sinapyl-alcohol and coniferyl-alcohol

To elucidate the influence of polysaccharides on hardwood lignification, dehydrogenative polymerization of monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), was attempted with recombinant cationic cell wall-bound peroxidase (rCWPO-C) and horseradish peroxidase (HRP) in measurement cells of a quartz crystal microbalance with dissipation (QCM-D). Hardwood cellulose nanofibers were anchored; hemicelluloses, xylan, partially acetylated xylan (AcXY), galactoglucomannan, and xyloglucan, and the enzymes were subsequently adsorbed onto the QCM-D sensor surface, enabling fabrication of artificial polysaccharide matrices. The largest amount of rCWPO-C is found to be adsorbed onto AcXY among all the polysaccharides, which affords the largest amount and size of spherical dehydrogenation polymers (DHPs) from both CA and SA. In contrast, no DHP and a small amount of DHPs are formed from SA and CA, respectively, by HRP catalysis in all of the polysaccharide matrices. This study demonstrates important functions of a real tree-derived peroxidase, rCWPO-C, and AcXY for hardwood lignification.

Topics: Cell Wall; Horseradish Peroxidase; Lignin; Peroxidase; Peroxidases; Polymerization; Polymers; Xylans

Lignin is an important component of the healing tissue in fruits. In this study, we treated muskmelon (Cucumis melo L. cv. "Manao") fruit with exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) to observe and analyze its effect on lignin synthesis and accumulation during healing. Results showed that SNP treatment enhanced the contents of endogenous NO and H

Topics: Alcohol Oxidoreductases; Cucumis melo; Fruit; Hydrogen Peroxide; Lignin; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Peroxidase; Phenols; Phenylalanine Ammonia-Lyase; Phenylpropionates; Plant Proteins

The ratio of syringyl (S) and guaiacyl (G) units in lignin has been regarded as a major factor in determining the maximum monomer yield from lignin depolymerization. This limit arises from the notion that G units are prone to C-C bond formation during lignin biosynthesis, resulting in less ether linkages that generate monomers. This study uses reductive catalytic fractionation (RCF) in flow-through reactors as an analytical tool to depolymerize lignin in poplar with naturally varying S/G ratios, and directly challenges the common conception that the S/G ratio predicts monomer yields. Rather, this work suggests that the plant controls C-O and C-C bond content by regulating monomer transport during lignin biosynthesis. Overall, our results indicate that additional factors beyond the monomeric composition of native lignin are important in developing a fundamental understanding of lignin biosynthesis.

Topics: Bioreactors; Catalysis; Chemical Fractionation; Gas Chromatography-Mass Spectrometry; Genetic Variation; Lignin; Magnetic Resonance Spectroscopy; Phenols; Phenylpropionates; Populus

Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.

Topics: Catalytic Domain; Coumaric Acids; Horseradish Peroxidase; Hydrogen Bonding; Molecular Docking Simulation; Molecular Dynamics Simulation; Oxidation-Reduction; Phenols; Phenylalanine; Phenylpropionates; Propionates; Protons; Thermodynamics; Water

This study was carried out to better understand the characteristic modification mechanisms of monolignols by enzyme system of Abortiporus biennis and to induce the degradation of monolignols. Degradation and polymerization of monolignols were simultaneously induced by A. biennis. Whole cells of A. biennis degraded coniferyl alcohol to vanillin and coniferyl aldehyde, and degraded sinapyl alcohol to 2,6-dimethoxybenzene- 1,4-diol, with the production of dimers. The molecular weight of monolignols treated with A. biennis increased drastically. The activities of lignin degrading enzymes were monitored for 24 h to determine whether there was any correlation between monolignol biomodification and ligninolytic enzymes. We concluded that complex enzyme systems were involved in the degradation and polymerization of monolignols. To degrade monolignols, ascorbic acid was added to the culture medium as a reducing agent. In the presence of ascorbic acid, the molecular weight was less increased in the case of coniferyl alcohol, while that of sinapyl alcohol was similar to that of the control. Furthermore, the addition of ascorbic acid led to the production of various degraded compounds: syringaldehyde and acid compounds. Accordingly, these results demonstrated that ascorbic acid prevented the rapid polymerization of monolignols, thus stabilizing radicals generated by enzymes of A. biennis. Thereafter, A. biennis catalyzed the oxidation of stable monolignols. As a result, ascorbic acid facilitated predominantly monolignols degradation by A. biennis through the stabilization of radicals. These findings showed outstanding ability of A. biennis to modify the lignin compounds rapidly and usefully.

