Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

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.     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Effects of fungal elicitor on lignin biosynthesis in cell suspension cultures of soybean

Soybean (Glycine max L.) cells cultured in B5 medium produce extremely low amounts of lignin. However, modification in the growth medium, by lowering the concentration of NO⁻₃ and PO²⁻₄, results in the lignification of these cells without affecting levels of cell wall-esterified 4-coumaric and ferulic acid. The production of an extracellular, macromolecular complex by the cultured soybean cells (Moore TS Jr 1973 Plant Physiol 51: 529-536) allows a rapid, nondestructive solubilization of the lignin which can be estimated by reaction with phloroglucinol in free solution. This system has been used to study the effects of fungal elicitor on the synthesis of lignin in soybean cells. The inclusion of very low levels of an elicitor fraction from the cell walls of Phytophthora megasperma in the medium in which lignification of the soybean cells occurs suppressed both the accumulation of extracellular lignin and phloroglucinol staining of the cell walls without affecting the levels of bound hydroxycinnamic acids. The activity profiles of phenylalanine ammonia-lyase (EC 4.3.1.5) and isoenzymes of 4-coumarate:CoA ligase (EC 6.2.1.12) were compared in lignifying and elicitor-treated cell cultures as was the activity of chalcone synthase, an enzyme of flavonoid biosynthesis. The measured activities of these enzymes in cell cultures treated with elicitor were considerably lower than in untreated cells.     

烟草4CL蛋白免疫荧光定位研究

4-香豆酸辅酶A连接酶(4CL)是维管植物木质素生物合成途径的关键酶,应用原核表达系统获得了毛白杨可溶性4CL1融合蛋白,以Ni2 -Agrose亲和柱层析纯化得到的SDS-PAGE电泳纯的毛白杨4CL1融合蛋白为抗原,免疫家兔获得毛白杨4CL1多克隆抗体,Western blotting鉴定表明兔抗毛白杨4CL1多克隆抗体具有高度特异性,免疫荧光定位发现普通烟草4CL1蛋白特异性地在木质部表达.为进一步应用木质部特异表达启动子定向调控木质素生物合成奠定了理论基础.     

Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.)

Genes encoding seven enzymes of the monolignol pathway were independently downregulated in alfalfa (Medicago sativa) using antisense and/or RNA interference. In each case, total flux into lignin was reduced, with the largest effects arising from the downregulation of earlier enzymes in the pathway. The downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase, hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate 5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional changes in lignin and wall-bound hydroxycinnamic acids consistent with the current models of the monolignol pathway. However, downregulating caffeoyl CoA 3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin composition differed in plants downregulated in either cinnamate 4-hydroxylase or phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were observed in the various transgenic lines. The current model for monolignol and ferulate biosynthesis appears to be an over-simplification, at least in alfalfa, and additional enzymes may be needed for the 3-O-methylation reactions of S lignin and ferulate biosynthesis.     

3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate: coenzyme A ligase from cultured cells of Centaurium erythraea

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. Received: 23 September 1996 / Accepted: 28 November 1996     

Hydroxycinnamates in lignification

Hydroxycinnamates incorporate into lignins by various mechanisms. The polysaccharide esters of ferulate, in particular, and the range of dehydrodiferulates and higher oligomers in grasses, participate in free-radical (cross-)coupling reactions during lignification to become integrally bound into the lignin polymer, resulting in extensive cross-linking between lignins and polysaccharides. Monolignol-hydroxycinnamate (primarily monolignol-p-coumarate) conjugates are primary building blocks for lignins, again in grasses (but analogously with monolignol acetates and p-hydroxybenzoates in other plants); radical coupling reactions of the monolignol moiety of the conjugate result in lignins with pendant p-coumarate units acylating a variety of lignin structures. Recent evidence suggests that even the hydroxycinnamic acids themselves can be monomers in lignification in wild-type and transgenic plants, undergoing radical cross-coupling reactions to incorporate into the polymer with interesting consequences. The compatibility of ferulate, in particular, with lignification suggests that plants able to utilize monolignol-ferulate conjugates in their primary monomer supply will be particularly well suited for subsequent chemical delignification, potentially improving processes for biomass conversion to biofuels, and for chemical pulping.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.     

4-Coumarate:Coenzyme A Ligase and Isoperoxidase Expression in Zinnia Mesophyll Cells Induced to Differentiate into Tracheary Elements

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.     

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees.

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.     

