An accurate and non-labor intensive method for the determination of syringyl to guaiacyl ratio in lignin

The syringyl to guaiacyl (S:G) ratio of hardwood lignin has long been identified as a significant parameter in delignification processes and more recent results have shown that it is also important in determining the amount of ethanol that can be obtained from fermentation of hydrolyzed wood. Acidolysis of Klason or acid insoluble lignin in dioxane/water/HCl was being investigated when syringyl and guaiacyl nuclei with a diketone-containing sidechain were observed as the major products. The area ratio of the two gas chromatogram peaks appeared to be indicative of the S:G ratio. After optimization of the method the relative standard deviation was found to be in the range of 0.3–3.76% for Klason lignin from a wide range of Eucalyptus grandis grown in South Africa. The method was then compared to nitrobenzene oxidation (NBO) using 13 poplars in a double-blind study. The respective S:G ratios were used to calculate percentages of S units and when these values were plotted against each other a linear correlation was obtained with a slope of approximately 1.0 (R² = 0.86). The largest discrepancy for any poplar was 6.9% (62% vs. 58% S units). Both methods convincingly demonstrated a significant decrease in lignin content with an increase in the S:G ratio. Discussion is presented on a series of reaction that could lead to the formation of the two diketones.     

Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) is considered necessary to activate the hydroxycinnamic acids for the biosynthesis of the coniferyl and sinapyl alcohols subsequently polymerized into lignin. To clarify the role played by 4CL in the biosynthesis of the guaiacyl (G) and syringyl (S) units characteristic of angiosperm lignin, we generated 4CL antisense Arabidopsis lines having as low as 8% residual 4CL activity. The plants had decreases in thioglycolic acid-extractable lignin correlating with decreases in 4CL activity. Nitrobenzene oxidation of cell walls from bolting stems revealed a significant decrease in G units in 4CL-suppressed plants; however, levels of S lignin units were unchanged in even the most severely 4CL-suppressed plants. These effects led to a large decrease in the G/S ratio in these plants. Our results suggest that an uncharacterized metabolic route to sinapyl alcohol, which is independent of 4CL, may exist in Arabidopsis. They also demonstrate that repression of 4CL activity may provide an avenue to manipulate angiosperm lignin subunit composition in a predictable manner.     

Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae

Cactaceae family has heterogeneity in the accumulation of lignocellulose due to the diversity of shapes and anatomy of the wood. Most studies focus on fibrous and dimorphic species; but the non-fibrous species are poorly studied. The aims of this work were to analyze the syringyl/guaiacyl ratio of lignin and its distribution in secondary xylem, especially in non-fibrous species. The syringyl/guaiacyl (S/G) ratio was quantified from 34 species of cacti by nitrobenzene oxidation of free-extractive wood. The distribution of lignocellulose in wood sections stained with safranin O/fast green was determined with epifluorescence microscopy. The S/G ratio was heterogeneous; most of the non-fibrous species had a higher percentage of syringyl, while the fibrous ones accumulate guaiacyl. Fluorescence emission showed that vessel elements and wide-band tracheids had similar tonalities. It is hypothesized that the presence of a higher percentage of syringyl in most cacti is part of the defense mechanism against pathogens, which together with the succulence of the stem represent adaptations that contribute to survival in their hostile environments.     

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.     

Altered lignin composition in phenylalanine ammonia-lyase-inhibited radish seedlings: implications for seed-derived sinapoyl esters as lignin precursors

We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 microM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.     

Conversion of guaiacyl to syringyl moieties on the cinnamyl alcohol pathway during the biosynthesis of lignin in angiosperms

Aglycons derived from 4-O-β-D-glucosides of both caffeyl and 5-hydroxyconiferyl alcohols were incorporated into guaiacyl (G) and syringyl (S) units in the lignin of newly formed xylem of several angiosperms. It is likely that these aglycons enter the cinnamyl alcohol pathway as intermediates in the introduction of methoxyl groups onto aromatic rings, and serve as precursors for the biosynthesis of lignin. The S/G ratio in this pathway was coincident with the ratio in the cell wall lignin of each tree. Our results indicate that the cinnamyl alcohol pathway involves the same mechanisms as the cinnamic acid and cinnamyl CoA pathways and they suggest that this novel pathway might be part of a metabolic grid in the biosynthesis of lignin. Received: 8 September 1999 / Accepted: 4 October 1999     

Effects on Lignin Structure of Coumarate 3-Hydroxylase Downregulation in Poplar

The lignin structural ramifications of coumarate 3-hydroxylase (C3H) downregulation have not been addressed in hardwoods. Such information is required to accompany an assessment of the digestibility and bioenergy performance characteristics of poplar, in particular. Structurally rich 2D NMR methods were applied to the entire lignin fraction to delineate lignin p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) levels and linkage distribution changes (and to compare with traditional degradative analyses). C3H downregulation reduced lignin levels by half and markedly increased the proportion of H units relative to the normally dominant G and S units. Relative stem H unit levels were up by ???100-fold to ???31?%, almost totally at the expense of G units; differences in the lignin interunit linkage distributions were more subtle. The H level in the most drastically C3H-downregulated transgenic poplar falls well beyond the H:G:S compositional bounds of normal angiosperms. The response observed here, in poplar, differs markedly from that reported for alfalfa where the S:G ratio remained almost constant even at substantial H levels, highlighting the often differing responses among plant species.     

