Evidence for the biosynthetic pathway from sinapic acid to syringyl lignin using labeled sinapic acid with stable isotope at both methoxy groups in Robinia pseudoacacia and Nerium indicum

A tracer experiment using synthesized labeled lignin precursors was designed to confirm the actual biosynthetic pathway for syringyl lignin. Tetradeuteroferulic acid-[8-D, 3-OCD(3)] and heptadeuterosinapic acid-[8-D, 3,5-OCD(3)] were synthesized and fed to shoots of robinia (Robinia pseudoacacia) and oleander (Nerium indicum) trees. The incorporation of each labeled precursor into lignin was traced by gas chromatography-mass spectrometry. The synthesized sinapic acid, in which both methoxy groups were labeled, was useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeuterosinapic acid was fed, syringyl units containing seven deuterium labels were detected. The results of this study support the traditionally accepted pathway that sinapic acid is converted to sinapyl alcohol via sinapoyl-CoA in robinia and oleander.     

Behavior of monolignol glucosides in angiosperms

To examine the behavior of cinnamaldehyde and cinnamyl alcohol glucosides in lignifying tissues, angiosperms were subjected to tracer experiments using radioisotope-labeled and stable isotope-labeled glucosides. The aglycone from coniferaldehyde glucoside was efficiently incorporated into guaiacyl and syringyl lignin as a cinnamyl alcohol unit. The aglycone from coniferin was also incorporated into guaiacyl and syringyl lignin. However, some of the coniferin-derived aglycone that was incorporated into lignin passed through a cinnamaldehyde form prior to dehydrogenative polymerization. When coniferin was administered together with coniferaldehyde glucoside, syringyl units were rarely synthesized from coniferin via the cinnamyl alcohol stage, whereas numerous syringyl units were synthesized from coniferaldehyde glucoside. These observations suggest that the coniferaldehyde form is crucial for the biosynthesis of syringyl lignin in angiosperms.     

Occurrence of naturally acetylated lignin units

This work examines the occurrence of native acetylated lignin in a large set of vascular plants, including both angiosperms and gymnosperms, by a modification of the so-called Derivatization Followed by Reductive Cleavage (DFRC) method. Acetylated lignin units were found in the milled wood lignins of all angiosperms selected for this study, including mono- and eudicotyledons, but were absent in the gymnosperms analyzed. In some plants (e.g., abaca, sisal, kenaf, or hornbeam), lignin acetylation occurred at a very high extent, exceeding 45% of the uncondensed (alkyl-aryl ether linked) syringyl lignin units. Acetylation was observed exclusively at the gamma-carbon of the lignin side chain and predominantly on syringyl units, although a predominance of acetylated guaiacyl over syringyl units was observed in some plants. In all cases, acetylation appears to occur at the monomer stage, and sinapyl and coniferyl acetates seem to behave as real lignin monomers participating in lignification.     

Detection and determination of p-coumaroylated units in lignins.

The derivatization followed by reductive cleavage (DFRC) method cleaves alpha- and beta-ethers in lignins but leaves lignin gamma-esters intact. When applied to grasses, which contain p-coumarate esters on their lignins, esterified monolignol derivatives are released. Saturation of the p-coumarate double bond occurs during DFRC, so the released products are 4-acetoxycinnamyl 4-acetoxyphenylpropionates. Synthesis of the esters allowed determination of response factors for the released products. Maize and bamboo lignins released 221 and 38 micromol/g of p-coumarate-derived esters. The sinapyl ester was much more abundant than the coniferyl one. The bamboo and maize lignin S/G ratios in the conjugates were 12 and 38 times greater than those of the normal monomers released by DFRC, evidence of a strong selectivity for acylation of syringyl units. Of three possible biochemical mechanisms for incorporating p-coumarates into lignin, evidence is mounting that the process involves incorporation of preacylated monolignols into the normal lignification process.     

Chemical characterization of lignin and lipid fractions in kenaf bast fibers used for manufacturing high-quality papers

The chemical composition of lignin and lipids of bast fibers from kenaf (Hibiscus cannabinus) used for high-quality paper pulp production was studied. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) of fibers showed a lignin with a high syringyl/guaiacyl ratio (5.4) and minor amounts of p-hydroxyphenyl units. Simultaneously, sinapyl and coniferyl acetates were also identified, indicating that this lignin is partially acetylated. p-Hydroxycinnamic acids were found in only trace amounts. The main lipids identified by GC/MS of extracts from kenaf fibers were series of long-chain n-fatty acids, waxes, n-alkanes, and n-fatty alcohols. Free and esterified sterols and triterpenols, steroid hydrocarbons, and steroid and triterpenoid ketones, as well as steryl glycosides, were also found. Finally, the fate of the main constituents of kenaf fibers in alkaline pulping was also investigated.     

