The People's Tree
ORNL teams seek to make the poplar tree an affordable raw material for biofuels and bioproducts.
In the days of the Roman Empire, people met under trees with leaves that fluttered noisily in the slightest breeze. These trees came to be known as arbor populi, or "the people's tree." The poplar tree, also known as cottonwood and aspen, certainly stands up to the name.
For centuries, foresters have treasured black cottonwoods (Populus trichocarpa) because they can be sold for lumber, plywood and pulp for paper. Today, scientists also believe the species holds in its woody fibers the fuel needed to shift a significant segment of America's foreign oil dependency to local, renewable energy sources in the not-so-distant future.
When Oak Ridge National Laboratory managed the Bioenergy Feedstock Development Program for the Department of Energy from 1978 to 2001, the ORNL team chose to focus on the improvement and management of just a few "energy crops" rather than a multitude of grass and tree species. They selected switchgrass and two tree species—willow and poplar—for carbon sequestration and bioenergy studies.
Researchers gathered data on these rapidly growing plants and applied breeding techniques for crop improvement to increase plant productivity and the drought tolerance of these energy crops. Their study determined the best crop management practices, such as the minimum amount of water, fertilizer and pesticides needed as well as when and how often these should be applied.
Since the completion of the human genome sequencing project, DOE's Joint Genome Institute in California has been determining the order of DNA bases in hundreds of onecelled microorganisms. Three years ago, Stan Wullschleger, a plant physiologist and leader of plant molecular ecology research at ORNL, and plant geneticist Jerry Tuskan in Wullschleger's group proposed that a multinational partnership sequence the first tree genome.
Sequencing the Populus genome
"It is difficult to move forest biology forward by studying trees in a greenhouse or forest," Tuskan says. "We proposed to DOE that we could accelerate the development of bioenergy crops by sequencing the genome of the black cottonwood."
One way ORNL promoted the decoding of the hybrid poplar was through the formation of the International Populus Genome Consortium to support the effort. DOE provided funding for the project of sequencing the Populus genome, which was completed in less than two years by JGI and an ORNL team led by Tuskan. Tuskan then led a multinational team of 250 scientists from 34 nations in analyzing the genetic blueprint of the first tree genome. Tuskan worked closely with scientists at JGI and universities in the United States, Austria, Canada, Belgium, Finland, France, Germany and Sweden. The analysis of the complete sequence of the genome of a female black cottonwood tree that grew along the Nisqually River in Washington was published September 15, 2006, in Science magazine.
"We think we have captured most of the 45,500 genes on 19 chromosomes in the Populus genome," Tuskan says, pointing out that in comparison the human genome is now thought to have 22,000 to 28,000 genes on 23 chromosomes. Populus has 485 million DNA base pairs, making the tree four times larger than Arabidopsis thaliana, a mustard weed and the first plant to be sequenced.
"One of the four algorithms 'trained' to find genes in Populus was developed at ORNL," Tuskan adds. "Oak Ridge researchers annotated the sequence to predict the functions of genes, including those associated with stem growth, biomass production and other bioenergy-relevant abilities. Our task now is to use that information to accelerate the domestication of the poplar tree for biofuels and biomaterials development."
"The poplar genome project is a major step toward the day when the production of biofuels becomes a major element in the world's energy supply," says ORNL Director Jeff Wadsworth.
ORNL researchers are among the world's leaders in woody plant genomics, says Brian Davison, the Laboratory's chief scientist for systems biology and biotechnology. He explains that ORNL plant scientists have a unique opportunity to "design" poplars for carbon sequestration and bioenergy. "The Lab's researchers have identified carbon partitioning genes and carbon allocation genes," he says. "These genes determine the relative amounts of carbon from atmospheric carbon dioxide that are stored in a tree's roots, trunk, branches and leaves."
