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Haiying Liang 1 , Nicole Brown 2 , John Carlson 2 , Ming Tien 2

A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In Poplar Cell Walls. Haiying Liang 1 , Nicole Brown 2 , John Carlson 2 , Ming Tien 2 1 Clemson University, Clemson, SC 29634

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Haiying Liang 1 , Nicole Brown 2 , John Carlson 2 , Ming Tien 2

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  1. A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In Poplar Cell Walls Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634 2The Pennsylvania State University, University Park, PA 16802

  2. Energy Crisis Don Tate III AMERICAN-STATESMAN ILLUSTRATION, from http://www.statesman.com/business/content/business/other/gas.html

  3. Haiying Liang1, Nicole Brown2, John Carlson2, Ming Tien2 1 Clemson University, Clemson, SC 29634 2The Pennsylvania State University, University Park, PA 16802 A Novel Approach to Facilitate Accessibility of Cellulose and Hemicellulose by Introducing a Tyrosine Rich Peptide Gene In Poplar Cell Walls

  4. About Lignin A major component of wood -raw material for pulp and paper production -a good renewable energy source A component of lignocellulosic material -feedstock for livestock 2nd most abundant terrestrial biopolymer -approx. 30% of the organic carbon in the biosphere

  5. Lignin Degradation is of Central Importance in Biomass Utilization Undesired material in pulp and paper industries and biomass utilization Difficult to degrade, no nutritional value Mosier et al. 2005

  6. Approaches Being Taken lignin network crystallinity of cellulose Expensive and environment-unfriendly • Schematic from Mosier et al. (2005) showing goals of pretreatment of lignocellulosic material

  7. Biotechnology • Decrease lignin content • Modify lignin monomer composition: • G (guaiacyl) S (syringyl)

  8. PAL COMT C4H 4CL monolignol synthesis CCR CAD

  9. Essential Role of Lignin in Cell Wall • Essential component of cell wall: • - imparts rigidity to plants • - conducts water & solutes to different parts of plants • - provides physical barrier to invading pests

  10. Hypothesis Free radical coupling between lignol subunits and TYR will result in a lignin structure that can be partially hydrolyzed with proteases. Representative structure of peptide-cross-linked lignin via phenolic tyrosines.

  11. Strategies • Design TYR-rich peptide genes differing in length and sequence • Express transgene in lignifying tissue in poplar • Characterize transgenic plants:-Plant fitness • -Lignin structure and lignin-tyrosine bonding in plants • -Small scale pulping and ethanol production tests

  12. Strategy 1: Gene Design Binary Vector (Modified from pBI101) Tyr rich gene (13%) PAL promoter--CBG-leader Expression of PAL2-GUS gene fusion in poplar in the actively lignifying phloem and xylem cells (PAL: Phenylalanine Ammonia-Lyase) For secretion into the cell wall during lignification (Pinus contorta coniferin-specific β-glucosidase)

  13. Transformation in Poplar Ogy (P. deltoides Marsh. × P. nigra L.) 2W 2ZZ 2-II 2X M 4-2 Wt Example of PCR screening with transgene-specific primers 2Kb Transgenic lines 1Kb 1 Kb Wild type DNA Plasmid DNA Example of genomic southern hybridization

  14. Strategy 2: Gene Expression Real-Time PCR

  15. Strategy 3: Characterization Histochemical Staining of Lignin Potassium permanganate Phloroglucinol Wt 2-5V 4-4 2EE Potassium permanganate Phloroglucinol 2-I 2X 2YY 2Z 2P

  16. Strategy 3: CharacterizationKlason Lignin Content Analysis

  17. Further Studies • Localization of TYR-rich peptide with antibody • Pathogen susceptibility analysis • Tensile strength, lignin structure, etc. Peptide showing salt bridging

  18. Strategy 3: Characterizing Lignin-Protein Structure and Interactions • Isolated Lignin • MWL isolated from stems • HSQC and TOCSY (Hu et al.) • Compare to 13C-Tyrosine treated MWL fractions (from control plants)

  19. Strategy 3: Characterizing Lignin-Protein Structure and Interactions • HSQC of MWL • (Hu et al., Nature Biotechnol, 1999)

  20. Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale • Solid samples • Method—CP/MAS NMR • 15N Tyrosine tissues • Plants supplied K15NO3 (Englesberger et al. 2006) • Cross polarization studies • Basis: < 10 Å proximity • Different 15N chemical shifts should be detected for the various amino acids Morais et al. 1999, J. Braz. Chem. Soc. 

  21. Strategy 3: Characterizing Lignin-Protein Interactions at the Nanoscale • Lignin-Protein Interactions • Solid state NMR (CP/MAS) • Variable Contact Time Cross Polarization • Proton spin-lattice relaxation time in the rotating frame (HT1r) • Common HT1rindicates nanoscale homogeneity, while different HT1r indicates nanoscale phase separation

  22. Strategy 3: Thermal Characterization • DMA/DSC • Lignin Glass Transition Region • Before and after plasticization (DMA) • Control and modified plants • Extracted MWL

  23. Strategy 3: Bioenergy Studies • Pulping efficiency and ethanol production from modified tissues • Does protease pre-treatment of tyrosine-rich transgenic poplar plants impact these processes? Preliminary digestibility assay using protease K

  24. Acknowledgments • Alan Benesi, Penn State NMR Director • DOE & Huck Institutes for the Life Sciences at • Penn Statefor $$$

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