1.1.1 Hemicellulose Biosynthesis - Mannans & Xylans

Team Members and Roles

Curtis Wilkerson, MSU project lead
Ken Keegstra, MSU project lead
David Cavalier, MSU scientist
Linda Danhof, MSU technician
Benjamin Fode, MSU post-doc
Jacob Jensen, MSU post-doc
Barbara Reca, MSU post-doc
Yan Wang, MSU post-doc
Brandon Guelette, MSU technician

1.1.2 Mutant lines for cell wall digestibility.

Altering the function of cell wall-related genes will affect the composition and characteristics of the plant cell wall. GLBRC's Thrust 1 aims to improve the biomass feedstock to be used for bioenergy conversion processes. Together with colleagues in Thrust 2, we assayed how digestibility, or the amount of sugar released by the cell wall after enzymatic lysis, varies throughout a collection of plants with potentially altered cell walls.

Arabidopsis mutants with disruptions in cell wall-associated genes are being screened for differences in digestibility. Nearly all lines should be knockout mutants, abolishing the function of the targeted gene product. Therefore, digestibility and other phenotypes observed in these lines can most likely be attributed to the disrupted gene. Further efforts are needed to integrate data with those of other thrusts to be able to translate the function of the candidate genes into applicable biofeedstocks. 

We have screened 1200 insertion lines and have identified 110 lines with altered digestibility in unique tissue types and stages of development, (80 that increase, and 30 that decrease the amount of sugar released).  We are currently replicating these results, and 17 lines have been confirmed. These lines are being further characterized for cell wall composition, in collaboration with colleagues in Thrust 2.

Team Members and Roles 

Sara Patterson, UW project lead
Carl-Erik Tornqvist, UW graduate student
Tanya Falbel, UW post-doc

1.1.3 Identification and characterization of genes and gene products involved in secondary cell wall biosynthesis and deposition.

Team Members and Roles

Sebastian Bednarek, UW project lead
Patrick Masson, UW project lead
Marisa Otegui, UW project lead
Christina Ondzighi-Assoume, UW post-doc
David Rancour, UW scientist
Julian Verdonk, UW post-doc
Hannetz Roschzttardtz, UW post-doc
Sathya Jali, UW post-doc
Gary Baisa, UW post-doc

1.1.4 Cellulose Biosynthesis.

Plant cell walls are composed of independent, interacting polysaccharide networks of cellulose, hemicellulose, and pectin. Cellulose is synthesized at the plasma membrane by rosette complexes. Hemicelluloses and pectins, on the other hand, are synthesized in the plant Golgi apparatus, packaged into secretory vesicles, and secreted to the plasma membrane for subsequent deposition into the cell wall. While great progress has been made toward the identification of genes involved in cell wall polysaccharide biosynthesis, far less is known about the trafficking machinery involved in polysaccharide secretion. We aim to identify novel factors that participate in and/or regulate cell wall polysaccharide secretion and biosynthesis, using cotton fiber development as a model.   

During cotton ovule development there is a rapid increase in the secretion and deposition of cell wall polymers between 4 and 6 days post-anthesis (dpa). Within this developmental time period, the cotton Golgi swell and produce a large number of secretory vesicles. Differential proteomics of cotton Golgi at 4 and 6 dpa has generated an extensive list of greater than 800 proteins that increase in abundance during these developmental stages.  While many of these 6dpa abundant proteins are known to be involved in polysaccharide biosynthesis and secretion (i.e. nucleotide sugar interconverting enzymes, glycosyltransferases, and transport related proteins), most have been annotated as genes of unknown function.  To identify novel proteins which facilitate and/or modulate polysaccharide transport, we have selected a subset of these 6dpa abundant unknown proteins.  We have cloned the Arabidopsis orthologs of these cotton candidates as fluorescent protein fusions and a majority of them have been shown to be localized to endomembranes by confocal laser scanning microscopy.  Cell wall analyses of t-DNA insertion lines of these candidates have identified several lines with altered cell wall phenotypes.  This research allows a unique way to investigate cellular mechanisms responsible for the secretion of cell wall components and will assist the development of new strategies for improving plant biomass yield and digestibility.