Chemical composition of abaca (Musa textilis) leaf fibers used for manufacturing of high quality paper pulps

The chemical composition of leaf fibers of abaca (Musa textilis), which are commonly used for high-quality paper pulp production, was thoroughly studied. The results revealed that the lignin content was 13.2% of the total fiber. The analysis of abaca fibers by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS) released predominantly compounds arising from lignin and p-hydroxycinnamic acids, with high amounts of 4-vinylphenol. The latter compound was demonstrated to arise from p-coumaric acid by pyrolysis of abaca fibers in the presence of tetramethylammonium hydroxide, which released high amounts of p-coumaric acid (as the methyl derivative). Products from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) propanoid units, with a predominance of the latter (H:G:S molar ratio of 1.5:1:4.9), were also released after Py-GC/MS of abaca fibers. Sinapyl and coniferyl acetates, which are thought to be lignin monomer precursors, were also found in abaca. The extractives content of the abaca fiber (0.4%) was low, and the most predominant compounds were free sterols (24% of total extract) and fatty acids (24% of total extract). Additionally, significant amounts of steroid ketones (10%), triglycerides (6%), omega-hydroxyfatty acids (6%), monoglycerides (4%), fatty alcohols (4%), and a series of p-hydroxycinnamyl (p-coumaric and ferulic acids) esterified with long chain alcohols and omega-hydroxyfatty acids were also found, together with minor amounts of steroid hydrocarbons, diglycerides, alpha-hydroxyfatty acids, sterol esters, and sterol glycosides.     

In search of a maize ideotype for cell wall enzymatic degradability using histological and biochemical lignin characterization

Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and M?ule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.     

NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.     

Lignins and ferulate-coniferyl alcohol cross-coupling products in cereal grains

Plant cell walls containing suberin or lignin in the human diet are conjectured to protect against colon cancer. To confirm the existence of authentic lignin in cereal grain dietary fibers, the DFRC (derivatization followed by reductive cleavage) method was applied to different cereal grain dietary fibers. By cleavage of diagnostic arylglycerol-beta-aryl (beta-O-4) ether linkages and identification of the liberated monolignols, it was ascertained that lignins are truly present in cereal grains. From the ratios of the liberated monolignols coniferyl alcohol and sinapyl alcohol, it is suggested that lignin compositions vary among cereals. Furthermore, dimeric cross-coupling products, comprising ferulate and coniferyl alcohol, were identified in most cereal fibers investigated. These ferulate 4-O-beta- and 8-beta-coniferyl alcohol cross-coupled structures indicate radical cross-coupling of polysaccharides to lignin precursors via ferulate.     

Highly acylated (acetylated and/or p-coumaroylated) native lignins from diverse herbaceous plants

The structure of lignins isolated from the herbaceous plants sisal ( Agave sisalana), kenaf ( Hibiscus cannabinus), abaca ( Musa textilis) and curaua ( Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the gamma-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are gamma-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of beta- O-4' linkages (up to 94% of all linkages), and a strikingly low proportion of traditional beta-beta' linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of beta-beta' homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions.     

Structural motifs of syringyl peroxidases are conserved during angiosperm evolution

The most distinctive variation in the monomer composition of lignins in vascular land plants is that between the two main groups of seed plants. Thus, whereas gymnosperm (softwood) lignins are typically composed of guaiacyl (G) units, angiosperm (hardwood) lignins are largely composed of similar levels of G and syringyl (S) units. However, there are some studies that suggest that certain angiosperm peroxidases are unable to oxidize sinapyl alcohol, and a coniferyl alcohol shuttle has been proposed for oxidizing S units during the biosynthesis of lignins. With this in mind, a screening of the presence of S peroxidases in angiosperms (including woody species and forages) was performed. Contrarily to what might be expected, the intercellular washing fluids from lignifying tissues of 25 woody, herbaceous, and shrub species, belonging to both monocots and dicotyledons, all showed both S peroxidase activities and basic peroxidase isoenzymes analogous, with regard the isoelectric point, to the Zinnia elegans basic peroxidase isoenzyme, the only S peroxidase that has been fully characterized. These results led to the protein database in the search for homologies between angiosperm peroxidases and a true eudicot S peroxidase, the Z. elegans peroxidase. The findings showed that certain structural motifs of S peroxidases are conserved within the first 15 million years of angiosperm history, because they are found in peroxidases from the two major lineages of flowering plants, eumagnoliids and eudicotyledons, of note being the presence of these peroxidases in Amborella and Nymphaeales, which represent the first stages of angiosperm evolution. These phylogenetic studies also suggest that guaiacyl peroxidases apparently constitute the most "evolved state" of the plant peroxidase family evolution.     

