Source: USDA, ARS, Midwest Area Office submitted to NRP
NON-DEGRADATIVE DISSOLUTION OF WOOD FIBER: BASIS FOR NEW AND IMPROVED ANALYTICAL METHODS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0196541
Grant No.
2003-35103-13834
Cumulative Award Amt.
$180,000.00
Proposal No.
2003-02319
Multistate No.
(N/A)
Project Start Date
Aug 15, 2003
Project End Date
Aug 14, 2005
Grant Year
2003
Program Code
[73.0]- (N/A)
Recipient Organization
USDA, ARS, Midwest Area Office
1201 W. Gregory Drive
Urbana,IL 61801
Performing Department
(N/A)
Non Technical Summary
The heterogeneous nature of plant cell walls limits the ease with which a variety of analytical methods can be applied (in research aimed at improved fiber utilization) We have modified a cellulose-dissolving ionic liquid system and developed a new organic system to completely dissolve finely milled wood fiber (and plant cell walls in general) at room temperature! Preliminary work has demonstrated its efficacy on pine, aspen, kenaf, corn, and a mutant pine. Acetylation produces fully acetylated cell walls with no apparent degradation, that are fully soluble in common organic solvents. NMR spectroscopy readily reveals the traditional lignin structures amongst the dominant polysaccharide resonances. Complete derivatization reactions in under an hour have been demonstrated, already promising to revolutionize certain analyses. The objectives of this proposal are to exploit the power of cell wall solubilization to improve and develop a range of analytical methods. We will detail extraordinarily diagnostic solution-state NMR of whole cell wall samples, and use the methods to analyze the residual lignin components from various previous studies, particularly from valuable mutant and transgenic plants. We will develop revised lignin analysis protocols and propose new analytical methods for lignins and polysaccharide analysis. This work will advance the knowledge of the chemistry and biochemistry of wood fiber, and provide a sound basis for wide-ranging research into its improved utilization.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061699100030%
2061699200070%
Goals / Objectives
Exploit the power of cell wall solubilization to improve and develop a range of analytical methods. In particular: develop methods for NMR of the whole wall fraction; re-examine (the whole lignin fraction in) valuable mutants and transgenics; develop NMR syringyl:guaiacyl method; improve phenolic methylation methods for thioacidolysis, the DFRC method, and hydroxycinnamates and hydroxybenzoates analysis; attempt to improve phenolic-OH determination.
Project Methods
a. Optimize the cell wall dissolution process and determine the limits on how finely-divided cell walls need to be for solubilization. b. Test survivability of a range of lignin and polysaccharide models through the dissolution process. c. Derivatization of the Cell Wall; test completeness of important acylation and etherification reactions, with model compounds. d. NMR of the Whole Cell Wall, directly on underivatized material, and following suitable derivatization. Samples will include at least pine, aspen, corn, and kenaf. e. NMR of Enzyme Lignins and Residues. The whole cell walls (where available), the enzyme lignins (where available) and the enzyme-lignin residues (following dioxane:water extraction of the MWL fraction, always kept and available) of a whole range of valuable normal and transgenic plant samples will be characterized as fully as possible; for the first time, it will be possible to make observations on the composition and structure of the entire lignin fraction, an enormously significant advance. f. Improvements to Analytical Methods including thioacidolysis, DFRC, phenol analysis, syringyl:guaiacyl determination, and hydroxycinnamates analyses.

Progress 10/01/03 to 09/30/04

Outputs
This project is based upon the development of an incredibly useful system for solubilizing the entire cell wall fraction of finely divided plant materials, potentially allowing far more rapid and complete analysis of cell walls than previously. The new cell wall dissolution method was featured as a cover article and a technical advance in "The Plant Journal" and has featured as an USDA-CSREES-NRI Cover Story. Progress is currently hampered by insufficient access to crucial high-sensitivity cryoprobe NMR instrumentation. However, the first high-resolution solution-state 3D NMR study on the entire wall component, revealing incredible detail, has just been published. Small-scale methods are being developed for analysis of valuable transgenics available only in tiny amounts. With collaborators in Germany, we are exploring improvements to methods for determining cell wall cross-linking by diferulates, utilizing aspects of the dissolution technology. Other methods are under development.

Impacts
The methods developed here allow for the entire cell wall component to be analyzed without requiring tedious fractionation. The potential is therefore to "screen" plant materials for lignin patterns much more quickly; previously lignin isolation could take many weeks whereas the solubilization can be done in 2 days. 3D NMR can be used to "edit" out the components of interest. The advantage also of being able to analyze the entire lignin component instead of just the fraction normally obtained avoids fractionation and representativity problems. It is therefore anticipated that the methods developed here will allow more rapid detailed analysis of normal and transgenic plant materials of considerable value to researchers.

Publications

  • Ralph, J. and Lu, F. 2004. Cryoprobe 3D NMR of acetylated ball-milled pine cell walls. Org. Biomol. Chem. 2:2714-2715.
  • Morreel, K., Ralph, J., Kim, H., Lu, F., Goeminne, G., Ralph, S. A., Messens, E. and Boerjan, W. 2004. Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem. Plant Physiol. 136:3537-3549.