task ably completed by a slew of outstanding collaborators worldwide with expertise in biotechnological methods—but who lacked the diagnostic structural tools to determine what the plant was doing in response.

Ralph’s team quickly learned that lignification was somewhat flexible. “We figured that we could engineer lignin well beyond the previously held bounds,” says Ralph. As various pathways and chemical possibilities danced in their heads, it struck them: What if, during lignification, they could persuade the plant to slip in a few monomers that had easily broken chemical bonds? If they did it right, lignin would retain its structural value to the plant, but be easier to deal with chemically.

“In the course of our conversation we realized that if plants could do this, it could really revolutionize how readily you could make paper,” recalls Grabber. Says Ralph: “It’s almost impossible to tell which one of us actually verbalized it first—it is one of those great outcomes of the group dynamic.”

Lu’s particular genius was synthesizing the various complex chemicals needed, particularly a novel monomer-conjugate called coniferyl ferulate. It was the key to the zip-lignin—the teeth of the zipper. “He’s got to be one of the best in terms of making molecules,” says Grabber.

They were thrilled by such a revelation, but, in retrospect, they soon realized it was sort of an obvious idea—one suggested by the underlying chemistry and biochemistry of a pathway that was becoming increasingly well understood. Yet it was a discovery of huge potential value. They dropped into stealth mode and began to work on it. They finished important research and stuck it in drawers—signature research, the kind that, when finally published, would capture journal covers. And yet they sat on it, quietly chipping away for nearly a decade.

It helped that there was a flurry of controversy in the field—what Chemical & Engineering News called “the lignin war.” “Part of the reason we could sit on it was that, at the time, making these kinds of molecules was so far-fetched,” says Grabber. “Probably if we had talked about it, people would have laughed at us.”

But as the idea for zip-lignin grew in principle, it became stronger. Lu, Hatfield and colleague Jane Marita MS’97 PhD’01 found that balsa trees and a fiber crop known as kenaf produced very small amounts of coniferyl ferulate. But even as the idea seemed more and more feasible, Hatfield and Marita couldn’t isolate the gene needed to manufacture coniferyl ferulate because of its very low expression in these plants.

And they got stuck. “At the beginning we were thinking that this is just a fantastic idea, but we really didn’t have that much confidence,” says Lu. “Maybe John [Ralph] had more confidence than me.” So they just kept at it. “Every step you think, yes, we are closer, closer, closer.”

In 2008 Ralph moved his work from the Dairy Forage Research Center into UW labs, with research projects under the recently formed Great Lakes Bioenergy Research Center (GLBRC). The center, launched with a $125 million grant from the U.S. Department of Energy that has since been renewed, was just one manifestation of the money and intellectual heft infusing biofuels research—and for zip-lignin it was a lucky move.

During the center’s first full meeting, Curtis Wilkerson, a plant biologist at GLBRC partner Michigan State University, was sitting in the audience when Ralph took his turn at the podium.

Wilkerson is a cell wall specialist. Though lignin is a third of the wall’s carbon and is essential to the way plants conduct water, he confesses he’d never given it much thought. In a room full of cell wall specialists, Ralph would “likely be the only person talking about lignin,” he says. “It just split that way a long time ago. People like myself had very little exposure to what John was thinking.”

It was this kind of academic silo that a place like GLBRC was supposed to breach. Ralph talked about putting ester bonds into lignins and his team’s long search for the elusive enzyme. Wilkerson saw a solution. Due to recent technical advances, the price of determining all of the expressed enzymes in a plant had become more refined and much less expensive. He offered to use these recent developments to try to find the missing enzyme to enable zip-lignin.

From the previous work, Wilkerson knew essentially the size and shape of the puzzle piece he was looking for. He began, quite literally with Google, trolling through the scientific literature looking for a plant that made a lot of coniferyl ferulate. The Chinese medicinal “dong quai” or Chinese angelica (Angelica sinensis) soon emerged as a candidate. Its roots contained about 2 percent coniferyl ferulate.

