Lignin Composition and Structure Differs between Xylem, Phloem and Phellem in Quercus suber L

Abstract

The composition and structure of lignin in different tissues-phellem (cork), phloem and xylem (wood)-of Quercus suber was studied. Whole cell walls and their respective isolated milled lignins were analyzed by pyrolysis coupled with gas chromatography/mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and derivatization followed by reductive cleavage (DFRC). Different tissues presented varied p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) lignin compositions. Whereas lignin from cork has a G-rich lignin (H:G:S molar ratio 2:85:13), lignin from phloem presents more S-units (H:G:S molar ratio of 1:58:41) and lignin from xylem is slightly enriched in S-lignin (H:G:S molar ratio 1:45:55). These differences were reflected in the relative abundances of the different interunit linkages. Alkyl-aryl ethers (β-O-4') were predominant, increasing from 68% in cork, to 71% in phloem and 77% in xylem, as consequence of the enrichment in S-lignin units. Cork lignin was enriched in condensed structures such as phenylcoumarans (β-5', 20%), dibenzodioxocins (5-5', 5%), as corresponds to a lignin enriched in G-units. In comparison, lignin from phloem and xylem presented lower levels of condensed linkages. The lignin from cork was highly acetylated at the γ-OH of the side-chain (48% lignin acetylation), predominantly over G-units; while the lignins from phloem and xylem were barely acetylated and this occurred mainly over S-units. These results are a first time overview of the lignin structure in xylem, phloem (generated by cambium), and in cork (generated by phellogen), in agreement with literature that reports that lignin biosynthesis is flexible and cell specific.

Keywords: DFRC; NMR; Py-GC/MS; Quercus suber; cork; milled lignin; phloem; xylem.

Figure 1

Cross-sectional image of a Quercus suber stem disc presenting cork, phloem, and xylem (scale bar = 1 cm), and transverse microscopic sections of cork, phloem, and xylem tissues (scale bar = 100 μm). scl, clusters of sclereids; f, fibers; v, vessel; r, rays; p, parenchyma.

Figure 2

Py-GC/MS chromatograms of the milled lignin preparations isolated from the different parts of Q. suber (A) cork, (B) phloem, and (C) xylem. The identities and relative abundances of the released lignin-derived compounds are listed in Table 2.

Figure 3

Side-chain (δ_C/δ_H 50–90/2.5–6.0) and aromatic/unsaturated (δ_C/δ_H 100–155/6.0–8.0) regions in the 2D HSQC NMR spectra of the whole cell-walls from the different parts of Q. suber (A,D) cork, (B,E) phloem and (C,F) xylem. The signal assignments are presented in Table 3 and the main lignin structures identified are depicted in Figure 5.

Figure 4

Side-chain (δ_C/δ_H 50–90/2.5–6.0) and aromatic/unsaturated (δ_C/δ_H 100–155/6.0–8.0) regions in the 2D HSQC NMR spectra of the milled lignin preparations isolated from the different parts of Q. suber (A,D) cork, (B,E) phloem, and (C,F) xylem. The signal assignments are presented in Table 3 and the main lignin structures identified are depicted in Figure 5.

Figure 5

Main structures present in the lignins from Q. suber cork, phloem and xylem: A, β-O-4′ alkyl-aryl ethers; A_ox, α-oxidized β–O–4′ aryl ethers; A′, β-O-4′ alkyl-aryl ethers with acylated γ-OH; B, phenylcoumarans; C, resinols; D, dibenzodioxocins; E, open β–1′ structures; F spirodienones; I, cinnamyl alcohol end-groups; I′, γ-acylated cinnamyl alcohol end-groups; J, cinnamaldehyde end-groups; FA, ferulate moieties; H, p-hydroxyphenyl units; G, guaiacyl units; S, syringyl units; S′, oxidized syringyl units unit bearing a carbonyl group at Cα.

Figure 6

Chromatograms of the DFRC′ degradation products from the milled lignin preparations isolated from the different parts of Q. suber (A) cork, (B) phloem, and (C) xylem. cG, tG, cS, and tS are the normal cis- and trans-coniferyl (guaiacyl) and sinapyl (syringyl) alcohol monomers (as their dipropionylated derivatives). cG_ac, tG_ac, cS_ac and tS_ac are the natively γ-acetylated cis- and trans-coniferyl (guaiacyl) and sinapyl (syringyl) alcohol monomers (as their phenol propionylated derivatives).