Team Members and Roles

Federica Brandizzi, MSU project lead
Curtis Wilkerson, MSU project lead
James Johnson, UW graduate student
Saunia Withers, MSU technician
Starla Zemelis, MSU technician
Sang-Jin Kim, MSU post-doc

1.1.6 Poplar: Xylem Specific Promoters.

Wood is gaining popularity as a source of fermentable sugars for liquid fuel production. Secondary wall of wood consists of a complex mixture of cellulose, hemicellulose, and lignin. Proportional variability within the mixture of the three major components varies depending on the species of feedstock used, growing site, climate, age and the part of the plant harvested. The essentially uncontrolled variability of biomass properties presents process design and operating challenges for the production of bioenergy from woody feedstocks. The goal of this project is to develop biotechnological means for optimization of woody biomass feedstocks for ‘low-input production’ systems and the efficiency of biological or chemical conversion of biomass. 

To achieve this goal, we will do the following:

1. Identify the key regulatory genes and proteins that control xylogenesis and secondary wall biosynthesis
2. Functionally test the wall biosynthesis candidate genes in transgenic poplars
3. Develop biotechnology tools that allows us to create woody biomass feedstocks with altered cell walls that are more easily digestible thereby releasing higher quantities of fermentable sugars.

Team Members and Roles

Kyung-Hwan Han, MSU project lead
Hyun-Tae Kim, MSU post-doc
Won Chan Kim, MSU post-doc
Joo Yeoi Kim, MSU post-doc

1.1.9 Plants designed for improved processing.

Two approaches are being targeted to explore biomass crop improvement for more efficient conversion. The first capitalizes on mechanistic insight gained from intense structural studies into plant cell wall chemistry/biochemistry, and from examining current and emerging cell-wall-pathway transgenics, from collaborators, to alter lignin levels and/or structure, to enhance cellulose levels or alter crystallinity. It represents an approach toward crops superior for biomass conversion to ethanol. A second approach is distinctly game-changing. We are pursuing the next steps in work that has highlighted a means to introduce ester-‘zips’ into the backbone of the lignin polymer. This approach is aimed at overcoming the recalcitrance of biomass toward processing by altering the lignin to be more readily removed from the polysaccharide components (in required pretreatments), and can provide enormous energy savings.

Team Members and Roles

John Ralph, UW project lead
Curtis Wilkerson, MSU project lead
Jorge Rencoret Pazo, UW post-doc
Fachuang Lu, UW scientist
Hoon Kim, UW scientist
Saunia Withers, MSU technician
Yimin Zhu, UW scientist
Sasha Ricaurte, MSU graduate student

1.3.4 Enhancing Energy Density of Biofuel Crops.

Fatty acids and their derivatives (oils) have the highest energy content of any products produced by plants.  The ideal biofuel feedstocks of the future provide highest possible energy density per agricultural area and versatility towards different options for energy conversion and fuel production.  Plants can produce oils such as triacylglycerols or wax esters in a number of different tissues or on their surface.  These oils have an energy density very similar to that of petroleum based fuels.  The goal of this project is to enhance the energy density of above ground vegetative tissues and underground storage organs by engineering triacylglycerol or wax ester content.  This strategy is compatible with the processing of residual lignocellulosic biomass by fermentation or with chemical conversion of total biomass and thereby synergistically enhances the energy value of the biofuel crop.

To accomplish this goal, we use high-throughput sequencing to identify novel enzymes and regulatory factors from plants that produce oils in vegetative tissues or that produced oil of high fuel value.  Genes for these factors will be introduced into perennial grasses suitable as biofuel crops or into high-yield storage organs such as the rutabaga.  We generate transgenic models and crop plants to test the utility of these genes and to design new biofuel crops.  We focus on regulatory factors controlling triacylglycerol biosynthesis in plant embryos and adapt them to the use in vegetative tissues and storage organs.  We identify genes encoding enzymes for the biosynthesis of advanced biofuel compounds such as acetyl-triacylglycerols or wax esters.  We explore transporters for the secretion of high energy compounds to the plant surface sequestering these compounds from degrading enzymes.  We also use flux analysis methods to gain a mechanistic understanding of bottlenecks of triacylglycerol biosynthesis such as the export of fatty acids from plastids.  Our sophisticated flux analysis of metabolism in transgenic plants provides a guide to further improvements.

