INVESTIGATOR(S): Chapple, C.; Meilan, R.; Ladisch, M.
INSTITUTION: Purdue University
NON-TECHNICAL SUMMARY: High gasoline prices, global warming, national security, and the limitations of global petroleum resources have reinvigorated worldwide interest in renewable resources as a feedstock for liquid transportation fuels, particularly those derived from cellulose. As a perennial woody plant, hybrid poplar (genus Populus) offers several advantages with regard to cellulosic biofuel production including rapid growth rates, the ability to cycle nutrients, a wide geographic distribution, genetic diversity, amenability to genetic engineering, and abundant genomic resources. The phenolic cell wall polymer lignin constitutes a significant barrier to biomass conversion but, at the same time, it is essential to normal plant growth and development. Recent advances in our understanding of how lignin monomers are synthesized provide us with an opportunity to modify the content and composition of the lignin polymer. The research to be conducted will enable us to rationally assess the cost savings that could result from using genetically engineered poplar, instead of corn, as a feedstock for producing biofuels.
OBJECTIVES: 1) Generation of transgenic poplar up- or down-regulated for four enzymes known to impact lignin quantity and quality; 2) Development of metabolic profiling methods for poplar and their application to greenhouse- and field-grown wild-type and transgenic plants; 3) Morphometric analysis of transgenic lines grown in field plots; and 4) Cell wall deconstruction analysis of wild-type and lignin-modified transgenic lines.
APPROACH: Obj. 1) The expression of four enzymes in the lignin biosynthetic pathway will be up-and/or down-regulated. For each DNA construct, poplar cDNA will be synthesized from young shoot RNA using reverse transcriptase and PCR-amplified with gene-specific primers developed based on conserved regions within the genes' sequences identified from the poplar genome, the Arabidopsis genome, and other plant sequences. All constructs will be transformed into clone NM-6 (Populus nigra x P. maximowiczii) using an Agrobacterium-mediated transformation protocol. Obj. 2) Transformants will be tested for changes in lignin composition by a battery of lignin analyses (i.e., Klason lignin, pyrolysis GC-MS, and DFRC analysis). At the same time, HPLC and GC-MS will be used to assay total cell extracts and cell-wall hydrolysates from these plants to determine whether perturbations in phenylpropanoid pathway gene expression have led to alterations in free and/or cell wall-esterified phenolic compounds. Obj. 3) Morphometric analyses will be conducted on all lines in the field trial to ensure the transgenes have no deleterious effects on phenotype. All plants will be visually examined at least twice during the first and second growing seasons, including at least once during leaf senescence in the fall and bud flush in the spring. Pairs of ramets with any unusual phenotypes will be photographed and measured for height, diameter, branch length, crown width, and changes in phenology. Obj. 4) Pretreatment will be carried out in batch-tube reactors. After pretreatment, an aliquot of the slurry in the tube will be collected and processed for image analysis, and the rest of the slurry in the tube will be processed by enzymatic hydrolysis using the NREL LAP009 enzyme digestibility procedure with minor modifications. The liquid separated from the solids in each condition will be filtered and analyzed via HPLC. These analyses will permit us to determine the impact of lignin modification on cell wall degradability.
Name: Chapple, C.