Knowledge of the poplar's genetic code will enable plant geneticists to modify poplars to grow more wood and a higher fraction of cellulose and hemicellulose. Aboveground tree biomass is 26 percent lignin, 20 percent hemicellulose and 44 percent cellulose.
Tuskan's colleagues found 93 protein-coding genes associated with the production of cellulose, hemicellulose and lignin, the building blocks of the tree's cell walls. Enzymes can degrade cellulose and hemicellulose into sugars. These sugars, in turn, can be fermented into alcohol, which is distilled to yield fuel-quality ethanol.
Making cellulosic ethanol affordable
Now that researchers have learned how to make ethanol from cellulose, the next challenge is to make the process affordable for commercial use. To help lower the cost of converting cellulose from trees to ethanol, Tuskan and his ORNL colleagues are analyzing the Populus genome to identify genes that could be disabled or amplified to create a tree with more desirable traits. The researchers would like to increase the poplar's productivity and its ability to tolerate stresses such as drought and extremely cold weather, as well as resist attacks by insects and pathogens.
Accelerated domestication of the poplar tree is desirable to create a cost-effective feedstock plant tailored for economical ethanol production. Tuskan cites corn as an illustration. American cornfields today have 25,000 corn stems per acre. At the beginning of the 20th century, only 10,000 stems could be grown on an acre because the corn leaves extended laterally instead of vertically, as they do today.
"Older varieties of corn also have a phytochrome gene that allows the plant to detect light reflected from its neighbor," Tuskan says. "Plants grown too close together did not grow well. Through many decades of breeding and selection, the phytochrome gene in corn has been disabled so that the plant barely senses its neighbors."
Domestication of corn took a century. ORNL plant scientists seek to domesticate the poplar in 15 years. In five years they hope to produce trees with new properties, and within 10 years they plan to establish plantations of genetically modified poplars as energy crops in approved field tests.
For carbon sequestration, researchers have been genetically redesigning poplars to shuttle more carbon into the roots for long-term storage in the soil, slowing the buildup of climate-altering atmospheric carbon dioxide. For bioenergy, the goal is to disable or amplify poplar genes so that less carbon goes into stem height and branch growth and more carbon goes into the tree trunk's radial growth. By understanding how to manipulate bioenergy-relevant genes and molecular "dimmer" switches that turn protein-coding genes on and off or up and down, Tuskan's team hopes to produce managed tree systems with more biomass in the right places. Within the next three to five years, the ORNL researchers believe they can create a genetically modified poplar tree with a shorter, thicker trunk, or stem, and fewer branches.
Results from ORNL's bioenergy feedstock program indicate that an annual yield of 10 tons of wood per acre of poplars is both achievable and practical. "Our goal is to get 20 tons per acre per year of biomass from trees using less water and nutrients," says Tim Tschaplinski, an ORNL plant physiologist and biochemist. "We want these trees to be able to grow in most regions of the United States, even under drought conditions, and to be harvested in six to seven years.
"We hope to reconstruct the architecture of the hybrid poplar and accelerate its domestication," Tschaplinski adds. "The key features of a highly productive poplar include a narrow crown with fewer lower branches that crowd its neighbors and reduce productivity. We prefer more branching at the top of the tree's crown for the capture of light for photosynthesis. Crowns shed these upper branches after only a few years."
A shorter, broader tree with fewer branches is easier and cheaper to harvest and contains more cellulose that can be converted to ethanol. Reconstructed trees should lower the cost of producing and harvesting feedstock from forest plantations, making the tree more attractive for biorefinery operations.
Shorter is better
Research results obtained by Udaya Kalluri suggest that the plant science group at ORNL is on the right path. Kalluri has been focusing on genes known to be associated with elongation of the poplar stem. The genetic blueprint of Populus indicates that the tree has 39 auxin response factor (ARF) genes. These unique sequences of DNA bases control the auxin signal response in plants.