Topics: Acrolein; Ascorbic Acid; Basidiomycota; Benzaldehydes; Culture Media; Lignin; Molecular Structure; Molecular Weight; Phenols; Phenylpropionates; Polymerization; Reducing Agents

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.

Topics: Amino Acid Substitution; Cell Wall; Cloning, Molecular; Coumaric Acids; Crystallography, X-Ray; Escherichia coli; Gene Expression; Gene Library; Lignin; Methyltransferases; Mutation; Phenols; Phenylpropionates; Plant Proteins; Plasmids; Populus; Propionates; Protein Engineering; Recombinant Proteins; Structural Homology, Protein; Substrate Specificity

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

Topics: Alcohol Oxidoreductases; Disease Resistance; Enzyme Activation; Genetic Variation; Genotype; Host-Pathogen Interactions; Lignin; Phenols; Phenylpropionates; Plant Diseases; Plant Growth Regulators; Plant Leaves; Saccharum; Ustilaginales

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20-33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls.

Topics: Animals; Bacteria; Bioengineering; Catechin; Cell Wall; Fermentation; Gallic Acid; Hydrogen-Ion Concentration; Lignin; Peroxidase; Phenols; Phenylpropionates; Quercetin; Rumen; Zea mays

In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H(2)O(2), respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of β-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of β-O-4 linkages during DHP synthesis was influenced by peroxidase type.

Topics: Biocatalysis; Coprinus; Glycine max; Horseradish Peroxidase; Hydrogen; Hydrogen Peroxide; Molecular Weight; Peroxidase; Phenols; Phenylpropionates; Polymerization

Two methyl esters of fatty acids, namely octadecanoic acid methyl ester (methyl stearate) and hexadecanoic acid methyl ester (methyl palmitate), in addition to four cinnamyl alcohol derivatives, sinapyl alcohol, coniferyl alcohol, p-coumaryl alcohol and coniferyl alcohol 4-O-glucoside (coniferin), were isolated from callus cultures of Wedelia prostrata. The structure of coniferin was established by spectroscopic and chemical methods, while the other compounds were identified by gas chromatography-mass spectrometry and thin layer chromatography in comparison with standards.

Topics: Cell Culture Techniques; Chromatography, Thin Layer; Cinnamates; Coumaric Acids; Esters; Gas Chromatography-Mass Spectrometry; Methanol; Palmitates; Phenols; Phenylpropionates; Plant Shoots; Propanols; Propionates; Stearates; Wedelia

Single-conformation spectroscopy of the three lignin monomers (hereafter "monolignols") p-coumaryl alcohol (pCoumA), coniferyl alcohol (ConA), and sinapyl alcohol (SinA) has been carried out on the isolated molecules cooled in a supersonic expansion. Laser-induced fluorescence excitation, dispersed fluorescence, resonant two-photon ionization, UV-UV hole-burning, and resonant ion-dip infrared spectroscopy were carried out as needed to obtain firm assignments for the observed conformers of the three molecules. In each case, two conformers were observed, differing in the relative orientations of the vinyl and OH substituents para to one another on the phenyl ring. In pCoumA, the two conformers have S(0)-S(1) origins nearly identical in size, split from one another by only 7 cm(-1), in close analogy with previous results of Morgan et al. on p-vinylphenol ( Chem. Phys. 2008 , 347 , 340 ). ConA, with its methoxy group ortho to the OH group, also has two low-energy conformers forming a syn/anti pair, in this case with the OH group locked into an orientation in which it forms an intramolecular H-bond with the adjacent methoxy group. The electronic frequency shift between the two conformers is dramatically increased to 805 cm(-1), with the dominant conformer of ConA (with S(0)-S(1) origin at 32 640 cm(-1)) about 5 times the intensity of its minor counterpart (with S(0)-S(1) origin at 33 444 cm(-1)). The presence of an OH···OCH(3) intramolecular H-bond is established by the shift of the OH stretch fundamental of the OH group to 3599 cm(-1), as it is in o-methoxyphenol ( Fujimaki et al. J. Chem. Phys. 1999 , 110 , 4238 ). Analogous single-conformation UV and IR spectra of o-methoxy-p-vinylphenol show a close similarity to ConA and provide a basis for a firm assignment of the red-shifted (blue-shifted) conformer of both molecules to the syn (anti) conformer. The two observed conformers of SinA, with its two methoxy group straddling the OH group, have S(0)-S(1) origins split by 239 cm(-1) (33 055 and 33 294 cm(-1)), a value between those in pCoumA and ConA. A combination of experimental data and calculations on the three monolignols and simpler derivatives is used to establish that the conformational preferences of the monolignols reflect the preferences of each of the ring substituents separately, enhanced by the presence of the intramolecular OH···OCH(3) H-bond. Taken as a whole, the presence of multiple flexible substituents locks in certain preferred orientations

