Lignin is one of the major components of woody plant cell walls, and it gives rigidity to plants and allows water conduction. Lignin molecules in angiosperms are composed of guaiacyl (G) and syringyl (S) units, which are derived from coniferyl alcohol and sinapyl alcohol, respectively [6]. A difference in the ratio of G to S units in different morphological regions of some angiosperms has been shown by UV microscopy [2,3,10], bromination-TEM-EDXA [13], and the chemical characterization of various tissue fractions [1,7]. In addition, the selective radio labeling of specific units in lignin and visualization of the labeled units by microautoradiography is useful for studying the formation and structure of lignin with respect to its location in wood tissues [11,12,17]. These studies demonstrated that G lignin is deposited mainly in the early stage of xylem differentiation on vessel walls and the compound middle lamella of the fiber wall, while S lignin is deposited on fiber secondary cell walls in the later stage of differentiation.

Recent studies suggest that the precursor of syringyl lignin, sinapyl alcohol, is biosynthesized via feruloyl–CoA thioester, coniferaldehyde, and sinapaldehyde without forming sinapic acid [4,5,8,14]. However, we previously demonstrated that exogenous ferulic acid was incorporated into G and S lignin, and exogenous sinapic acid was also incorporated into S lignin in robinia [9,15]. This suggests that sinapic acid is an intermediate of syringyl lignin. Sinapyl alcohol is formed from ferulic acid via the aromatic ring modifying reaction (the hydroxylation and the methylation) and the reducing reactions (the reduction of acids to alcohols), whereas it formed from sinapic acid only via the reducing reductions. It is important to clarify the differences in the regions where sinapic and ferulic acid are incorporated in differentiating xylem in order to elucidate the enzymatic systems responsible for modifying the aromatic ring and reducing the side chain.

In this study, we prepared different stage of continuous sections of newly formed xylem from robinia fed labeled ferulic and sinapic acids, to reveal where these compounds are incorporated into S lignin in the differentiating xylem. We estimated the distribution of the labeled lignin by using the derivatization followed by reductive cleavage method (DFRC), followed by gas chromatography–mass spectrometry (GC-MS). The difference in the in situ incorporation of the labeled precursors is discussed in the regulation of sinapyl alcohol biosynthesis in the differentiating xylem.

2.1 Synthesis of labeled precursors

[8-¹³C, 3-OCD₃]-ferulic acid and [8-¹³C, 3,5-OCD₃]-sinapic acid (Fig. 1) were synthesized as described previously [9,15].

2.2 Plant material and administration of precursors

The upper parts of 2-year-old shoots of robinia (Robinia pseudoacacia L.) trees that were growing on the campus of the Nagoya University were cut off in July 2000. A small depression was made at the top of the remaining stem and was filled with 30 ml of a 2-mM aqueous solution of each precursor dissolved in a phosphate buffer (pH. 7.01, 25 °C). After 9 days, each shoot was harvested and was soaked in 80% ethanol. And then 50-μm-thick tangential sections were cut from the surface of the bark-free xylem with a sliding microtome. All section was treated by ethanol-benzene (1:2) for 8 h to remove the labeled precursor and low molecular phenolic compounds. The analyses of lignin by the DFRC method were performed using these sections, as shown in Fig. 2 and described previously [15].

2.3 Analysis of lignin

Fig. 3 shows the distribution of the DFRC degradation products (G_DFRC, S_DFRC) in the robinia given labeled ferulic acid. DFRC method only produces these products from 8-O-4′ structures in lignin [17]. Compared with non-labeled shoots, the lignin composition was not affected by the administration of ferulic acid (data not shown). The low yields of the sections near the cambium indicated that these tissues were in the initial stage of lignification. G lignin began to form in the early stage of cell-wall lignification, and the proportion of S lignin increased gradually as lignification proceeded. This indicates that lignin composition is clearly regulated by the differentiation of cell wall in robinia. Previous studies using microautoradiography demonstrated that G lignin was deposited in the early stage of xylem differentiation on vessel walls, followed by the deposition of S lignin on the fiber cell wall, in magnolia, lilac, and beech [12,17]. Our results for robinia were similar to theirs.

Fig. 4 shows the partial mass spectra of each DFRC product (G_DFRC and S_DFRC) in controls (A) and robinia fed with labeled ferulic (B) or sinapic (C) acid. Each figure shows where the labeled precursor was incorporated into lignin. The molecular ion peaks for G_DFRC and S_DFRC monomers are at m/z 264 and 294, respectively. In general, aromatic acetates easily lose a ketene group (m 42). Therefore, the mass spectra of G_DFRC and S_DFRC have base peaks (m) at m/z 222 and 252, respectively. As described previously [15], when fed with [8-¹³C, 3-OCD₃]-ferulic acid, mass peaks were detected at m/z 226 (222+4; m+4) in G_DFRC and at m/z 256 (252+4; m+4) in S_DFRC (Fig. 4B). When fed with [8-¹³C, 3,5-OCD₃]-sinapic acid, a mass peak was detected at m/z 259 (252+7; m+7) in S_DFRC (Fig. 4C). Examination of the areas of the m+4, m+7, and m peaks on selected ion-monitoring (SIM) chromatograms allowed us to estimate the amount of labeled lignin subunits in each section. Fig. 5 shows the distribution of the yields of labeled DFRC products in each section of robinia fed with labeled ferulic (A) or sinapic (B) acid. When fed with [8-¹³C, 3-OCD₃]-ferulic acid, labeled G_DFRC was located 250–350 and 550–700 μm from the cambium. The distribution of labeled G_DFRC increased gradually as cell-wall lignification proceeded, and the labeled S_DFRC was located 450–600 μm from the cambium. When fed with [8-¹³C, 3,5-OCD₃]-sinapic acid, labeled S_DFRC was located 150–250 and 450–700 μm from the cambium. Little of the label from sinapic acid was incorporated into G_DFRC in any section.

Labeled ferulic acid has begun to incorporate into G lignin in the early stages of cell-wall lignification and the incorporations increased as cell-wall lignification proceeded. And the labeled ferulic acid is also incorporated into S lignin during the late stage of lignification. These localizations corresponded with the heterogeneous deposition of lignin moieties. Labeled sinapic acid was incorporated into S lignin in the early and late stages of lignification. However, the deposition of S lignin occurred mainly in the later stage of lignification. Thus, the incorporation of labeled sinapic acid into syringyl lignin in the early stage would be abnormal conversion.

These results indicated that the exogenous ferulic acid was incorporated in the normal lignin biosynthetic process, including the regulation of coniferyl and sinapyl alcohol biosynthesis, whereas the exogenous sinapic acid was not. The conversion of ferulic acid to sinapyl alcohol goes via the aromatic ring modifying reaction (the hydroxylation and the methylation) and the reducing reactions (the reduction of acids to alcohols). The conversion of sinapic acid to sinapyl alcohol goes only via the reducing reactions (the reduction of acids to alcohols). Therefore, the regulation of the syringyl lignin biosynthetic process would occur in the aromatic ring modifying reaction (the conversion from guaiacyl to syringyl moiety) in the differentiating xylem.

In robinia, sinapyl alcohols are generated mainly in the later stage in the differentiating xylem. For the regulation of the sinapyl alcohol biosynthesis, the aromatic ring-modifying reactions including the hydroxylation and methylation (the conversion from guaiacyl to syringyl moiety) are more important than the reducing reactions (the reduction from sinapic acid to sinapyl alcohol).