Lignin composition and structure in young versus adult Eucalyptus globulus plants

Abstract

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4' and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1' structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (β-O-4' and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.

Figure 1.

Py-GC-MS chromatogram of the E. globulus wood samples at different growth stages. The numbers refer to the lignin-derived compounds, whose identities and relative abundances are listed in Table II. Letters refer to the carbohydrate-derived compounds: a, hydroxyacetaldehyde; b, (3H)-furan-2-one; c, (2H)-furan-3-one; d, furfural; e, 2-hydroxymethylfuran; f, 2,3-dihydro-5-methylfuran-2-one; g, 5-methyl-2-furfuraldehyde; h, (5H)-furan-2-one; i, 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one; j, 2-hydroxy-3-methyl-2-cyclopenten-1-one; k, 5-hydroxymethyl-2-furfuraldehyde; l, levoglucosane.

Figure 2.

Py-GC-MS chromatogram of the MWLs isolated from the E. globulus wood samples at different growth stages. The identities and relative abundances of the released compounds are listed in Table II.

Figure 3.

HSQC NMR spectra (δ_C/δ_H 45–135/2.5-8.0 ppm) of the E. globulus wood samples at different growth stages after forming a gel in DMSO-d₆:pyridine-d₅ (4:1). See Table III for lignin signal assignment and Figure 5 for the main lignin structures identified. The assignments of the carbohydrate signals are listed in Table IV.

Figure 4.

HSQC NMR spectra (δ_C/δ_H 45–135/2.5-8.0 ppm) of the MWLs isolated from the E. globulus wood samples at different growth stages. See Table III for lignin signal assignment and Figure 5 for the main lignin structures identified. Olefinic cross-signals of unsaturated fatty acid structures (U_F) were also identified.

Figure 5.

Main substructures present in the E. globulus wood lignin during tree growth. A, β-Ether structure formed by β-O-4′ linkages. B, Resinol structure including β-β′ linkages. C, Phenylcoumaran structure including β-5′ linkages. D, Spirodienone structure including β-1′ linkages. E, Open β-1′ structure. F, C_α-oxidized β-O-4′ structure. I, Cinnamyl alcohol end group. H, p-Hydroxyphenyl unit. G, Guaiacyl unit. S, Syringyl unit. S′, Oxidized syringyl unit bearing a carbonyl at Cα.

Figure 6.

Carbohydrate anomeric regions (δ_C/δ_H 90–110/3.5-6.0 ppm) of HSQC NMR spectra of the E. globulus wood samples at different growth stages after forming a gel in DMSO-d₆:pyridine-d₅ (4:1). The assignments of the carbohydrate signals are listed in Table IV.

Figure 7.

Chromatogram of the thioacidolysis degradation products (after Raney-nickel desulfurization) of a representative MWL from E. globulus wood (at 1 month of growth) as trimethylsilyl derivatives. The numbers refer to the compounds (monomers and dimers) listed in Table VI, and the corresponding chemical structures are shown in Figure 8. I.S. refers to octadecane used as an internal standard.

Figure 8.

Structures of monomeric and main dimeric compounds obtained after thioacidolysis and Raney-nickel desulfurization of the MWLs isolated from the E. globulus wood samples at different growth stages. All mass spectral data of the compounds (as trimethylsilyl derivatives) are listed in Table VI.