NMR Assignment for Diaryl Ether Structures (4-O-5 Structures) in Pine Wood Lignin.

A 4-O-5-tetramer lignin model compound carrying ß-O-4 linkages on each of the side-chain moieties was synthesized, as well as 4-O-5-coupled dehydrodiconiferyl alcohol. By comparison with their NMR data, two cross-signals in the HSQC spectrum of pine milled wood lignin recorded in DMSO-d6 were assigned to H2/C2 and H6/C6 correlations on the aromatic rings of 4-O-5-linked units. Although the H2/C2 correlation peak appeared in the same region as syringyl units, nitrobenzene oxidation of the pine lignin did not yield any syringyl-type product, but did release a 4-O-5-type product.

Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii.

Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.

Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants.

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3'H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.

Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis.

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies.

Mass spectrometry-based fragmentation as an identification tool in lignomics.

The ensemble of all phenolics for which the biosynthesis is coregulated with lignin biosynthesis, i.e., metabolites from the general phenylpropanoid, monolignol, and (neo)lignan biosynthetic pathways and their derivatives, as well as the lignin oligomers, is coined the lignome. In lignifying tissues, the lignome comprises a significant portion of the metabolome. However, as is true for metabolomics in general, the structural elucidation of unknowns represents the biggest challenge in characterizing the lignome. To minimize the necessity to purify unknowns for NMR analysis, it would be desirable to be able to extract structural information from liquid chromatography-mass spectrometry data directly. However, mass spectral libraries for metabolomics are scarce, and no libraries exist for the lignome. Therefore, elucidating the gas-phase fragmentation behavior of the major bonding types encountered in lignome-associated molecules would considerably advance the systematic characterization of the lignome. By comparative MS(n) analysis of a series of molecules belonging to the ß-aryl ether, benzodioxane, phenylcoumaran, and resinol groups, we succeeded in annotating typical fragmentations for each of these bonding structures as well as fragmentations that enabled the identification of the aromatic units involved in each bonding structure. Consequently, this work lays the foundation for a detailed characterization of the lignome in different plant species, mutants, and transgenics and for the MS-based sequencing of lignin oligomers and (neo)lignans.

Differences in the Mechanisms of MnO2 Oxidation between Lignin Model Compounds with the p-Hydroxyphenyl, Guaiacyl, and Syringyl Nuclei.

The purpose of this study was to examine how the rate and mechanism of MnO2 oxidation differ between the p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) types of simple nonphenolic lignin model compounds as well as the p-ethylphenyl (E) type compounds. The oxidation was conducted using an excess amount of MnO2 in a sulfate buffer solution at a pH value of 1.5 at room temperature. MnO2 oxidized at least the G and S nuclei, although it commonly oxidizes alcohols present at the benzyl position. The oxidation rates of the benzyl alcohol derivatives were in the order of G- > S- ≫ H- > E-type, which suggests that the rates are determined by the electronic effects of their methoxy and ethyl functional groups on not only their benzyl positions but also their aromatic π-electron systems. The kinetic isotope effect was observed in the MnO2 oxidations of the same derivatives deuterated at their benzyl hydroxymethyl groups. The observed magnitudes were in the order of E- ≫ H- > G- ≫ S-type, suggesting that the contribution of oxidation of their aromatic nuclei, which is another reaction mode of the oxidation of their benzyl positions, increases in the reverse order.

Lignin-Biosynthetic Study: Reactivity of Quinone Methides in the Diastereopreferential Formation of p-Hydroxyphenyl- and Guaiacyl-Type ß- O-4 Structures.

p-Quinone methides are involved in lignin biosynthesis as transient intermediates, and the aromatization step has a great impact on the chemical structure of the resulting lignin. A series of quinone methides (QMs) were synthesized and allowed to react with water in pH 3-7 buffers at 25 °C to mimic the formation of p-hydroxyphenyl- and guaiacyl-type (H- and G-type, respectively) ß- O-4 structures in gymnosperm-plant cell walls. Water addition occurred in 3-methoxy-substituted QMs (G-type QMs) with half-lives of 1.4-15 min. In contrast, nonsubstituted QMs (H-type QMs) were very labile; they were aromatized to ß- O-4 products with half-lives of only 10-40 s. The rapid aromatization in H-type QMs may provide an advantage over G-type species for efficiently driving the lignin-polymerization cycle, which possibly contributes to the development of highly lignified compression wood. In the water-addition reaction, the threo isomers of the ß- O-4 products were stereopreferentially formed more than the erythro isomers from both G- and H-type QMs ( erythro/ threo ratios of 24:76 and 50:50, respectively). The proportion of erythro isomers was higher at lower-pH conditions. This pH-dependent trend agrees with findings from a previous study on 3,5-dimethoxy-substituted (syringyl-type, S-type) QMs; thus, this pH-dependent trend is common in H-, G-, and S-type lignin-related QMs. Higher threo-selectivity was obtained by changing the ß-etherified aromatic rings from G- to H-type. A similar but weaker effect was also observed by changing the QM moiety from G- to H-type.

