From EMSL: “Developing Better Biomass Feedstock”

Environmental Molecular Sciences Laboratory (EMSL)

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Multi-omics unlocking the workings of plants

Biomass holds great promise as a fuel source to generate renewable energy to help the United States achieve energy independence. Kim Hixson is applying what she’s learned from a lowly weed to bioengineer better biomass feedstock.

Hixson, an EMSL senior research scientist, is collaborating with Norman Lewis, a regents professor and director of the Institute of Biological Chemistry at Washington State University in Pullman. Together they are using EMSL’s multi-omics capabilities to better understand how manipulating the genes in one plant can be applied to other plants to improve their potential as biofuel and biochemical feedstock.

“Our research is hypothesis driven, but there’s also a lot of discovery,” says Hixson. “The more we study biology, the more we see how interconnected things are in nature.”

More Than a Weed

Central to this research is Arabidopsis, a model plant system. A small flowering plant related to cabbage, Arabidopsis makes for an ideal plant model. Its genome has been sequenced and is easy to genetically modify. Hixson calls it the “lab rat of the plant world.”

Thale cress (Arabidopsis thaliana)

Arabidopsis is a small flowering plant related to cabbage and a model organism used to research plant biology. The plant on the left is wild-type Arabidopsis at five weeks. The plant on the right is Arabidopsis at five weeks with ADT-related genes knocked out, reducing the levels of lignin.

Arabidopsis has a six-member isoenzyme family of arogenate dehydratases, or ADTs. These enzymes are involved in the catalytic reactions that turn arogenic acid, a metabolite, into phenylalanine, an essential metabolite which is incorporated directly into proteins or is further modified into other chemicals such as flavonoids, coumarins, anthocyanins and lignin.

Hixson’s research found hundreds of different changes occurring in the plant due to the modifications in the ADT composition. The collaborators at WSU discovered that out of the six ADTs in Arabidopsis, five ADTs are seemingly linked to the production of phenylalanine utilized in the phenylpropanoid pathway, which is involved in lignin production. Lignin gives plants their recalcitrance; it’s hydrophobic and difficult to degrade. Lignin is the structural material that makes the sugars in plants difficult to extract when making biofuels.

“It is well known that a significant amount of the carbon dioxide that Arabidopsis fixes goes into the phenylpropanoid pathway and ultimately ends up as lignin,” says Hixson. “This is very important from the perspective of turning plant material into biofuel.”

Hixson’s Arabidopsis studies earned her an American Chemical Society Withycombe-Charalambous Graduate Student Symposium Award. At the symposium she presented some of her findings from the multi-omics analysis of the gene knockouts conducted at EMSL. The researchers analyzed several knockout mutants of ADTs, including single, double, triple and quadruple knockout mutants, with each mutant strain producing varying degrees of lignin reduction. They found that knocking out multiple ADTs and specific ADTs leads to a measured reduction of lignin in the plants.

By knocking out different combinations of the ADT-related genes, the researchers produced plants with various levels of lignin. In the most extreme case where they knocked out four ADT-related genes, the plant was unable to hold its own weight and became vine-like.

“We knew there were a lot of changes going on in this plant, but we didn’t know at the molecular level what those changes were,” says Hixson. “The questions about what pathways are being changed and what potential points of regulation are being up-regulated or repressed are precisely what transcriptomics coupled with proteomics can answer.”

Other findings from Hixson’s research showed knocking out ADT genes alters the photosynthesis machinery and pathways in the mutant plants. For reasons not completely understood, knocking out ADT genes causes the mutant plant systems to produce more photosynthetic machinery, potentially fixing more carbon, but an overall increase in plant mass was not observed. Using transcriptomics and proteomics techniques at EMSL, the researchers were able to look at the other pathways and genes that were changed in the mutant plants. They found the photorespiration pathways were also up-regulated. While more carbon was potentially being fixed, more of it was likely being lost or released back into the atmosphere through the photorespiration pathways.

“This is potentially a very useful discovery,” says Hixson. “In future bioengineering attempts we may need to incorporate strategies to counteract carbon loss via photorespiration which would potentially improve the rates of biomass growth in ADT-altered plants used for biofuels or biochemicals.”