The team used knowledge about the likely type of enzyme they were searching for and successfully identified the gene and its enzyme that could produce coniferyl ferulate. The whole search took less than six months.

Would you believe an essential tool for the genetic engineering of poplars is a hole punch? That’s the word from Shawn Mansfield, a molecular biochemist at the University of British Columbia, who took the zip-gene from the Angelica and made it work in poplar, a popular tree in the biomass and forest products industry.

Working from Wilkerson’s gene, the first job was figuring out how to tag the new protein so that it fluoresced during imaging. While not necessary to the function of the genetically modified plant, it essentially allows the scientists to check their work: see where the protein is, how much is there, and if it is behaving as a protein should.
Mansfield’s lab also had to find a way to turn the gene on at the right time and place. It could make all the coniferyl ferulate one wanted, but if it wasn’t made at the right time and tissue, there would be no zip-lignin.

After perfecting these finer points, the gene is inserted into a special bacterium—and then the hole punch finally comes into play. Disks punched from poplar leaves are mixed with bacteria that have been inoculated with a special chemical that stimulates the bacteria to share their DNA around. Then the leaf disks are put in a special growth medium. As many as 12 shoots might emerge off of a single disk, but the lab would select and nurture only one shoot from each disk.

In the end they had about 15 successful transgenic candidates that they grew in the greenhouse and then shipped off to Wilkerson and Ralph for further study. Final selection was made based on the amount of fluorescent yellow the trees gave off, and from a newly devised analytical method developed by Lu and Ralph that was particularly diagnostic for the incorporation of the zip monomer into the lignin polymer.

The team knew that genetically modified organisms are not popular or easily talked about—never mind the exploding trees. The idea of reworking a fundamental building block of the plant world will breed resistance.

Ralph argues that this is already part of nature’s vocabulary: they’ve found their building blocks within the plant kingdom, including mutants that do similar things. And now that they know what they are looking for, Steven Karlen, a member of Ralph’s group, is continuing to find more evidence that Mother Nature is doing it herself. “We managed to persuade plants to do this,” Ralph says. “Chances are that nature has already attempted it and you could actually get there by breeding.”

It’s no surprise that Mansfield, who created the final transgenic tree, argues that there is a role for this kind of technology. “We as scientists should be wise in advocating for the proper use of it,” he cautions. “I would never force it on anybody. I would never try to sway people to think that it is the end-all or be-all for everything.”
But given the growing human population and rising CO2 levels, something like zip-lignin has a definite use in reducing the carbon footprint by reducing processing energy and chemical loads. “That means there are less environmental pollutants that need to be cleaned up afterwards,” Mansfield says.

“Our ecological footprint can be much reduced using these kinds of transgenic trees,” he argues. “The caveat is that we need to be very smart about where and how we plant them.”

Not many things in the natural world can take apart lignin, but any homeowner with a deck knows that fungi are up to the task. A recent analysis of mushroom genomes suggests that fungi evolved this ability about 300 million years ago. This is about the end of the Carboniferous era, when earth’s coal production began to slow down. Coincidence? Perhaps not. Now that wood could rot, it probably slowed the burial of organic carbon via tree trunks and other lignin-rich plants.

Could the discovery of zip-lignin signal another transition, and hasten our move away from fossil fuels laid down in the Carboniferous?

Tim Donohue likes to think so. He likens biofuels now to the early oil industry, when oil was simply being turned into liquid fuel while the by-products were burned or dumped. It took a few decades for inventors to capitalize on this now valuable stream of raw materials to build the modern chemical industry.

“Lignin is about 25 to 30 percent of carbon in the plant. So if we’re going to catalyze an industry that makes clean energy and chemicals from plant biomass, figuring out what to do with the lignin is going to be key,” Donohue says.

