Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/10/2006
Publication Date: 1/1/2007
Citation: Mao, J., Holtman, K.M., Scott, J.T., Kadia, J.F., Schmidt-Rohr, K. 2007. Differences between Lignin in Unprocessed Wood, Milled Wood, Mutant Wood, and Extracted Lignin Detected by 13C Solid-State NMR. Journal of Agricultural and Food Chemistry. 59 (26): 9677-9686. (2007). Interpretive Summary: Solid-state NMR has the potential to be a useful tool for routine analysis of whole plant materials. Screening large sets of plants for structural changes in response to gene manipulation can be achieved non-destructively since the technique does not require isolation of cell wall components for analysis. Depending upon the plant type, core sampling or selective pruning of plants can be utilized to monitor growth and pinpoint deviations from typical cell wall structure. Historically, solid-state NMR has provided relatively little structural detail due to the heterogeneous nature of the plant cell wall. This manuscript describes several newly developed pulse sequences that have now been applied to the analysis of intact wood core samples and target quantitation of changes in specific structural moieties. A loblolly pine exhibiting a naturally occurring mutation has been used as an example of the utility of these techniques.
Technical Abstract: Solid-state 13C nuclear magnetic resonance (NMR) has been applied to an array of intact and isolated wood samples in order to identify potential structural changes induced by tree age, milling, lignin extraction, or naturally occurring mutations. Included in this study were mature loblolly pine milled by two different techniques and a set of juvenile wild-type and CAD (coniferyl aldehyde dehydrogenase)-deficient loblolly pines. Special attention has been paid to C=O (ketone/quinone and aldehyde) as well as nonpolar alkyl groups; these can be observed with little overlap and at low concentrations (2 in 1000 C) using an improved method of spinning-sideband suppression after gated decoupling. By utilizing these and other spectral-editing techniques, many of which are novel to the field of wood chemistry, it was verified that carbonyl structures are present in intact, unmodified wood and that there are more keto groups than aldehydes. Their concentrations increase from juvenile- to mature-wood lignin. The brief Wiley milling process does not cause changes in C=O concentrations, whereas “conventional” milling for one week lead to increases in C=O concentrations. Rotary milling with porcelain balls for 6 weeks (P-6 milling) results in even higher C=O concentrations; this difference is also reflected in their corresponding lignin samples, with more C=O in lignin extracted from P-6 milled wood. The extraction process does not increase the C=O fraction in the lignin. Spectra of whole wood from wild-type and 99% CAD-deficient mutant loblolly pines prove that significant amounts of aldehyde and dihydroconiferyl alcohol residues are present in the mutant wood, confirming published solution-state NMR spectra of the corresponding lignin samples. This application demonstrates the utility of solid-state NMR for routine assay for changes in the lignin structure of genetically modified plants.