Lignification in cell suspension cultures of Pinus taeda. In situ characterization of a gymnosperm lignin

Pinus taeda suspension cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed only primary cell wall formation and essentially no lignification, as determined by histochemical, ultrastructural, chemical, and NMR spectroscopic analyses. However, these cultures maintained a functional phenylpropanoid pathway as demonstrated by formation of the lignans (-)-matairesinol and (-)-pinoresinol. Administration of [1-13C]Phe to these cultures, followed by solid-state carbon-13 NMR spectral analysis of their cell walls, demonstrated that the phenylalanine incorporated into the cell wall matrix was primarily as protein, rather than lignin. Successive transfer of the 2,4-dichlorophenoxyacetic acid-grown cultures to alpha-naphthaleneacetic acid-containing medium induced cell wall thickening concomitant with lignification. The presence of lignin was confirmed by histochemical, ultrastructural, chemical, biochemical, and NMR spectroscopic analyses. Specific labeling of the lignin polymer in situ with [1-13C]-, [2-13C]-, and [3-13C]Phe and analysis of the cell wall preparations by solid-state carbon-13 NMR spectroscopy permitted the first direct determination of the in situ bonding patterns in a gymnosperm lignin. Several dominant interunit linkages were observed, including beta-O-aryl, furanofuran, phenylcoumarin, and phenolic-linked monolignols, consistent with those predicted but hitherto not proven. Finally, milled wood lignin derivatives prepared from these 13C-specifically enriched lignin tissues gave a relatively high fidelity copy of the native lignin.