People in the industry used to joke that you could do a lot of things with lignin except make money from it. But that may be changing. “The economics and profitability of the industry will be very different if lignin can be turned into valuable compounds,” says Donohue.

One of the early efforts to make use of lignin was in Rothschild, Wisconsin, at a company now known as Borregaard LignoTech. When processed properly, lignin has many uses, from the manufacture of vanilla flavor to additives for concrete. There is even a small amount of it in the battery of your car that allows it to keep recharging.

Jerry Gargulak is research manager at Borregaard LignoTech, and learned about zip-lignin recently in his capacity as a scientific advisor to the GLBRC. Despite its many uses, Gargulak and his colleagues dream about a time when lignin can replace carbon black in tires and be used to build carbon fibers and structural plastics.

Zip-lignin and the ideas behind it could bring this day closer. “It gives us a technology that might yield a more interesting lignin-derived starting material,” Gargulak says. “It could potentially lead to a lot of innovation downstream in lignin technology.” But he emphasizes, “There are a lot of i’s to be dotted and t’s to be crossed.”

This story is just beginning. Zip-lignin has a patent and has excited industrial interest that could be worth significant dollars. Ralph and his colleagues continue working to further refine the process, increasing the percentage of zippable bonds in poplar and also inserting the gene into more plants, such as corn and Brachypodium, both grasses.
And in the basement of the shiny new Wisconsin Energy Institute building, where the GLBRC is based, two massive new nuclear magnetic resonance (NMR) spectrometers work 24/7, providing a level of detail into lignin that Ralph has never had before.

“We spend a lot of time looking at these Rorschach test–like figures,” Ralph says of the information generated from the NMR. “The detail in them is unbelievable. These things have been revolutionizing what we do.”

Uganda: The Benefits of Biogas

Generating enthusiasm for a new kind of technology is key to its long-term success. Rebecca Larson, a CALS professor of biological systems engineering, has already accomplished that goal in Uganda, where students at an elementary school in Lweeza excitedly yell “Biogas! Biogas!” after learning about anaerobic digester systems.

Larson, a UW–Extension biowaste specialist and an expert in agricultural manure management, designs, installs and upgrades small-scale anaerobic digester (AD) systems in developing countries. Her projects are funded by the Wisconsin Energy Institute at UW–Madison and several other sources. Community education and outreach at schools and other installation sites are an important part of these efforts.

Children get excited by the “magic” in her work, she says. “It’s converting something with such a negative connotation as manure into something positive,” Larson notes. In an AD system, this magic is performed by bacteria that break down manure and other organic waste in the absence of oxygen.

The resulting biogas, a form of energy composed of methane and carbon dioxide, can be used directly for cooking, lighting, or heating a building, or it can fuel an engine generator to produce electricity.

Larson’s collaborators in Uganda include Sarah Stefanos and Aleia McCord, graduate students at the Nelson Institute for Environmental Studies who joined forces with fellow students at Makarere University in Kampala to start a company called Waste 2 Energy Ltd.
Along with another company, Green Heat Uganda, which has built a total of 42 digesters, Waste 2 Energy has helped install four AD systems since 2011.

“Most of these digesters are locally built underground dome systems at schools and orphanages,” Larson explains. Lweeza’s elementary school is a perfect example.

The AD systems use food waste, human waste from pit latrines and everything in between. The biogas generated by the digester is run through a pipeline to a kitchen stove where the children’s meals are prepared. Compared to traditional charcoal cooking, the AD systems greatly reduce the school’s greenhouse gas emissions.

Larson and her team are now focusing on enhancing the efficiency and environmental benefits of these systems. Their goals are to improve the digester’s management of human waste, reduce its water needs, increase the amount of energy it produces and generate cheap fertilizer to boost food crop yields.

“Our overall goal is to create a closed-loop and low-cost sustainability package that addresses multiple local user needs,” Larson says.