Auxin is a major hormone that controls many functions in plants, such as plant development, vertical growth, radial growth, branching and root development, Kalluri explains. Auxin regulates diverse developmental aspects in the plant by conveying the signals from one part of the plant to another.
Kalluri has targeted different ARF genes believed to be responsible for vertical growth. She down-regulated, or decreased the function of, each gene by designing a DNA construct that, when expressed in the plant, causes RNA interference (RNAi). Each expressed gene sends messenger RNA with instructions to the cell's protein-making machinery. In RNA interference certain molecules trigger destruction of RNA from a targeted gene so its encoded protein is never produced.
"We choose an RNAi sequence that has a certain size and DNA base composition specific to the gene we want to disable," Kalluri says. "Then we design RNAi constructs that Agrobacteria introduce into poplar tissue. The infected cells that each contain the RNAi construct divide further, producing a callus-like mass of transformed cells.
"We grow the genetically modified plants in our greenhouse," Kalluri adds. "When the saplings are a certain height, we carry out phenotypic evaluation of the genetically modified poplars and determine how their visible features differ from those of poplar trees of the same age."
After three months in the ORNL greenhouse, a wild-type poplar sapling grows to be about three feet tall with a long, slender stem. In dramatic contrast, a genetically modified tree of the same age is only a foot high and has a stocky, leaf-covered stem.
Kalluri and her colleagues showed that genetically modified trees can achieve greater radial growth and less vertical growth than the wild-type poplars. "We have identified some of the genes responsible for the targeted set of traits needed for accelerated domestication of the poplar," she says, adding that the two-year project has been renewed for three more years because of successful results.
Identifying the right genes
Some hybrid poplars are extremely productive so long as water availability is high. The ORNL strategy is to identify and regulate the expression of the poplar's native gene that makes the tree drought sensitive and increase the activity of genes that enable the tree to pull more water out of the soil.
"We are looking at upregulating, or overexpressing, only genes already present in native poplars," Tschaplinski says. "We will not introduce foreign genes from other species into our plants. Eventually, we will conduct tests to determine which poplar trees designed to be more drought tolerant are as productive as trees without this genetic modification."
Other traits that the ORNL group hopes to incorporate in the genetically modified poplar are cold tolerance, increased ability to take nitrogen from the soil so less fertilizer is needed and branching at the right heights to increase productivity. "Trees tend to grow tall and thin in a crowded environment," he says.
Tschaplinski and collaborators are interested in the chemicals that the poplar makes in response to external stimuli to protect the tree against pathogens and predators. The researchers study poplars grown in plantations at Grand Rapids and Thief River Falls in Minnesota and at Oregon State University in Corvallis. Leaf and root samples from the trees are ground and the plants' chemicals, called metabolites, are extracted. The metabolites are then analyzed.
Using gas chromatography and mass spectrometry, Tschaplinski has identified 58 compounds in the poplar's metabolome, including many carbon-containing phenolic compounds. He is also interested in identifying changes in the chemical content of trees that have been genetically modified for DOE bioenergy and carbon sequestration projects.
As part of a sequestration project, Tschaplinski is creating a genetically modified poplar that makes higher levels of phenolics and other compounds in its roots that are more resistant to breakdown by soil microbes that emit carbon dioxide to the air. "Our hypothesis is that these phenolic compounds are recalcitrant to microbial degradation and, therefore, will stick around in the soil longer," he says.
ORNL is the world's only laboratory involved in identifying, analyzing and modifying poplar genes that code for the production of metabolites of importance to DOE's missions. In addition to the agency's focus on biofuel sources, DOE is also interested in funding studies of how to genetically modify poplar and switchgrass to enhance production in biorefineries of bio-based constituents of commodity chemicals needed to make plastics and nylon substitutes. Of special interest are chemical constituents that are too difficult or expensive to make commercially using petrochemicals.
Should researchers prove successful in making the poplar a solution to America's energy needs, the tree will indeed renew its claim as the "people's tree."—Carolyn Krause
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