Topics: Coumaric Acids; Molecular Conformation; Phenols; Phenylpropionates; Propionates; Quantum Theory; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Stereoisomerism

Cationic cell wall peroxidase (CWPO_C) from poplar tree (Populus alba L) was heterologously expressed in Escherichia coli as an inclusion body. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The condition for this procedure was optimized by varying the refolding pH, and the concentrations of the oxidizing agent (GSSG), denaturing agent (GndCl), and hemin, respectively. The optimal conditions for refolding CWPO_C were 100 mM Tris-HCl at pH 8.5, 0.6mM GSSG, 5 μM hemin, 0.6 M GndCl and 5 mM CaCl₂. The fact that the absorbance spectrum was identical to that of wild CWPO_C from poplar tree suggests that the protein folding, heme insertion and iron coordination were correctly archived. The binding affinity and turnover rate values of refolded CWPO_C were compared with those of HRP_C. k(cat)/K(m) for sinapyl alcohol of CWPO_C was over 170 times higher than that of HRP_C, on the while k(cat)/K(m) for coniferyl alcohol showed similar values for both peroxidase. The kinetic parameters showed that refolded CWPO_C possesses a very unique property of S-peroxidase, preferentially oxidizes sinapyl alcohol rather than coniferyl alcohol. The successful expression of CWPO_C in E. coli provides a valuable tool to elucidate the structure and functional relationship of S-peroxidase, which plays an important role in the lignification of angiosperm woody plant cell walls.

Topics: Benzothiazoles; Enzyme Assays; Escherichia coli; Genetic Vectors; Glutathione Disulfide; Hemin; Horseradish Peroxidase; Hydrogen-Ion Concentration; Inclusion Bodies; Oxidation-Reduction; Peroxidase; Phenols; Phenylpropionates; Plant Proteins; Populus; Protein Binding; Protein Refolding; Solubility; Sulfonic Acids; Transformation, Genetic

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

Topics: Carboxylic Ester Hydrolases; Chromatography, Liquid; Coumaric Acids; DNA Primers; Hydroxylation; Immunoprecipitation; Kinetics; Lignin; Mass Spectrometry; Membrane Proteins; Microscopy, Confocal; Molecular Structure; Multiprotein Complexes; Phenols; Phenylpropionates; Plasmids; Populus; Propionates; Trans-Cinnamate 4-Monooxygenase; Xylem; Yeasts

In order to determine the mechanism of the earlier copolymerization steps of two main lignin precursors, sinapyl (S) alcohol and coniferyl (G) alcohol, microscale in vitro oxidations were carried out with a PRX34 Arabidopsis thaliana peroxidase in the presence of H(2)O(2). This plant peroxidase was found to have an in vitro polymerization activity similar to the commonly used horseradish peroxidase. The selected polymerization conditions lead to a bulk polymerization mechanism when G alcohol was the only phenolic substrate available. In the same conditions, the presence of S alcohol at a 50/50 S/G molar ratio turned this bulk mechanism into an endwise one. A kinetics monitoring (size-exclusion chromatography and liquid chromatography-mass spectrometry) of the different species formed during the first 24h oxidation of the S/G mixture allowed sequencing the bondings responsible for oligomerization. Whereas G homodimers and GS heterodimers exhibit low reactivity, the SS pinoresinol structure act as a nucleating site of the polymerization through an endwise process. This study is particularly relevant to understand the impact of S units on lignin structure in plants and to identify the key step at which this structure is programmed.