Team Members and Roles

Christoph Benning, MSU project lead
John Ohlrogge, MSU project lead
Yair Shachar-Hill, MSU project lead
Sanjaya Sanjaya, MSU post-doc
Aruna Kilaru, MSU post-doc
Mike Pollard, MSU scientist
Que Kong, MSU post-doc
Tina Martin, MSU technician
Leann Matta, MSU technician
Wilson Chen, MSU scientist
Jeffrey Simpson, MSU graduate student
Xia Cao, MSU post-doc
Lisa Carey, MSU technician
Wei Ma, MSU post-doc

1.4.4 Switchgrass Using Maize as Model Discovery Engine.

Perennial grasses are excellent candidates as dedicated biofuel feedstocks because they generally have relatively low inputs, high energy output to energy input ratio, long-term persistence which reduces establishment costs and needs for tillage, relatively high rates of carbon sequestration, and are highly resistant to soil erosion.  Since 1992, switchgrass has been the focus of considerable DOE-funded research, leading to a wealth of agronomic and genomic information to leverage genetic improvement programs. 

Our project is aimed at improving switchgrass as a biofuel feedstock, primarily by increasing biomass yield and conversion efficiency.  Biomass yield is the single factor with the greatest limitation to economic efficiency of switchgrass feedstock production, largely because production costs, which are tightly linked to biomass yield, are still too high to generate reliable profits.  Conversion efficiency can be significantly improved by modifications to switchgrass cell walls, such as decreases in lignin concentration, based on endogeneous genetic variability.

The project uses a combination of field-based selection for increased biomass yield and decreased lignin concentration, development of molecular markers associated with these traits, and systems biology approaches to modeling selection strategies.  We are using a combination of candidate gene analysis, association analysis, and genomewide selection to identify markers useful for genetic improvement of switchgrass populations per se and hybrids between diverse upland and lowland ecotypes.

Team Members and Roles

Michael Casler, UW project lead
Shawn Kaeppler, UW project lead
Natalia de Leon, UW project lead
Robin Buell, MSU project lead
Kevin Childs, MSU post-doc
Heidi Kaeppler, UW professor/faculty
Haining Lin, MSU post-doc
Lori Kroiss-Schneider, UW post-doc
Candice Hansey, MSU post-doc
Aruna Nandety, UW post-doc

1.4.5 Gene Discovery for Ethanol Traits in Maize

Team Members and Roles

Shawn Kaeppler, UW project lead
Natalia de Leon, UW project lead
Timothy Beissinger, UW graduate student
Dustin Eilert, UW technician
Nicholas Haase, UW graduate student
Rajandeep Sekhon, UW post-doc
Julianne Smith, UW technician
Brieanne Vaillancourt, MSU technician

1.5.1 Biomass trait analyses of Brachypodium accessions and mutants.

The grass Brachypodium distachyon is emerging as an important model system for bioenergy crop grasses such as switchgrass and Miscanthus owing to its small genome size, small stature, short generation time, transformability, and self-fertilization. We are taking a two-pronged approach to identify novel biomass trait genes and allele variants in Brachypodium, with the long-term goal of translating these findings into the improvement of bioenergy crops through breeding and transformation. 

Firstly, we are surveying a genetically diverse collection of wild type Brachypodium accessions for a variety of traits relevant to biomass production including cell wall hydrolytic enzyme digestibility, nutrient utilization, and flowering time, the last of which has a profound affect on plant biomass yield. Not only are these data relevant for identifying gene variants that could be employed to improve bioenergy crops, they are also essential for understanding how different Brachypodium genetic backgrounds could modify the phenotypes of novel mutations. Secondly, to identify mutations affecting the enzymatic breakdown of plant biomass, we are screening through large collections of Brachypodium EMS mutagenized plants using an HPLC-based cell wall digestibility assay. We anticipate finding novel genes affecting plant cell wall composition and structure that can be manipulated to improve the economics and efficiency of plant biomass processing to biofuels.

Team Members and Roles

John Sedbrook, ILSTU project lead
Rick Amasino, UW project lead
Cynthia Cass, ILSTU post-doc
Megha Phutane, ILSTU graduate student
Chris Schwartz, UW post-doc
Daniel Woods, UW graduate student
Deborah Petrik, ILSTU graduate student

1.7.1 Metabolic flux analysis of cell wall.

Team Members and Roles

Yair Shachar-Hill, MSU project lead
Wilson Chen, MSU scientist