Significant increases in pulping efficiency in C4H-F5H-transformed poplars: improved chemical savings and reduced environmental toxins

The gene encoding ferulate 5-hydroxylase (F5H) was overexpressed in poplar (Populus tremula x Populus alba) using the cinnamate-4-hydroxylase (C4H) promoter to drive expression specifically in cells involved in the lignin biosynthetic pathway and was shown to significantly alter the mole percentage of syringyl subunits in the lignin, as determined by thioacidolysis. Analysis of poplar transformed with a C4H-F5H construct demonstrated significant increases in chemical (kraft) pulping efficiency from greenhouse-grown trees. Compared to wild-type wood, decreases of 23 kappa units and increases of >20 ISO brightness units were observed in trees exhibiting high syringyl monomer concentrations. These changes were associated with no significant modification in total lignin content and no observed phenotypic differences. C4H-F5H-transformed trees could increase pulp throughputs at mills by >60% while concurrently decreasing chemicals employed during processing (chemical pulping and bleaching) and, consequently, the amount of deleterious byproducts released into the environment.     

Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta' and 8-beta' and some 8-O-4' and 8-5' cross-coupled structures. Some 8-beta' structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.     

Sisal fibers: surface chemical modification using reagent obtained from a renewable source; characterization of hemicellulose and lignin as model study

Sisal fibers have one of the greatest potentials among other lignocellulosic fibers to reinforce polymer matrices in composites. Sisal fibers have been modified to improve their compatibility with phenolic polymer matrices using furfuryl alcohol (FA) and polyfurfuryl alcohols (PFA) that can be obtained from renewable sources. The modification corresponded first to oxidation with ClO 2, which reacts mainly with guaiacyl and syringyl units of lignin, generating o- and p-quinones and muconic derivatives, followed by reaction with FA or PFA. The FA and PFA modified fibers presented a thin similar layer, indicating the polymer character of the coating. The untreated and treated sisal fibers were characterized by (13)C CP-MAS NMR spectrometry, thermal analysis, and scanning electron microscopy. Furthermore, for a better understanding of the reactions involved in the FA and PFA modifications, the sisal lignin previously extracted was also submitted to those reactions and characterized. The characterization of isolated lignin and hemicellulose provides some information on the chemical structure of the main constitutive macrocomponents of sisal fibers, such information being scarce in the literature.     

Physicochemical and structural characterization of alkali soluble lignins from oil palm trunk and empty fruit-bunch fibers.

Six alkali soluble lignin fractions were extracted from the cell wall materials of oil palm trunk and empty fruit-bunch (EFB) fibers with 5% NaOH, 10% NaOH, and 24% KOH/2% H(3)BO(3). All of the lignin fractions contained rather low amounts of associated neutral sugars (0.8-1.2%) and uronic acids (1.1-2.0%). The lignin fractions isolated with 5% NaOH from the lignified palm trunk and EFB fibers gave a relatively higher degree of polymerization as shown by weight-average molecular weights ranging between 2620 and 2840, whereas the lignin fractions isolated with 10% NaOH and 24% KOH/2% H(3)BO(3) from the partially delignified palm trunk and EFB fibers showed a relatively lower degree of polymerization, as shown by weight-average molecular weights ranging between 1750 and 1980. The results obtained by alkaline nitrobenzene oxidation showed that all of the lignin preparations contained a high proportion of noncondensed syringyl units with small amounts of noncondensed guaiacyl and fewer p-hydroxyphenyl units. The lignin fraction extracted with 5% NaOH from the lignified EFB fiber was mainly composed of beta-O-4 ether-linked units. Small amounts of 5-5', beta-5, and beta-beta' carbon-carbon linkages were also found to be present between the lignin structural units. Further studies showed that uronic, p-hydroxybenzoic, and ferulic acids in the cell walls of palm fibers were esterified to lignin.     