Induction of phenylpropanoid and tyramine metabolism in pectinase- or pronase-elicited cell suspension cultures of tobacco (Nicotiana tabacum)

The induction of the phenylpropanoid pathway and of tyramine metabolism was monitored in cell suspension cultures of Nicotiana tabacum treated with cell wall-degrading enzymes, in an attempt to correlate the synthesis of hydroxycinnamic acid amides of tyramine with the formation of wall-bound phenolic polymers. Treatment with commercial pectinase (from Penicilium occitanis ) induced a rapid rise in phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate:CoA ligase (EC 6.2.1.12), tyramine hydroxycinnamoyltransferase (EC 2.3.1.110) and peroxidase (EC 1.11.1.7) activities, and a concomitant decline in cinnamyl alcohol dehydrogenase (EC 1.1.1.195) activity. The induction of the phenylpropanoid pathway and of the synthesis of cinnamoyl-tyramines preceded the death of a large proportion of the elicited cells. When the cultures were treated with pronase (from Streptomyces griseus ), most cells remained alive and the induction of enzymes of the phenylpropanoid pathway lasted for several days, resulting in an accumulation of cinnamoyltyramines in the cells and in the culture medium. Treatment with pronase induced an increase in the activity of moderately anionic isoperoxidases which were also induced in pectinase-treated cells. Cinnamyl alcohol dehydrogenase activity remained stable in pronase-elicited cells, which rapidly accumulated thioglycolic acid-extractable phenolic polymers in their cell walls. The accumulation of these polymers coincided with the induction of 4-coumarate:CoA ligase but preceded the rise in tyramine hydroxycinnamoyltransferase and peroxidase activities.     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the beta-position, in grass cell walls.

A suspension in dichloromethane-water (18:1, v/v) of various fractions containing hydroxycinnamic acid ester-ether bridges between lignin and polysaccharides prepared from cell walls of matured oat (Avena sativa L.) intemodes, and a solution of their acetates in the same solvent, were treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). This reagent selectively cleaves benzyl ether and ester linkages of negatively charged aromatic nuclei. The sample treated with DDQ was directly hydrolysed either under mild (1 M NaOH, overnight at 37 degrees C) or severe (4 M NaOH, for 2 h at 170 degrees C) conditions. The hydroxycinnamic acids released in the hydrolysate were methylated with diazomethane and analysed quantitatively using gas chromatography. Significant portions of ether linkages between hydroxycinnamic acids and lignin were cleaved with DDQ, which suggests that most of the hydroxycinnamic acids were ether-linked at the benzyl position, and not the beta-position, of the lignin side chain as previously claimed.     

Formation of syringyl-rich lignins in maize as influenced by feruloylated xylans and <Emphasis Type="Italic">p</Emphasis>-coumaroylated monolignols

Abstract Grass cell walls are atypical because their xylans are acylated with ferulate and lignins are acylated with p-coumarate. To probe the role and interactions of these p-hydroxycinnamates during lignification, feruloylated primary cell walls isolated from maize cell suspensions were lignified with coniferyl and sinapyl alcohols and with varying levels of p-coumarate esters. Ferulate xylan esters enhanced the formation of wall-bound syringyl lignin more than methyl p-coumarate, however, maximal concentrations of syringyl lignin were only one-third that of guaiacyl lignin. Including sinapyl p-coumarate, the presumed precursor of p-coumaroylated lignins, with monolignols unexpectedly accelerated peroxidase inactivation, interfered with ferulate copolymerization into lignin, and had minimal or adverse effects on cell wall lignification. Free phenolic groups of p-coumarate esters in isolated maize lignin and pith cell walls did not undergo oxidative coupling with each other or with added monolignols. Thus, the extensive formation of syringyl-rich lignins and the functional role of extensive lignin acylation by p-coumarate in grasses remains a mystery.     

Monoclonal antibodies to p-coumarate

p-Coumaric acid is one of the predominant phenolic acids acylating the cell walls of grasses; p-coumarates are mainly esterified by lignins and arabinoxylans. Here we describe the production and characterisation of two monoclonal antibodies against p-coumarates.The 5-O-pCou-Ara(1 → 4)Xyl was chemically synthesized and conjugated to a carrier protein. Two interesting antibodies were obtained, hereinafter named INRA-COU1 and INRA-COU2. The specificity of these monoclonal antibodies has been evaluated using competitive-inhibition assays with different oligosaccharides and phenolic compounds. INRA-COU1, recognized free p-coumaric acid or p-coumarate esters. INRA-COU1 did not react with any of the other hydroxycinnamic acids and related compounds found in plants. INRA-COU2, only recognizes esterified p-coumarate. These antibodies were used to study the localization of p-coumarates in the cell walls of grasses. Immunocytochemical analyses indicated noticeable amounts of p-coumarate in the cell walls of the aleurone layer of wheat grain, in the epiderm of cereal straw, and in the exoderm of wheat root.The use of these antibodies will contribute to a better understanding of the organisation and developmental dynamics of cell walls in Graminaceae.