Topics: Arabidopsis; Electrophoresis, Polyacrylamide Gel; Lignin; Peroxidases; Phenols; Phenylpropionates; Polymerization

Recalcitrance of lignocellulosic biomass to sugar release is a central issue in the production of biofuel as an economically viable energy source. Among all contributing factors, variations in lignin content and its syringyl-guaiacyl monomer composition have been directly linked with the yield of fermentable sugars. While recent advances in genomics and metabolite profiling have significantly broadened our understanding of lignin biosynthesis, its regulation at the pathway level is yet poorly understood. During the past decade, computational and mathematical methods of systems biology have become effective tools for deciphering the structure and regulation of complex metabolic networks. As increasing amounts of data from various organizational levels are being published, the application of these methods to studying lignin biosynthesis appears to be very beneficial for the future development of genetically engineered crops with reduced recalcitrance. Here, we use techniques from flux balance analysis and nonlinear dynamic modeling to construct a mathematical model of monolignol biosynthesis in Populus xylem. Various types of experimental data from the literature are used to identify the statistically most significant parameters and to estimate their values through an ensemble approach. The thus generated ensemble of models yields results that are quantitatively consistent with several transgenic experiments, including two experiments not used in the model construction. Additional model results not only reveal probable substrate saturation at steps leading to the synthesis of sinapyl alcohol, but also suggest that the ratio of syringyl to guaiacyl monomers might not be affected by genetic modulations prior to the reactions involving coniferaldehyde. This latter model prediction is directly supported by data from transgenic experiments. Finally, we demonstrate the applicability of the model in metabolic engineering, where the pathway is to be optimized toward a higher yield of xylose through modification of the relative amounts of the two major monolignols. The results generated by our preliminary model of in vivo lignin biosynthesis are encouraging and demonstrate that mathematical modeling is poised to become an effective and predictive complement to traditional biotechnological and transgenic approaches, not just in microorganisms but also in plants.

Topics: Algorithms; Biocatalysis; Computer Simulation; Coumaric Acids; Enzymes; Kinetics; Lignin; Metabolic Networks and Pathways; Models, Biological; Phenols; Phenylpropionates; Plants, Genetically Modified; Populus; Propionates; Systems Theory

Grasses are a predominant source of nutritional energy for livestock systems around the world. Grasses with high lignin content have lower energy conversion efficiencies for production of bioenergy either in the form of ethanol or to milk and meat through ruminants. Grass lignins are uniquely acylated with p-coumarates (pCA), resulting from the incorporation of monolignol p-coumarate conjugates into the growing lignin polymer within the cell wall matrix. The required acyl-transferase is a soluble enzyme (p-coumaroyl transferase, pCAT) that utilizes p-coumaroyl-CoenzymeA (pCA-CoA) as the activated donor molecule and sinapyl alcohol as the preferred acceptor molecule. Grasses (C3and C4) were evaluated for cell wall characteristics; pCA, lignin, pCAT activity, and neutral sugar composition. All C3 and C4 grasses had measurable pCAT activity, however the pCAT activities did not follow the same pattern as the pCA incorporation into lignin as expected.

Topics: Acyltransferases; Cell Wall; Chromatography, High Pressure Liquid; Coumaric Acids; Lignin; Mass Spectrometry; Molecular Structure; Phenols; Phenylpropionates; Poaceae; Sorghum; Substrate Specificity; Zea mays

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

Topics: Alcohol Oxidoreductases; Chromosome Mapping; Cloning, Molecular; Escherichia coli; Genes, Plant; Kinetics; Lignin; Oryza; Phenols; Phenotype; Phenylpropionates; Phylogeny; Plant Proteins; Recombinant Fusion Proteins; Seeds