The beauty of the project is that all these needs can be met by simply adding two new components to the existing systems: heating elements and a solid-liquid separator.

To help visualize the impact of the fertilizer, Larson set up demonstration plots that compare crop yields with and without it. Down the road, a generator could be added to the system to provide electricity in a country where only 9 percent of the population currently has access.

As a next step, Larson hopes to replicate the project’s success in Bolivia. She is finalizing local design plans with Horacio Aguirre-Villegas, her postdoctoral fellow in biological systems engineering, and their collaborators at the Universidad Amazonica de Pando in Cobija.

Give: Hands-On Fieldwork

Before last summer, Vera Swanson’s only exposure to plant sciences had been through classes in introductory biology. That changed big-time when Swanson, a junior majoring in environmental sciences and Russian, signed on to intern at the CALS-based Arlington Agricultural Research Station as a crop scout.

Crop scouts are used in agricultural management to diagnose stress factors in a field—such elements as potentially negative soil and climate conditions, the presence of pests, and threatened crop performance—and determine which management practices are appropriate for the goals of a specific plot. As part of her training, Swanson spent copious hours learning to identify weeds by walking through the fields and the Weed Garden, which displays dozens of invasive plants accompanied by their names.

Swanson paired her internship, which was run through the Department of Agronomy, with an independent research project involving biofuel crops being tested at Arlington. For that work Swanson drew on her growing knowledge of weeds to test the effect of three biofuel crop systems—native prairie, switchgrass and continuous corn—on the soil’s weed seed bank, or the viable seeds present in the soil and its surface. The project involved working one-on-one with research scientists in Randy Jackson’s grassland ecology lab. Jackson is running the crop trials through his affiliation with the UW’s Great Lakes Bioenergy Research Center, housed in the Wisconsin Energy Institute.

The intense focus on plants got Swanson thinking a lot more about soil. “It is such a finite resource, yet so much of what we depend on comes from it—our food, clothing and the materials that we build with,” says Swanson.

It also got her more interested in food systems, to the point where she chose to make horticulture a disciplinary focus within her major and a possible new career direction. “I’d love to work for an organization where I would be able to complement my interests in agriculture, development and language within a global context,” she says.

Swanson’s path exemplifies the power of “beyond classroom” experiences to dramatically shape, and in many cases transform, a student’s education and career goals. These experiences—which include internships, research projects, study abroad, honors thesis stipends, field courses and more—are the hallmark of a CALS education.

“They’re a big part of what makes CALS CALS—and they offer our students a major advantage in both their personal and professional development,” says Sarah Pfatteicher, the college’s associate dean for academic affairs. “Our goal is to ensure that each student can participate in at least four of these important opportunities.”

To help support the CALS Student Experience Fund, visit: http://go.wisc.edu/student-experience

Plant Prowess

It may look jury-rigged, but it’s cutting-edge science.

In a back room in the university’s Seeds Building, researchers scan ears of corn—three at a time—on a flatbed scanner, the kind you’d find at any office supply store. After running the ears through a shelling machine, they image the de-kerneled cobs on a second scanner.

The resulting image files—up to 40 gigabytes’ worth per day—are then run through a custom-made software program that outputs an array of yield-related data for each individual ear. Ultimately, the scientists hope to link this type of information—along with lots of other descriptive data about how the plants grow and what they look like—back to the genes that govern those physical traits. It’s part of a massive national effort to deliver on the promise of the corn genome, which was sequenced back in 2009, and help speed the plant breeding process for this widely grown crop.

“When it comes to crop improvement, the genotype is more or less useless without attaching it to performance,” explains Bill Tracy, professor and chair of the Department of Agronomy. “The big thing is phenotyping—getting an accurate and useful description of the organism—and connecting that information back to specific genes. It’s the biggest thing in our area of plant sciences right now, and we as a college are playing a big role in that.”

