Browsing by Author "Eberhardt, Thomas Leonard"
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- Characterization of lignin deposition in Pinus taeda L. cell suspension culturesEberhardt, Thomas Leonard (Virginia Tech, 1992-03-05)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.
- Studies on lignin biosynthesis and structureEberhardt, Thomas Leonard (Virginia Tech, 1988-02-15)Beech (Fagus grand1folia Ehrh.> bark contains appreciable quantities of Z- (cis) con1feryl and Z-s1napyl alcohols and not the corresponding E- (trans) alcohols. Previous rad1otracer experiments suggested that the Z-coniferyl alcohol in beech bark is formed by isomerization of E-coniferyl alcohol which proceeds either directly or through the corresponding aldehydes. In the work conducted in this thesis, is has been found that crude cinnamy1 alcohol dehydrogenase isolated from beech bark shows a strong substrate preference for E-coniferyl alcohol (as opposed to Z-coniferyl alcohol) thereby suggesting that the E to Z isomerization described occurs directly at the alcohol level. Administration of (2-¹⁴C) ferul1c acid to feland wheat (Triticum aest1yum L.) over extended durations (21 days) and subsequent isolation of the lignin from the root tissue as its acetal derivative demonstrated the incorporation of the labelled feru1ic acid into the lignin component of the tissue. Through sim1lar administrations of (1-¹³C, 2-¹³C and 3-¹³C) ferul1c acid and subsequent analysis of the root tissues by solid state ¹³C nuclear magnetic resonance (NMR) spectroscopy, it was possible to determine the bonding patterns of lignin in situ. The lignin component of each ¹³C feru1ic acid enriched root tissue was then isolated as its acetal derivative and analyzed by solution state ¹³C NMR. Through comparison it was shown that the enhanced resonances observed in the solution state ¹³C NMR spectra of the ¹³C ferulic acid enriched acetal lignins corresponded to the enhanced resonances in the respective spectra of the intact root tissues. This indicated that minimal changes to the lignin bonding patterns occurred during the isolation procedure. The dominant presence of ¹³C NMR resonances corresponding to hydroxycinnamic acid functionalities in the solid and solution state NMR spectra demonstrates the important role of hydroxyc1nnamic acids in wheat root lignin. However, no evidence of the formation of dimers such as 4,4'-dihydroxytruxillic acid was noted. Thus, such structures do not represent an important bonding pattern in wheat root lignin.