The properties of syringyl, guaiacyl and p-hydroxyphenyl artificial lignins

1. Artificial lignins have been produced on potato parenchyma. 2. The methoxyl-free lignin and 4-hydroxy-3-methoxy (guaiacyl) lignins could be estimated by the sulphuric acid method but the 4-hydroxy-3,5-dimethoxy (syringyl) lignins could not. 3. Permanganate oxidation of isolated p-coumaric lignin gave 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and small amounts of hydroxytrimesic acid and 4-hydroxyphthalic acid. Ferulic lignin gave vanillic acid and 5-carboxyvanillic acid and also small amounts of 4-hydroxybenzoic acid and dehydrodivanillic acid. The sinapic lignin gave traces of syringic acid and of 4-hydroxybenzoic acid. 4. The p-coumaric lignin is a highly condensed polymer. The ferulic lignin is partly uncondensed and partly condensed through the 5-position like gymnosperm lignin. The sinapic lignin shows no evidence of condensation and is probably an ether-linked polymer.     

Capillary zone electrophoresis of syringyl and guaiacyl monomers resulting from lignin thioacidolysis

A capillary zone electrophoretic method has been developed for the quantitative determination of syringyl (S) and guaiacyl (G) monomers resulting from lignin thioacidolysis. The effects on the separation of altering a number of parameters have been investigated, resulting in an efficient and rapid separation. Analysis times were about 10 min instead of 50 min for the conventional GC methods, with no requirement for a derivatisation step prior to analysis. Relative standard deviations ranged between 8 and 12% for the absolute determination of S and G monomers, and between 1.4 and 3.5% for the S/G ratio.     

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.     

Regulation of Syringyl to Guaiacyl Ratio in Lignin Biosynthesis

p -hydroxyphenyl (H)-, guaiacyl (G)- and syringyl (S) propane, in situ is described. New pathways that regulate the ratio of S to G moieties operating at the stages of cinnamoyl CoA, cinnamyl aldehyde and cinnamyl alcohol are introduced. The roles of monolignol glucoside in the lignification of tree xylem are discussed. The results of gene manupulations that alter the lignin structures are also introduced. Received 15 September 2001/ Accepted in revised form 16 October 2001     

Chemical Composition of Hypodermal and Endodermal Cell Walls and Xylem Vessels Isolated from Clivia miniata (Identification of the Biopolymers Lignin and Suberin)

The occurrence of the biopolymers lignin and suberin was investigated with hypodermal (HCW) and endodermal cell walls (ECW) and xylem vessels (XV) isolated from Clivia miniata Reg. roots. Both biopolymers were detected in HCW and ECW, whereas in XV, typical aliphatic suberin monomers were missing and only representative lignin monomers such as guaiacyl (G) and syringyl (S) units could be detected. The absolute amounts of lignin were about one order of magnitude higher compared with suberin in both HCW and ECW. The ratios of the two aromatic lignin units (G/S) decreased from 39 in XV and 10 in HCW to about 1 in ECW, indicating significant differences in lignin structure and function between the three investigated samples. Additionally, compared with the detectable lignin-derived aromatic units G and S, significantly higher amounts of esterified p-coumaric acid-derived aromatic monomers were obtained with HCW, but not with ECW. This is interpreted as a functional adaption of HCW toward pathogen defense at the root/soil interface. The final aim of this study was to provide a thorough chemical characterization of the composition of HCW, ECW, and XV, which in turn will form the basis for a better understanding of the relevant barriers toward the passive, radial, and apoplastic diffusion of solutes from the soil across the root cortex into the root cylinder.     

The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units?

Lignin is a complex polymer formed by the oxidative polymerization of hydroxycinnamyl alcohol derivatives termed monolignols. The major monolignols in dicotyledonous angiosperm lignin are monomethylated guaiacyl (G) units derived from coniferyl alcohol, and dimethylated syringyl (S) units derived from sinapyl alcohol. The biochemical pathways leading to the formation of monolignols feature successive hydroxylation and O-methylation of the aromatic ring and conversion of the side chain carboxyl to an alcohol function. The current view of the monolignol biosynthetic pathway envisages a metabolic grid leading to G and S units, through which the successive hydroxylation and O-methylation reactions may occur at different levels of side chain oxidation. The present article assesses biochemical and genetic evidence for and against such a model, including recent data on the methylation reactions of monolignol biosynthesis in alfalfa. We draw attention to portions of the currently accepted monolignol pathway that may require revision, and suggest an alternative model in which metabolic channeling allows for independent pathways to G and S lignin.     

Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained.     

Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa. impacts on lignin structure and implications for the biosynthesis of G and S lignin

Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in beta-5 linked dimers of G units. Accumulation of soluble caffeic acid beta-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis.     

