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-dichlor ophenoxyacetic acid showed only primary cell wall formation and essent ially no lignification, as determined by histochemical, ultrastructura l, chemical, and NMR spectroscopic analyses. However, these cultures m aintained a functional phenylpropanoid pathway as demonstrated by form ation of the lignans (-)-matairesinol and (-)-pinoresinol. Administrat ion of [1-C-13]Phe to these cultures, followed by solid-state carbon-1 3 NMR spectral analysis of their cell walls, demonstrated that the phe nylalanine incorporated into the cell wall matrix was primarily as pro tein, rather than lignin. Successive transfer of the 2,4-dichloropheno xyacetic acid-grown cultures to alpha-naphthaleneacetic acid-containin g medium induced cell wall thickening concomitant with lignification. The presence of lignin was confirmed by histochemical, ultrastructural , chemical, biochemical, and NMR spectroscopic analyses. Specific labe ling of the lignin polymer in situ with [1-C-13]-, [2-C-13]-, and [3-C -13]Phe and analysis of the cell wall preparations by solid-state carb on-13 NMR spectroscopy permitted the first direct determination of the in situ bonding patterns in a gymnosperm lignin. Several dominant int erunit linkages were observed, including beta-O-aryl, furanofuran, phe nylcoumarin, and phenolic-linked monolignols, consistent with those pr edicted but hitherto not proven. Finally, milled wood lignin derivativ es prepared from these C-13-specifically enriched lignin tissues gave a relatively high fidelity copy of the native lignin.