No surprise there. Since the college’s founding, plant scientists at CALS have been tackling some of the biggest issues of their day. Established in 1889 to help fulfill the University of Wisconsin’s land grant mission, the college focused on supporting the state’s fledgling farmers, helping them figure out how to grow crops and make a living at it. At the same time, this practical assistance almost always included a more basic research component, as researchers sought to understand the underlying biology, chemistry and physics of agricultural problems.

That approach continues to this day, with CALS plant scientists working to address the ever-evolving agricultural and natural resource challenges facing the state, the nation and the world. Taken together, this group constitutes a research powerhouse, with members based in almost half of the college’s departments, including agronomy, bacteriology, biochemistry, entomology, forest and wildlife ecology, genetics, horticulture, plant pathology and soil science.

“One of our big strengths here is that we span the complete breadth of the plant sciences,” notes Rick Lindroth, associate dean for research at CALS and a professor of entomology. “We have expertise across the full spectrum—from laboratory to field, from molecules to ecosystems.”

This puts the college in the exciting position of tackling some of the most complex and important issues of our time, including those on the applied science front, the basic science front—and at the exciting new interface where the two approaches are starting to intersect, such as the corn phenotyping project.

“The tools of genomics, informatics and computation are creating unprecedented opportunities to investigate and improve plants for humans, livestock and the natural world,” says Lindroth. “With our historic strength in both basic and applied plant sciences, the college is well positioned to help lead the nation at this scientific frontier.”

It’s hard to imagine what Wisconsin’s agricultural economy would look like today without the assistance of CALS’ applied plant scientists.

The college’s early horticulturalists helped the first generation of cranberry growers turn a wild bog berry into an economic crop. Pioneering plant pathologists identified devastating diseases in cabbage and potato, and then developed new disease-resistant varieties. CALS agronomists led the development of the key forage crops—including alfalfa and corn—that feed our state’s dairy cows.

Fast-forward to 2015: Wisconsin is the top producer of cranberries, is third in the nation in potatoes and has become America’s Dairyland. And CALS continues to serve the state’s agricultural industry.

The college’s robust program covers a wide variety of crops and cropping systems, with researchers addressing issues of disease, insect and weed control; water and soil conservation; nutrient management; crop rotation and more. The college is also home to a dozen public plant-breeding programs—for sweet corn, beet, carrot, onion, potato, cranberry, cucumber, melon, bean, pepper, squash, field corn and oats—that have produced scores of valuable new varieties over the years, including a number of “home runs” such as the Snowden potato, a popular potato chip variety, and the HyRed cranberry, a fast-ripening berry designed for Wisconsin’s short growing season.

While CALS plant scientists do this work, they also train the next generation of researchers—lots of them. The college’s Plant Breeding and Plant Genetics Program, with faculty from nine departments, has trained more graduate students than any other such program in the nation. Just this past fall, the Biology Major launched a new plant biology option in response to growing interest among undergraduates.

“If you go to any major seed company, you’ll find people in the very top leadership positions who were students here in our plant-breeding program,” says Irwin Goldman PhD’91, professor and chair of the Department of Horticulture.

Among the college’s longstanding partnerships, CALS’ relationship with the state’s potato growers is particularly strong, with generations of potato growers working alongside generations of CALS scientists. The Wisconsin Potato and Vegetable Growers Association (WPVGA), the commodity group that supports the industry, spends more than $300,000 on CALS-led research each year, and the group helped fund the professorship that brought Jeff Endelman, a national leader in statistical genetics, to campus in 2013 to lead the university’s potato-breeding program.

“Research is the watchword of the Wisconsin potato and vegetable industry,” says Tamas Houlihan, executive director of the WPVGA. “We enjoy a strong partnership with CALS researchers in an ongoing effort to solve problems and improve crops, all with the goal of enhancing the economic vitality of Wisconsin farmers.”

