Takuya Akiyama

The Effects on Lignin Structure of Overexpression of Ferulate 5-Hydroxylase in Hybrid Poplar1

Poplar (Populus tremula × 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 C4H∷F5H transgenics 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 β-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (β-β) and spirodienone (β-1) contents, but with virtually no phenylcoumarans (β-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the γ-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.

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 ∼65% P (from wild-type levels of ∼1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional13C-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 β-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of β-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 range13C-1H correlation experiments. For example, although β-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.

Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels in DMSO-d6

Although finely divided ball-milled whole cell walls do not completely dissolve in dimethylsulfoxide (DMSO), they readily swell producing a gel. Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) of this gel, produced directly in the NMR tube, provides an interpretable structural fingerprint of the polysaccharide and lignin components of the wall without actual solubilization, and without structural modification beyond that inflicted by the ball milling and ultrasonication steps. Since the cellulose is highly crystalline and difficult to swell, the component may be under-represented in the spectra. The method however provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. With the new potential for chemometric analysis using the 2D NMR fingerprint, this method may find application as a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

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.

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 β-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.

Erythro/threo ratio of β-O-4-5 structures as an important structural characteristic of lignin. Part 4: Variation in the erythro/threo ratio in softwood and hardwood lignins and its relation to syringyl/guaiacyl ratio

Abstract The proportion of erythro- and threo-forms of β-O-4-structures in lignin was elucidated by ozonation analysis of 21 wood species, and the relationship to the syringyl and guaiacyl composition was investigated. For all hardwood species, the erythro-form of β-O-4-structures predominated, although the extent varied widely, depending on wood species. In contrast, the proportion and amount of erythro- and threo-forms were very similar in all softwood species. The proportion of the erythro-form was greater in species with a higher methoxyl content in the lignin (correlation coefficient, R2=0.83). The S/V ratio (molar ratio of syringaldehyde and syringic acid to that of vanillin and vanillic acid) obtained by nitrobenzene oxidation was also strongly correlated with the proportion of the erythro-form (R2=0.99). Accordingly, the syringyl/guaiacyl ratio is closely related to the erythro/threo ratio. This stereochemical characteristic of β-O-4-structures is discussed in relation to the process of lignin formation.

Erythro/threo ratio of β-O -4 structures as an important structural characteristic of lignin. I: Improvement of ozonation method for the quantitative analysis of lignin side-chain structure

Ozonation as a quantitative tool to analyze the stereo structures of arylglycerol-β-aryl ether linkages was examined using wood meal, milled wood lignin, and a lignin model compound, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (veratrylglycerol-β-guaiacyl ether, VG). The procedure was improved. When mild postreduction was conducted for ozonation products, the total yield of erythronic and threonic acids from this model compound was 74%, which is 15% higher than the yield without postreduction. A decrease in the recovery of these two acids under prolonged ozonation treatment was successfully suppressed by postreduction. Theerythro/threo ratio of VG determined by the ozonation method with postreduction is in good agreement with the ratio determined by1H-nuclear magnetic resonance. Excellent reproducibility of the yield was obtained by adopting a procedure that included trimethylsilylation of ammonium salts of ozonation products using a dimethylsulfoxide-hexamethyldisilazane-trimethylchlorosilane mixture and subjecting it to gas chromatography analysis. It was concluded that arylglycerol-β-aryl ether structures comprise at least 35% of the C3-C6 structure in birch wood meal, with anerythro/threo ratio of 2.8.

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

Abstract The ratio of erythro and threo forms of β-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 β-O-4 structures during lignin formation.

Reactivity of Lignin with Different Composition of Aromatic Syringyl/Guaiacyl Structures and Erythro/Threo Side Chain Structures in β-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 β-O-4 type lignin substructure on the β-O-4 bond cleavage rate. It was known that the ratio of this reaction rate of the erythro to threo isomers of the dimeric β-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.