Characterization of lignin deposition in Pinus taeda L. cell suspension cultures

Pinus taeda L. suspension culture cells were used to develop a model system to study the process of lignification occurring during the early stages of cell wall formation and maturation. Chemical, biochemical and histochemical analyses of the P. taeda suspension cultures grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the growth regulator did not provide conclusive evidence for lignin deposition. On the other hand, cultures in which 2,4-D was substituted with α-naphthaleneacetic acid (NAA) were shown to lignify. During this induction of lignification, limited cell wall thickening occurred since transmission electron microscopy of the 2,4-D grown cells showed only primary walls while the average cell wall thickness of the NAA-grown cells was consistent with secondary (S₁) layer formation. Despite the possibility of only limited lignin deposition in the 2,4-0 grown cells, secondary metabolism had occurred as evidenced by reversed-phase and chiral chromatographic separations which revealed the ability of these cells to produce enantiomerically pure (-)-matairesinol. Administrations of [1-¹³C], [2-¹³C ] and [3-¹³C ] specifically labeled phenylalanines to the P. taeda suspension cultures in medium containing NAA allowed the determination of lignin bonding patterns in situ by solid-state ¹³C NMR spectroscopy of the resulting ¹³C enriched cells.

Aqueous and organic solvent extractions and protease treatment yielded ¹³C enriched cell walls for solid-state ¹³C NMR spectroscopic analyses of the cell wall bound lignin component. Subsequently, an isolated lignin derivative from these cell walls was analyzed by solution-state ¹³C NMR spectroscopy and verified the assignments made in the solid-state. Accordingly, the above experiments represent the first demonstration of lignin bonding patterns in situ in a Pinus species as well as a suspension culture. This culture system possesses great potential as a model to thoroughly study the early stages of lignification.