The regulation from guaiacyl to syringyl lignin in the differentiating xylem of Robinia pseudoacacia

13C- and deuterium (D)-labeled ferulic acid and sinapic acid ([8-(13)C, 3-OCD3]-ferulic acid and [8-(13)C, 3,5-OCD3]-sinapic acid) were administered to robinia (Robinia pseudoacacia L.) shoots. To estimate the distribution of the label from administrated ferulic or sinapic acid, continuous 50-microm-thick tangential sections cut from the cambium of robinia were subjected to lignin chemical analysis by the DFRC method. Labeled ferulic acid was incorporated into guaiacyl and syringyl lignin. The incorporation of labeled ferulic acid into syringyl units was observed only in the later stage of lignification. Labeled sinapic acid was incorporated into syringyl lignin in the early stage and the later stage of lignification. In general, syringyl lignin was deposited in the later stage of cell wall lignification. Thus, the incorporation of sinapic acid to syringyl lignin in the early stage of lignification was abnormal. Taken together, the aromatic ring-modifying reactions (the conversion from guaiacyl to syringyl moiety, including the hydroxylation and methylation) were more important for the regulation of the sinapyl alcohol biosynthesis than the reducing reactions (the reduction of acids to alcohols) in the differentiating xylem.     

Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

The influence of soil type and altered lignin biosynthesis on the growth and above and belowground biomass allocation of Populus tremuloides

Plants influence soil carbon (C) formation through the quality and quantity of C released to soil. Soil type, in turn can modify a plant’s influence on soil through effects on plant production, tissue quality and regulation of soil C decomposition and stabilization. Wild-type aspen and three transgenic aspen lines expressing reduced stem lignin concentrations and/or increased syringyl (S) to guaiacyl (G) ratio lignin were grown in greenhouse mesocosms containing a sandy loam, a silt loam, or a clay loam soil for 6 months in order to examine the effects of altered lignin biosynthesis and soil type on biomass partitioning (above vs. belowground) and soil C processes. Results indicated that soil type significantly affected plant performance. Aspen grown in soils with high sand/low clay content accumulated the most total biomass, while aspen grown in soils with high clay content accumulated the least total biomass. These reductions in growth combined with specific soil characteristics led to differences among soil types in soil C formation. Transformed aspen expressing high syringyl/guaiacyl (S/G) lignin accumulated less total plant C and subsequently accumulated less aspen derived C in soil. Reduced lignin content alone in aspen did not affect plant growth or soil C formation. There were significant soil type × genetic line interactions indicating that growth and soil C formation for transgenic and wild type aspen lines varied among the different soil types. Given these interactions, future investigation needs to include long-term field studies across a range of soil types before transgenic aspen are widely planted.     

Lignification in poplar plantlets fed with deuterium-labelled lignin precursors

Lignification was investigated in wild-type (WT) and in transgenic poplar plantlets with a reduced caffeic acid O-methyl-transferase (COMT) activity. Coniferin and syringin, deuterated at their methoxyl, were incorporated into the culture medium of microcuttings. The gas chromatography-mass spectrometry (GC-MS) analysis of the thioacidolysis guaiacyl (G) and syringyl (S) lignin-derived monomers revealed that COMT deficiency altered stem lignification. GC-MS analysis proved that the deuterated precursors were incorporated into root lignins and, to a lower extent, in stem lignins without major effect on growth and lignification. Deuterium from coniferin was recovered in G and S lignin units, whereas deuterium from syringin was only found in S units, which further establishes that the conversion of G to S lignin precursors may occur at the level of p-OH cinnamyl alcohols.     

Unpolluted fractionation of wheat straw by steam explosion and ethanol extraction

An unpolluted process of wheat straw fractionation by steam explosion coupled with ethanol extraction was studied. The wheat straw was steam exploded for 4.5 min with moisture of 34.01%, a pressure of 1.5 MPa without acid or alkali. Hemicellulose sugars were recovered by water countercurrent extraction and decolored with chelating ion exchange resin D412. The gas chromatography (GC) and high-performance liquid chromatography (HPLC) analysis results indicated that there were organic acids in the hemicellulose sugars and the ratio of monosaccharides to oligosaccharides was 1:9 and the main component, xylose, was 85.9% in content. The total recovery rate of hemicellulose was 80%. Water washed materials were subsequently extracted with ethanol. The optimum extraction conditions in this work were 40% ethanol, fiber/liquor ratio 1:50 (w/v), severity log(R)=3.657 (180 degrees C for 20 min), 0.1% NaOH. The lignin yield was 75% by acid precipitation and 85% ethanol solvent was recovered. The lignin was purified using Bj?rkman method. Infrared spectrometry (IR) results indicated that the lignin belonged to GSH (guaiacyl (G) syringyl (S) and p-hydroxyphenyl (H)) lignin and its purity rate reached 85.3%. The cellulose recovery rate was 94% and the results of electron spectroscopy for chemical analysis (ESCA) and infrared spectrometry (IR) showed that hemicellulose and lignin content decreased after steam explosion and ethanol extraction.