Stereoselective Formation of ß-O-4 Structures Mimicking Softwood Lignin Biosynthesis: Effects of Solvent and the Structures of Quinone Methide Lignin Models.

p-Quinone methide (QM) is formed as an intermediate during lignin biosynthesis. The aromatization of the QM by the attack of a nucleophile at the α-position of its side chain generates a phenolic hydroxy group in a growing polymer and creates stereoisomeric forms in the side chain. A series of ß-O-4-aryl ether QMs was reacted with water at 25 °C to replicate the formation of p-hydroxyphenyl (H) and guaiacyl (G) ß-O-4 structures in plant cell walls. Water addition occurred in 3-methoxy-substituted QMs (G-type QMs) with half-lives ( t1/2) between 13 and 15 min, at pH 7, in 50% water solution (dioxane-water, 1:1). The rate increased as the water concentration increased to 99% ( t1/2, 1.2-1.4 min). Similar solvent effects were observed for more reactive nonsubstituted QMs (H-type QMs with t1/2 of <1 min). Consequently, t1/2 of the H-type QMs was shorter than that of the G-type QMs under every solvent condition. Upon increasing the water concentration, the variation in the erythro/ threo ratios of the four dimeric ß-O-4 products increased. Interestingly, the effect of pH on the stereopreference, which was observed in 50% water solution, was small and became imperceptible as the water concentration increased to 99%, suggesting that the effect of the solvent, as well as the effect of the pH, plays an important role in understanding the reaction conditions in cell walls during lignin biosynthesis. The threo isomer was preferentially formed in the four dimeric ß-O-4 structures, which is inconsistent with the structural features of compression wood lignin rich in H-units. However, the erythro-selective formation was attained in an H-type QM at every pH studied (pH 3.5-7) by introducing a biphenyl structure into the ß-etherified ring moiety.

Ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin.

The ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin was investigated by ozonation analysis of wood meal taken from various positions in the stem of yellow poplar (Liriodendron tulipifera). The proportion of the erythro form was higher in tension wood than in opposite wood, and the methoxyl group content showed a similar trend. The proportion of the erythro form and the methoxyl group content in the 7 positions in the stem lignin was correlated (correlation coefficient R=0.98), suggesting that the type of aromatic ring, syringyl or guaiacyl, is one of the factors which stereochemically controls the ratio of erythro and threo forms of beta-O-4 structures during lignin formation.

Changes in lignin content of leaf litters during mulching.

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content. Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.

Reactivity of lignin with different composition of aromatic syringyl/guaiacyl structures and erythro/threo side chain structures in ß-O-4 type during alkaline delignification: as a basis for the different degradability of hardwood and softwood lignin.

The reactivity of lignin during alkaline delignification was quantitatively investigated focusing on the effect of the structural differences between syringyl and guaiacyl aromatic nuclei and between erythro and threo in the side chain of ß-O-4 type lignin substructure on the ß-O-4 bond cleavage rate. It was known that the ratio of this reaction rate of the erythro to threo isomers of the dimeric ß-O-4 type lignin model compound with two guaiacyl aromatic nuclei was ca. 4. However, the presence of a syringyl nucleus strongly influenced the rate, and the ratio of the syringyl type analogue was in the range between 2.7 and 8.0 depending on the reaction temperature. The effect of syringyl nucleus on the enhancement of the reaction rate appeared to be greater when the syringyl nucleus consists of the cleaving ether bond rather than being a member of the carbon framework.

The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar.

Poplar (Populus tremula x alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4HF5H transgenic's lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial beta-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (beta-beta) and spirodienone (beta-1) contents, but with virtually no phenylcoumarans (beta-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the gamma-positions of lignin side chains, were reduced by >50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes.

Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata.

The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata, a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p-hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13C-1H correlative NMR confirmed the increase in p-hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata.

Effects of coumarate 3-hydroxylase down-regulation on lignin structure.

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 approximately 65% P (from wild-type levels of approximately 1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional 13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional 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 beta-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of beta-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 13C-1H correlation experiments. For example, although beta-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.