We partially purified peroxidase isoform fractions from xylem extracts of a gymnosperm, Norway spruce (Picea abies (L.) Karst.), and an angiosperm, silver birch (Betula pendula Roth.), to determine the participation of xylem-localized peroxidases in polymerization of different types of lignin in vivo. Several peroxidase fractions varying in isoelectric point values from acidic to basic were tested for their ability to catalyze the oxidation of the monolignols coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol in vitro. All of the xylem peroxidases extracted from Norway spruce and most of those from silver birch showed the highest rate of oxidation with coniferyl alcohol in the presence of hydrogen peroxide. The exception was an acidic peroxidase fraction (pI 3.60-3.65) from silver birch that exhibited higher oxidation activity for sinapyl alcohol than for coniferyl alcohol. For the xylem enzyme fractions extracted from silver birch, the ability to oxidize the artificial phenolic substrate syringaldazine coincided with high specific activity for sinapyl alcohol. Therefore, we conclude that the acidic, neutral and basic xylem peroxidases of Norway spruce all function in the synthesis of guaiacyl-type lignin, whereas in silver birch the acidic peroxidases preferentially oxidize sinapyl subunits. The latter provides a mechanism for synthesis of guaiacyl-syringyl lignin typical of tracheid cell walls in angiosperm trees.

Topics: Betula; Biopolymers; Isoenzymes; Lignans; Lignin; Molecular Structure; Oxidation-Reduction; Peroxidases; Phenols; Phenylpropionates; Picea; Xylem

We previously showed that eight laccase genes (Lac 1-Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147-155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO(4) in the reaction mixtures increased in the range from 1 to 100 microM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.

Topics: Cell Line, Transformed; Cloning, Molecular; Copper Sulfate; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoenzymes; Laccase; Lignin; Nicotiana; Phenols; Phenylpropionates; Pinus taeda; Plants, Genetically Modified; Xylem

Coniferyl and sinapyl alcohols were prepared from commercially available coniferaldehyde and sinapaldehyde using borohydride exchange resin in methanol. This reduction is highly regioselective and exceptionally simple, making these valuable monolignols readily available to researchers lacking synthetic chemistry expertise.

Topics: Acrolein; Borohydrides; Phenols; Phenylpropionates

During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional beta-O-4-linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene.

Topics: Alcohol Oxidoreductases; Arabidopsis; DNA, Plant; Down-Regulation; Flowers; Gene Expression Regulation, Plant; Genes, Plant; Lignin; Molecular Sequence Data; Mutation; Phenols; Phenotype; Phenylpropionates; Protein Isoforms; Spectrophotometry, Infrared; Transcription, Genetic

The thermochemolytic behavior of 4-O-etherified cinnamyl alcohols and aldehydes in lignin was investigated in the presence of tetramethylammonium hydroxide (TMAH) (315 degrees C/4 s), using veratrylglycol-beta-(coniferyl alcohol) ether (1a), veratrylglycol-beta-(sinapyl alcohol) ether (1b), and veratrylglycol-beta-(coniferyl aldehyde) ether (2). The methylated products were monitored with gas chromatography-mass spectrometry. Dimers 1a and 1b provided the coniferyl and sinapyl alcohol dimethyl ethers consisting of three isomers, respectively. Coniferyl alcohol dimethyl ether isomers were also observed in the TMAH thermochemolysis pyrolysates of a bulk dehydrogenation polymer of coniferyl alcohol and a Japanese cedar (Cryptomeria japonica) wood. Coniferyl aldehyde methyl ether was not provided from TMAH thermochemolyses of coniferyl aldehyde, 2, a dehydrogenation polymer of coniferyl aldehyde, and the cedar wood. The former three provided veratryl aldehyde in a large abundance, instead of coniferyl aldehyde methyl ether. Sinapyl aldehyde provided 3,4,5-trimethoxybenzaldehyde in a large abundance and sinapyl aldehyde methyl ether in a trace abundance. The results showed that TMAH thermochemolysis is an effective tool to obtain information on cinnamyl alcohol end groups, but is not applicable to analysis of cinnamyl aldehyde end groups.

Topics: Acrolein; Aldehydes; Dimerization; Ethers; Gas Chromatography-Mass Spectrometry; Lignin; Methylation; Phenols; Phenylpropionates; Propanols; Quaternary Ammonium Compounds; Tracheophyta; Wood