Applying Their Findings to Poplars

Researchers collect samples of mutant poplars to undergo multi-omics analysis to determine if the genetic changes affect other pathways and functions in the tree.
The research has developed several lines of mutant Arabidopsis by altering the composition of the ADT genes, which ultimately decreases the amount of lignin these plants produce. Questions arise about how much ADT can be lessened and thus how much lignin can be reduced before a plant in a real world setting shows detrimental growth affects. Additionally it is important to understand how these changes alter other pathways and other systems within the plant, and how the changes are altering the plant system as a whole.

“These are really important questions, not so much in Arabidopsis, which is just a little weed,” says Hixson. “But we’ve started to incorporate some of the same knockouts into poplar trees, which show good potential as a biofuel feedstock.”

Poplar trees are native to the Pacific Northwest and widely used as a feedstock in the paper industry. The Department of Energy is interested in the poplar as a biofuel. Within the missions of EMSL and the DOE Office of Biological and Environmental Research is a charge to reduce the United States’ dependence on foreign oil and to develop technology for alternative fuel and chemical options.

Hixson hopes the manipulations in the Arabidopsis translate to other plants, such as poplars. Poplar has a larger and more complex genome than Arabidopsis. She will test the altered poplars at EMSL with multi-omics analysis to see how the transcriptome and proteome changes and if she sees the same types of response in the tree as she saw in the Arabidopsis.

“I expect a lot of things are going to be similar between the two plants,” Hixson says. “But they are different systems and it will be interesting to see the changes in the poplar compared to the Arabidopsis at the molecular level.”

The researchers have incorporated the ADT knockouts into poplar trees to test if the amount of lignin can be reduced and how far it can be reduced without damaging effects to the tree as a whole. Hixson recently collected samples of the mutant poplars from a test plot and greenhouse in western Washington. She will use EMSL’s multi-omics capabilities to determine if a change to one pathway affects other pathways and functions in the tree. According to Hixson, proteomics and transcriptomics identify what genes are being affected, either positively or negatively. This information will be useful when bioengineering poplars as a feedstock. The data will also be incorporated into her dissertation for her doctorate in molecular plant sciences from WSU.

Other Proposals: Where few Have Gone Before

The study with the poplar trees is an approved EMSL user project. Lewis is the principal investigator and Hixson is a collaborator. Lewis is also Hixson’s faculty advisor.

Hixson and Lewis have several research proposals they are hoping get approved. In collaboration with Mary Lipton, an integrative omics scientist at Pacific Northwest National Laboratory, and other scientists, they submitted a proposal to NASA. In this study several of the mutant Arabidopsis would be sent into outer space to test what happens to reduced-lignin plants in a microgravity environment.

“I really hope NASA approves it,” Hixson says. “The findings could be very interesting.”

In another submission, this one in response to an EMSL internal call, Hixson and Lewis are proposing to study red alder trees as an ideal biofuel source. A red alder grows almost as fast and dense as a poplar, but it forms specialized symbiotic relationships in its root system. These symbiotic relationships produce root nodules which can fix nitrogen, allowing red alders to thrive without added fertilizer and grow on marginal lands. Hixson believes red alder has the potential to be a highly valuable source for biomass feedstock or other wood-based materials.

For this study, they will apply what they learned from the Arabidopsis and poplar research. The proposal includes a full genetic characterization of the red alder and a multi-omics study of the tree’s association with two ubiquitous root symbionts.

“Our end goal is to gather enough information throughout multi-omics evaluations to be able to bioengineer the ideal biofuel and biochemical feedstock,” says Hixson. “We’re not there yet, but we’re working on it.”

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EMSL is a national scientific user facility that is funded and sponsored by DOE’s Office of Biological & Environmental Research. As a user facility, our scientific capabilities – people, instruments and facilities – are available for use by the global research community. We support BER’s mission to provide innovative solutions to the nation’s environmental and energy production challenges in areas such as atmospheric aerosols, feedstocks, global carbon cycling, biogeochemistry, subsurface science and energy materials.

A deep understanding of molecular-level processes is critical to gaining a predictive, systems-level understanding of the impacts of aerosols and terrestrial systems on climate change; making clean, affordable, abundant energy; and cleaning up our legacy wastes. Visit our Science page to learn how EMSL leads in these areas, through our Science Themes.

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