Over the decades, multi-disciplinary teams of CALS experts have coalesced around certain crops, including potato, pooling their expertise.

“Once you get this kind of core group working, it allows you to do really high-impact work,” notes Patty McManus, professor and chair of the Department of Plant Pathology and a UW–Extension fruit crops specialist.

CALS’ prowess in potato, for instance, helped the college land a five-year, $7.6 million grant from the U.S. Department of Agriculture to help reduce levels of acrylamide, a potential carcinogen, in French fries and potato chips. The multistate project involves plant breeders developing new lines of potato that contain lower amounts of reducing sugars (glucose and fructose) and asparagine, which combine to form acrylamide when potatoes are fried. More than a handful of conventionally bred, low-acrylamide potato varieties are expected to be ready for commercial evaluations within a couple of growing seasons.

“It’s a national effort,” says project manager Paul Bethke, associate professor of horticulture and USDA-ARS plant physiologist. “And by its nature, there’s a lot of cross-talk between the scientists and the industry.”

Working with industry and other partners, CALS researchers are responding to other emerging trends, including the growing interest in sustainable agricultural systems.

“Maybe 50 years ago, people focused solely on yield, but that’s not the way people think anymore. Our crop production people cannot just think about crop production, they have to think about agroecology, about sustainability,” notes Tracy. “Every faculty member doing production research in the agronomy department, I believe, has done some kind of organic research at one time or another.”

Embracing this new focus, over the past two years CALS has hired two new assistant professors—Erin Silva, in plant pathology, who has responsibilities in organic agriculture, and Julie Dawson, in horticulture, who specializes in urban and regional food systems.

“We still have strong partnerships with the commodity groups, the cranberries, the potatoes, but we’ve also started serving a new clientele—the people in urban agriculture and organics that weren’t on the scene for us 30 years ago,” says Goldman. “So we have a lot of longtime partners, and then some new ones, too.”

Working alongside their applied colleagues, the college’s basic plant scientists have engaged in parallel efforts to reveal fundamental truths about plant biology—truths that often underpin future advances on the applied side of things.

For example, a team led by Aurélie Rakotondrafara, an assistant professor of plant pathology, recently found a genetic element—a stretch of genetic code—in an RNA-based plant virus that has a very useful property. The element, known as an internal ribosome entry site, or IRES, functions like a “landing pad” for the type of cellular machine that turns genes—once they’ve been encoded in RNA—into proteins. (A Biology 101 refresher: DNA—>RNA—>Protein.)

This viral element, when harnessed as a tool of biotechnology, has the power to transform the way scientists do their work, allowing them to bypass a longstanding roadblock faced by plant researchers.

“Under the traditional mechanism of translation, one RNA codes for one protein,” explains Rakotondrafara. “With this IRES, however, we will be able to express several proteins at once from the same RNA.”

Rakotondrafara’s discovery, which won an Innovation Award from the Wisconsin Alumni Research Foundation (WARF) this past fall and is in the process of being patented, opens new doors for basic researchers, and it could also be a boon for biotech companies that want to produce biopharmaceuticals, including multicomponent drug cocktails, from plants.

Already, Rakotondrafara is working with Madison-based PhylloTech LLC to see if her new IRES can improve the company’s tobacco plant-based biofarming system.

“The idea is to produce the proteins we need from plants,” says Jennifer Gottwald, a technology officer at WARF. “There hasn’t been a good way to do this before, and Rakotondrafara’s discovery could actually get this over the hump and make it work.”

While Rakotondrafara is a basic scientist whose research happened to yield a powerful application, CALS has a growing number of scientists—including those involved in the corn phenotyping project—who are working at the exciting new interface where basic and applied research overlap. This new space, created through the mind-boggling advances in genomics, informatics and computation made in recent years, is home to an emerging scientific field where genetic information and other forms of “big data” will soon be used to guide in-the-field plant-breeding efforts.