Comprehensive study on the chemical structure of dioxane lignin from plantation Eucalyptus globulus wood

Results of a comprehensive study on the chemical structure of lignin from plantation Eucalyptus globulus Labill are presented. Lignin has been isolated by a modified mild acidolysis method and thoroughly characterized by functional group analysis, by a series of degradation techniques (nitrobenzene oxidation, permanganate oxidation, thioacidolysis, and Py-GC-MS), and (1)H and (13)C NMR spectroscopy. Plantation Eucalyptus globulus lignin was found to be of the S/G type with an extremely high proportion of syringyl (S) units (82-86%) and a minor proportion of p-hydrophenyl propane (H) units (roughly 2-3 mol %). Unknown C-6 substituted and 4-O-5' type syringyl substructures represent about 65% of lignin "condensed" structures. Eucalypt lignin showed high abundance of beta-O-4 (0.56/C(6)) structures and units linked by alpha-O-4 bonds (0.23/C(6)). The proportion of phenylcoumaran structures was relatively low (0.03/C(6)). Different kinds of beta-beta substructures (pino-/syringaresinol and isotaxiresinol types) in a total amount of 0.13/C(6) were detected. ESI-MS analysis revealed a wide molecular weight distribution of lignin with the center of gravity of mass distribution around 2500 u.     

Biodegradation of oak (Quercus alba) wood during growth of the shiitake mushroom (Lentinula edodes): a molecular approach

The chemical transformations that occur during growth of the shiitake mushroom (Lentinula edodes) on oak (Quercus alba) were investigated to improve mushroom cultivation and utilization of the spent substrate. Oak logs were decayed by L. edodes over 8 years, during which time they were sampled at six intervals (30, 40, 66, 76, 77, and 101 months). Fresh and decayed oak samples were analyzed using solid-state (13)C NMR and pyrolysis-gas chromatography-mass spectrometry as well as off-line thermochemolysis with tetramethylammonium hydroxide. Degraded oak exhibited lower carbon contents and increased oxygen content compared to the control. Solid-state (13)C NMR analysis revealed that polysaccharides were the major component of both fresh and decayed oak but that L. edodes mediated the preferential loss of cellulose and xylans as compared to lignin, which remained in an altered form. Several trends point toward the degradation of lignin, including a decrease in the proportion of syringyl units as compared to guaiacyl units and a reduction in side-chain length. An increase in guaiacyl and syringyl acid-to-aldehyde ratios occurred with growth, which suggested that the fungus had caused oxidation of Calpha-Cbeta bonds. The overall effect of L. edodes on oak is similar to that of many white-rot fungi, which simultaneously degrade all cell wall components.     

Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.     

Lignin structure in a mutant pine deficient in cinnamyl alcohol dehydrogenase

Cinnamyl alcohol dehydrogenase (CAD) activity is deficient in loblolly pine (Pinus taeda L.) harboring a mutated allele of the cad gene (cad-n1). We compared lignin structure of CAD-deficient and wild-type pines, both types segregating within full-sib families obtained by controlled crosses. The type and frequency of lignin building units and distribution of interunit bonds were determined from the GC-MS analysis of thioacidolysis monomers and dimers. While the lignin content was only slightly reduced, the lignin structure was dramatically modified by the mutation in both mature and juvenile trees. Lignins from CAD-deficient pine displayed unusually high levels of coniferaldehyde and dihydroconiferyl alcohol. In addition, biphenyl and biphenyl ether bonds were in large excess in these abnormal lignins. These results suggest that the CAD-deficient pines efficiently compensate for the shortage in normal lignin precursors by utilizing nontraditional wall phenolics to construct unusual lignins particularly enriched in resistant interunit bonds.     

Pyrolysis of lignin in the presence of tetramethylammonium hydroxide: a convenient method for S/G ratio determination

Pyrolysis-gas chromatography in the presence of tetramethylammonium hydroxide (TMAH) was applied to the determination of the ratio of the abundances of the syringyl beta-aryl ether subunits to those of the guaiacyl equivalents (S/G) in lignin. Diazomethane-methylated kenafs (Hibiscus cannabinus and Hibiscus sabdariffa) and beech (Fagus crenata) in situ lignins were employed. Relative abundances of pyrolysis products derived from the guaiacyl and syringyl beta-aryl ether subunits were determined. The S/G ratios for in situ lignins were obtained with average 3.1% relative standard deviation for a minimum of six repeated runs. The S/G ratios determined by pyrolysis in the presence of TMAH agreed well with those determined by thioacidolysis, with a significant linear regression (R(2) = 0.9867). The results showed that pyrolysis with TMAH is an effective tool for obtaining information on the S/G ratio for in situ lignins.