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.
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