1a.Objectives (from AD-416)
1) To provide the plant cell wall & biomass research communities with improved methods for polysaccharide & lignin structural profiling, based on complete cell wall solubilization & NMR..
2)To develop & streamline procedures to allow 20-30 samples/day to be profiled & to develop chemometric methods that allow this profile to predict digestibility & bioconversion efficiency. This project is being extended to provide more time to complete the studies cited above. The additional funds will be used to purchase supplies involved in the design, fabrication, and repair of milling equipment.

1b.Approach (from AD-416)
Improvements to the current dissolution/NMR methods will be sought:.
1)Provide the necessary database, via model compounds & isolated components, to characterize component polysaccharides & lignins in whole-cell-wall mixtures..
2)Optimize milling conditions for the various biomass sample types & seek alternative solutions that require less rigorous milling..
3)Attempt to develop improved rapid dissolution methods that can be performed directly in the NMR tube; determine solvent systems that do not interfere with the correlation contours from polysaccharide & lignin components..
4)Develop NMR methods that allow the crucial 1D-proton & 2D-HSQC NMR spectra to be acquired in under 1 hour (on the whole cell wall sample)..
5)Develop methods for databasing & quantifying the NMR cell wall spectra..
6)With collaborators, attempt to develop chemometrics methods that can be applied to 2D-NMR data.

3.Progress Report
This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Wisconsin-Madison. Additional details of research can be found in the report for the parent research project 3655-21000-033-00D: "Identify Cell Wall Factors Limiting Digestibility and Forage Utilization in Sustainable Dairy Farming." Among the accomplishments this year were the following: From the prior development of an incredibly useful system for solubilizing the entire cell wall fraction of finely divided plant materials, we continue to streamline and apply the method, including the development of chemometrics methods, on 2D NMR data (in collaboration with Umeå University, Sweden). It has been applied to the detailed analysis of pectin methylesterase-downregulated plants. A publication is in the early writing stages. With collaborators in Germany, we have discovered new ferulate trimers and tetramers implicated in cross-linking plant cell walls, portending an expanded role in wall cross-linking and consequent digestibility reduction. We have also utilized the most diagnostic methods developed here to deduce the nature of lignins in fruits and vegetables. With collaborators in Sweden, we have published on the nature of various new structures in lignins. Finally, we examined the production of syringyl-rich lignins in maize walls promoted by p-coumaroylated lignins; this work too was published.

Monitoring activities have included monthly progress meetings with all investigators, and monthly assessment of the finances with ARS administrative staff. This is the first submitted annual report (AD-421).