Sequencing the genome of an organism, for instance, “is almost trivial in both cost and difficulty now,” notes agronomy’s Bill Tracy. But a genome—or even a set of 1,000 genomes—is only so helpful.

What plant scientists and farmers want is the ability to link the genetic information inside different corn varieties—that is, the activity of specific genes inside various corn plants—to particular plant traits observed in the greenhouse or the field. The work of chronicling these traits, known as phenotyping, is complex because plants behave differently in different environments—for instance, growing taller in some regions and shorter in others.

“That’s one of the things that the de Leon and Kaeppler labs are now moving their focus to—massive phenotyping. They’ve been doing it for a while, but they’re really ramping up now,” says Tracy, referring to agronomy faculty members Natalia de Leon MS’00 PhD’02 and Shawn Kaeppler.

After receiving a large grant from the Great Lakes Bioenergy Research Center in 2007, de Leon and Kaeppler decided to integrate their two research programs. They haven’t looked back. With de Leon’s more applied background in plant breeding and field evaluation, plus quantitative genetics, and with Kaeppler’s more basic corn genetics expertise, the two complement each other well. The duo have had great success securing funding for their various projects from agencies including the National Science Foundation, the U.S. Department of Agriculture and the U.S. Department of Energy.

“A lot of our focus has been on biofuel traits, but we measure other types of economically valuable traits as well, such as yield, drought tolerance, cold tolerance and others,” says Kaeppler. Part of the work involves collaborating with bioinformatics experts to develop advanced imaging technologies to quantify plant traits, projects that can involve assessing hundreds of plants at a time using tools such as lasers, drone-mounted cameras and hyperspectral cameras.

This work requires a lot of space to grow and evaluate plants, including greenhouse space with reliable climate control in which scientists can precisely measure the effects of environmental conditions on plant growth. That space, however, is in short supply on campus.

“A number of our researchers have multimillion-dollar grants that require thousands of plants to be grown, and we don’t always have the capacity for it,” says Goldman.

That’s because the Walnut Street Greenhouses, the main research greenhouses on campus, are already packed to the gills with potato plants, corn plants, cranberries, cucumbers, beans, alfalfa and dozens of other plant types. At any given moment, the facility has around 120 research projects under way, led by 50 or so different faculty members from across campus.

Another bottleneck is that half of the greenhouse space at Walnut Street is old and sorely outdated. The facility’s newer greenhouses, built in 2005, feature automated climate control, with overlapping systems of fans, vents, air conditioners and heaters that help maintain a pre-set temperature. The older houses, constructed of single-pane glass, date back to the early 1960s and present a number of challenges to run and maintain. Some don’t even have air conditioning—the existing electrical system can’t handle it. Temperatures in those houses can spike to more than 100 degrees during the summer.

“Most researchers need to keep their plants under fairly specific and constant conditions,” notes horticultural technician Deena Patterson. “So the new section greenhouse space is in much higher demand, as it provides the reliability that good research requires.”

To help ameliorate the situation, the college is gearing up to demolish the old structures and expand the newer structure, adding five more wings of greenhouse rooms, just slightly north of the current location—out from under the shadow of the cooling tower of the West Campus Co-Generation Facility power plant, which went online in 2005. The project, which will be funded through a combination of state and private money, is one of the university’s top building priorities.

Fortunately, despite the existing limitations, the college’s plant sciences research enterprise continues apace. Kaeppler and de Leon, for example, are involved in an exciting phenotyping project known as Genomes to Fields, which is being championed by corn grower groups around the nation. These same groups helped jump-start an earlier federal effort to sequence the genomes of many important plants, including corn.

“Now they’re pushing for the next step, which is taking that sequence and turning it into products,” says Kaeppler. “They are providing initial funding to try to grow Genomes to Fields into a big, federally funded initiative, similar to the sequencing project.”

