Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure
John Ralph, Takuya Akiyama, Hoon Kim, Fachuang Lu, Paul F. Schatz, Jane M. Marita, Sally A. Ralph, M. S. Srinivasa Reddy, Fang Chen, Richard A. Dixon
Journal of Biological Chemistry
Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to ϳ65% P (from wild-type levels of ϳ1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional 13 C-1 H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S
... sitional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key ␤-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of ␤-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range 13 C-1 H correlation experiments. For example, although ␤-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants. unit in lignin or coniferyl alcohol monolignol; S, syringyl unit in lignin or sinapyl alcohol monolignol; DFRC, derivatization followed by reductive cleavage; ML, milled lignin; AL, acidolysis lignin; EL, enzyme lignin; HSQC, heteronuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; COSY, two-dimensional correlated spectroscopy; TOCSY, total correlation spectroscopy; COMT, caffeic acid O-methyl transferase; F5H, ferulate 5-hydroxylase; CAld-5H, coniferaldehyde 5-hydroxylase; 5HG, 5-hydroxyguaiacyl; ABSL, acetyl bromide-soluble lignin; ppm, parts/million. FIGURE 2. Partial short range 13 C-1 H (HSQC) correlation spectra (aromatic regions only) of milled lignins (ML) isolated from the wild-type control (a) and the most highly C3H-deficient line, C3H-4a (b). Traces of p-hydroxyphenyl (P) units are seen in the typically syringyl/guaiacyl (S/G) lignin in the wild-type alfalfa, whereas P-units entirely dominate the spectrum in the transgenic alfalfa. Semiquantitative volume integrals are given in Table 2 . Analogous spectra for the other lignin isolates (acidolysis lignins, AL, and enzyme lignins, EL) are provided in the supplemental material. Effects of C3H Down-regulation on Lignin 8848 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 13 • MARCH 31, 2006 by guest on July 25, 2018 http://www.jbc.org/ Downloaded from maryl alcohol efficiently cross-couples with the dominant guaiacyl and/or syringyl units. HMBC correlations from the C3H-deficient lignin (Fig. 4b) provide the first indications of the coupling and cross-coupling propensities of p-coumaryl alcohol and p-hydroxyphenyl units (see "Discussion"). The following appear to be evidenced quite clearly: (i) ␤-ether units A are of all three types, P, G and S, but with relatively low levels of the G-(especially) and S-units; (ii) the phenylcoumarans B are almost entirely P; (iii) FIGURE 3. Partial short range 13 C-1 H (HSQC) spectra (side chain regions) of milled lignins (ML) isolated from the wild-type control (a) and the most highly C3H-deficient line, C3H-4a (b). C3H deficiency and the incorporation of higher levels of p-coumaryl alcohol into the lignin produce substantial changes in the distribution of interunit linkage types. The absence of spirodienone S units in the transgenic alfalfa reveals that p-coumaryl alcohol does not apparently favor ␤-1 cross-coupling reactions. Several types of new dibenzodioxocins D are more readily seen at the lower contour levels in the more highly resolved partial spectrum in the inset. Note that the contour levels used to display the two spectra were chosen to highlight the structural similarities and differences; with no internally invariant peaks, interpretation of apparent visual quantitative differences needs to be cautious. The upper left corner photograph shows WT and C3H-4a transgenic plants at the WT flowering stage; pictures of the C3H-9a transgenic and histochemical staining are provided elsewhere (13). Volume integrals and semiquantitative data are given in Table 3 . Analogous spectra for the other lignin isolates (acidolysis lignins (AL) and enzyme lignins (EL)) are provided in the supplemental material. Interunit type designations A-D, S, X1, and X7 follow conventions established previously (1, 3, 58).