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     

The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems

The hypothesis that auxin (IAA) and gibberellic acid (GA₃) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA₃ (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA₃/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA₃ induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA₃/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.     

13C NMR cross polarization and magic angle spinning (CPMAS) and gas chromatography/mass spectrometry analysis of the products from a soda pulp mill effluent decolourised by two Streptomyces strains

Two Streptomyces strains, UAH 30 and UAH 51, have been shown to decolourise a paper-mill effluent obtained after semichemical alkaline pulping of wheat straw. Fractionation of the effluent decolourised by strains UAH 30 and UAH 51 showed that 60% and 80% respectively of the alkali-lignin fraction have been removed from the effluent after 7 days of growth. ¹³C NMR cross polarization and magic angle spinning (CPMAS) spectra of the alkali-lignin remaining in the effluent after decolourisation revealed a decrease in the relative amount of aromatic lignin units compared to that obtained from the untreated effluent along with a reduction in the ratio of syringyl:guaiacyl units. Gas chromatography/mass spectrometry analysis of the low-molecular-mass compounds extracted from the decolourised effluent revealed the presence of new aromatic lignin-related compounds that were not present in the untreated control effluent. This was linked to a general depolymerization of larger lignin molecules occurring during decolourisation by the two Streptomyces strains. Identification of low-molecular-mass aromatic compounds extracted from the decolourised effluent revealed only the presence of p-hydroxyphenyl units in effluents decolourised by the strain UAH 30 while p-hydroxyphenyl, guaiacyl and syringyl units were detected in effluents decolourised by Streptomyces strain UAH 51. The study indicates that, while decolourisation is a common feature of the two Streptomyces strains, the mechanisms involved in the degradation of the lignin fractions may be different and strain-specific. Received: 8 July 1996 / Received revision: 9 October 1996 / Accepted: 14 October 1996     

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.     

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.     

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.     

Lignin content versus syringyl to guaiacyl ratio amongst poplars

Two oxidation techniques that afford high yields of monomers and dimers were used to more accurately estimate the syringyl to guaiacyl (S:G) ratio of hardwood lignins. Permanganate oxidation of the woodmeal after a CuO pre-hydrolysis step gave poor results and this was attributed to preferential oxidation and degradation of syringyl nuclei by CuO. However, this procedure did provide a good estimate of the percentages of both S and G phenylpropane (C(9)) units that were uncondensed. When the total S and G products from nitrobenzene oxidation (NBO) of the uncondensed fractions were corrected, credible S:G ratios were obtained. These ratios were in good agreement with results from KMnO4 oxidation of dissolved kraft lignin without CuO pre-hydrolysis. The corrected NBO method was used to determine the S:G ratio of 13 poplars, and the values ranged from 1.01 to 1.68. Unlike results from other investigations, an excellent linear correlation (R(2) =0.846) was obtained for a decreasing lignin content (28% to 16.5%) with an increase in the S:G ratio.     

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice.     

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     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.     

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.     

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.     

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.     

Genotype- and age-dependent patterns of lignin and cellulose in regenerants derived from 80-year-old trees of black mulberry (<Emphasis Type="Italic">Morus nigra</Emphasis> L.)

Proliferating shoot cultures of black mulberry (Morus nigra), derived from axillary buds of two donor trees designated as Mn1 and Mn2, more than 80 years of age, were established in vitro. Subsequently, shoot-tips were used to induce both axillary and adventitious shoot regeneration following incubation on Murashige and Skoog medium containing 14 different treatments of various concentrations of plant growth regulators, including 6-benzyladenine (BA), thidiazuron (TDZ), and combinations of BA with indole-3-butyric acid (IBA) and TDZ with BA. The highest shoot proliferation of 5.3 shoots per explant for the Mn1 tree and 6.9 shoots per explant for the Mn2 tree were obtained when explants were incubated on a medium containing 0.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IBA. The maximum frequency of adventitious rooting was similar for both genotypes. Changes in lignin and cellulose content, macromolecular properties of dioxane and Klason lignins, lignin monomer composition, and macromolecular properties of cellulose were determined in 1-year-old and 3 year-old micropropagated plants, as well as in the donor trees. Lignin and cellulose properties were significantly dependent on the genotype, the age and the mutual interaction of both these factors. The syringyl to guaiacyl weight ratio in lignin rose with the age of the micropropagated plants. Moreover, the tensile strength of wood in 1-year-old plants was supported by a high cellulose degree of polymerization. The highest polydispersity index of cellulose was detected in 3-year-old plants.     

Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus DC

The biosynthetic pathways to monolignols in Magnolia kobus were investigated by feeding stems with a deuterium-labeled precursor. Pentadeutero [γ,γ-²H₂, OC²H₃] coniferyl alcohol was supplied to shoots of Magnolia kobus and the incorporation of the labeled precursor into lignin was traced by gas chromatography-mass spectrometry. In addition to the direct incorporation of the labeled precursor into guaiacyl units, we detected a significant amount of pentadeuterium-labeled syringyl units with two γ-deuterium atoms. The relative level of trideuterium-labeled syringyl monomers (the result of conversion via the cinnamic acid pathway, in which two γ-deuterium atoms are removed during enzymatic re-oxidation) was negligible. Our results provide conclusive evidence for a novel alternative pathway for generation of lignin subunits at the monolignol stage and they suggest that this new pathway might be important for regulation of the composition of lignin. Received: 21 August 1998 / Accepted: 30 September 1998     

A comparative study of the biomass properties of Erianthus and sugarcane: lignocellulose structure,alkaline delignification rate,and enzymatic saccharification efficiency

A comprehensive understanding of the structure and properties of gramineous lignocelluloses is needed to facilitate their uses in biorefinery. In this study, lignocelluloses from fractionated internode tissues of two taxonomically close species, Erianthus arundinaceus and sugarcane (Saccharum spp.), were characterized. Our analyses determined that syringyl (S) lignins were predominant over guaiacyl (G) or p-hydroxyphenyl (H) lignins in sugarcane tissues; on the other hand, S lignin levels were similar to those of G lignin in Erianthus tissues. In addition, tricin units were detected in sugarcane tissues, but not in Erianthus tissues. Distributions of lignin inter-monomeric linkage types were also different in Erianthus and sugarcane tissues. Alkaline treatment removed lignins from sugarcane tissues more efficiently than Erianthus tissues, resulting in a higher enzymatic digestibility of sugarcane tissues compared with Erianthus tissues. Our data indicate that Erianthus biomass displayed resistance to alkaline delignification and enzymatic digestion.     

Guaiacyl Lignin Associated with Vessels in Aspen Callus Cultures

Lignin from aspen (Populus tremuloides Michx.) tissue cultures containing only mature vessels and undifferentiated parenchymatous cells is exclusively of the guaiacyl type normally associated with gymnosperms. This supports the theory that the guaiacyl and syringyl lignin in angiosperm wood is compartmentalized, with guaiacyl lignin in vessels and syringyl lignin in fibers and ray cells.     

Photoregulation of the incorporation of guaiacyl units into lignins

When fed with [¹⁴C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled.When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaCl₂) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.Abbreviations S syringyl residue - G guaiacyl residue This is paper No. 4 in a series. For paper No. 3 see Grand and Ranjeva, in press