It’s a massive undertaking. Over 1,000 different varieties of corn are being grown and evaluated in 22 environments across 13 states and one Canadian province. Scientists from more than a dozen institutions are involved, gathering traditional information about yield, plant height and flowering times, as well as more complex phenotypic information generated through advanced imaging technologies. To this mountain of data, they add each corn plant’s unique genetic sequence.

“You take all of this data and just run millions and billions of associations for all of these different traits and genotypes,” says de Leon, who is a co-principal investigator on the project. “Then you start needing supercomputers.”

Once all of the dots are connected—when scientists understand how each individual gene impacts plant growth under various environmental conditions—the process of plant breeding will enter a new sphere.

“The idea is that instead of having to wait for a corn plant to grow for five months to measure a certain trait out in the field, we can now take DNA from the leaves of little corn seedlings, genotype them and make decisions within a couple of weeks regarding which ones to advance and which to discard,” says de Leon. “The challenge now is how to be able to make those types of predictions across many environments, including some that we have never measured before.”

To get to that point, notes de Leon, a lot more phenotypic information still needs to be collected—including hundreds and perhaps thousands more images of corn ears and cobs taken using flatbed scanners.

“Our enhanced understanding of how all of these traits are genetically controlled under variable environmental conditions allows us to continue to increase the efficiency of plant improvement to help meet the feed, food and fiber needs of the world’s growing population,” she says.

Sidebar:

The Bigger Picture

Crop breeders aren’t the only scientists doing large-scale phenotyping work. Ecologists, too, are increasingly using that approach to identify the genetic factors that impact the lives of plants, as well as shape the effects of plants on their natural surroundings.

“Scientists are starting to look at how particular genes in dominant organisms in an environment—often trees—eventually shape how the ecosystem functions,” says entomology professor Rick Lindroth, who also serves as CALS’ associate dean for research. “Certain key genes are driving many fantastically interesting and important community- and ecosystem-level interactions.”

How can tree genes have such broad impacts? Scientists are discovering that the answer, in many cases, lies in plant chemistry.
“A tree’s chemical composition, which is largely determined by its genes, affects the community of insects that live on it, and also the birds that visit to eat the insects,” explains Lindroth. “Similarly, chemicals in a tree’s leaves affect the quality of the leaf litter on the ground below it, impacting nutrient cycling and nitrogen availability in nearby soils.”

A number of years ago Lindroth’s team embarked on a long-term “genes-to-ecosystems” project (as these kinds of studies are called) involving aspen trees. They scoured the Wisconsin landscape, collecting root samples from 500 different aspens. From each sample, they propagated three or four baby trees, and then in 2010 planted all 1,800 saplings in a so-called “common garden” at the CALS-based Arlington Agricultural Research Station.

“The way a common garden works is, you put many genetic strains of a single species in a similar environment. If phenotypic differences are expressed within the group, then the likelihood is that those differences are due to their genetics, not the environment,” explains Lindroth.

Now that the trees have had some time to grow, Lindroth’s team has started gathering data about each tree—information such as bud break, bud set, tree size, leaf shape, leaf chemistry, numbers and types of bugs on the trees, and more.

Lindroth and his partners will soon have access to the genetic sequence of all 500 aspen genetic types. Graduate student Hilary Bultman and postdoctoral researcher Jennifer Riehl will do the advanced statistical analysis involved—number crunching that will reveal which genes underlie the phenotypic differences they see.

In this and in other projects, Lindroth has called upon the expertise of colleagues across campus, developing strategic collaborations as needed. That’s easy to do at UW–Madison, notes Lindroth, where there are world-class plant scientists working across the full spectrum of the natural resources field—from tree physiology to carbon cycling to climate change.

“That’s the beauty of being at a place like Wisconsin,” Lindroth says.

Want to help? The college welcomes your gift toward modernizing the Walnut Street Greenhouses. To donate, please visit: supportuw.org/giveto/WalnutGreenhouse. We thank you for your contribution.
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