O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction

The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. demonstrated the presence of 4- O-linked hydroxycinnamyl aldehyde end groups and 4- O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.     

Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls.

The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-beta-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by beta-0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with kappa(M) values in the mu(M) range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic "potholes" in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity.     

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD⁺-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (ΩKm) or gentamycin (ΩGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3′-O-phosphotransferase gene and the gentamycin- 3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by ΩKm or ΩGm, and the functional vdh gene was replaced by vdhΩKm or vdhΩGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhΩKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase. Received: 19 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Metabolic differences between gymnosperms and angiosperms in the formation of syringyl lignin

Sliced xylem tissue from shoots of both poplar and cherry reduces ferulic and sinapic acids to the corresponding aldehydes and alcohols, while tissue from gymnosperms such as Japanese red pine and ginkgo can reduce only ferulic acid. In young, less differentiated, xylem tissue and callus tissue of angiosperms the ability to reduce sinapic acid is markedly lower than that of the fully differentiated xylem.Both gymnosperm and angiosperm tissues reduced coniferyl and sinapyl aldehydes to the corresponding alcohols and, further, the peroxidases from both classes gave similar dehydrogenation polymers from a mixture of coniferyl and sinapyl alcohols. In agreement with these findings, sinapyl aldehyde and sinapyl alcohol, when fed to living plants and tissue cultures of gymnosperms, were shown to be readily converted to syringyl lignin which was not originally present.     

Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure of ATP A2 was solved to 1.45 Å resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.     

The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p‐coumaryl, coniferyl and sinapyl) alcohols in a reaction mediated by peroxidases (EC 1.11.1.7) and laccases (EC 1.10.3.2), yielding H, G and S units, respectively. Although both acidic and basic peroxidases can oxidize p‐coumaryl and coniferyl alcohol, only basic peroxidases are able to oxidize sinapyl alcohol. The AtPrx52 from Arabidopsis is a basic peroxidase that has been reported to be highly homologous to the basic peroxidase of Zinnia elegans, the only peroxidase which has been unequivocally linked to lignin formation. Here, we show how the suppression of AtPrx52 causes a change in lignin composition, mainly at the level of stem interfascicular fibers. Quantification of lignins in two different atprx52 knock‐out mutants revealed a decrease of lignin amount compared with wild type. The S/G ratio, obtained by both nitrobenzene oxidation and thioacidolysis, indicated a decrease in S units in the atprx52 mutants. As deduced from Wiesner and mainly Mäule staining, this reduction in S unit content appears to be restricted to the interfascicular fibers. Moreover, quantitative polymerase chain reaction analysis in atprx52 plants showed a general downregulation of genes involved in lignin biosynthetic pathway, as well as genes related to secondary cell wall. On the other hand, other routes from phenylpropanoid metabolism were induced. Taken together, our results indicate that AtPrx52 is involved in the synthesis of S units in interfascicular fibers at late stages of the lignification process.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.     

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

Basic peroxidases: The gateway for lignin evolution?

Lignins are cell wall phenolic heteropolymers which result from the oxidative coupling of three monolignols, p-coumaryl, coniferyl and sinapyl alcohol, in a reaction mediated by peroxidases. The most distinctive variation in the monomer composition of lignins in vascular plants is that found between the two main groups of seed plants. Thus, while gymnosperms lignins are typically composed of G units, with a minor proportion of H units, angiosperms lignins are largely composed of similar levels of G and S units. The presence of S units in angiosperm lignins raises certain concerns in relation with the step of lignin assembly due to the inability of most peroxidases to oxidize syringyl moieties. Zinnia elegans is currently used as a model for lignification studies: – first because of the simplicity and duality of the lignification pattern shown by hypocotyls and stems, in which hypocotyl lignins are typical of angiosperms, while young stem lignins partially resemble those occurring in gymnosperms. Secondly, because of the nature of the peroxidase isoenzyme complement, which is almost completely restricted to the presence of a basic peroxidase isoenzyme, which is capable of oxidizing both coniferyl and sinapyl alcohol, as well as both coniferyl and sinapyl aldehyde. In fact, the versatility of this enzyme is such that the substrate preference covers the three p-hydroxybenzaldehydes and the three p-hydroxycinnamic acids. The basic pI nature of this peroxidase is not an exceptional frame point in this system since basic peroxidases are differentially expressed during lignification in other model systems, show unusual and unique biochemical properties as regards the oxidation of syringyl moieties, and their down-regulation in transgenic plants leads to a reduction in lignin (G+S) levels. Basic peroxidase isoenzymes capable of oxidizing syringyl moieties are already present in basal gymnosperms, an observation that supports the idea that these enzymes were probably present in an ancestral plant species, pre-dating the early radiation of seed plants. It also suggests that the evolutionary gain of the monolignol branch which leads to the biosynthesis of sinapyl alcohol, and of course to syringyl lignins, was not only possible but also favored because the enzymes responsible for its polymerization had evolved previously. In this scenario, it is not surprising that these enzymes responsible for lignin construction appeared early in the evolution of land plants, and have been largely conserved during plant evolution. Abreviations: 4CL –p-hydroxycinnamate CoA ligase; C3H –p-coumarate-3-hydroxylase; C4H – cinnamate-4-hydroxylase; p-CA –p-coumaric acid; CAD – coniferyl alcohol dehydrogenase; CAld5H – coniferylaldehyde-5-hydroxylase; CCR –p-hydroxycinnamoyl-CoA reductase; CoI – compound I; CoII – compound II; G – guaiacyl unit; H –p-hydroxyphenyl unit; PAL – phenylalanine ammonia-lyase; S – syringyl unit.     

Potential of Rhodococcus strains for biotechnological vanillin production from ferulic acid and eugenol

The potential of two Rhodococcus strains for biotechnological vanillin production from ferulic acid and eugenol was investigated. Genome sequence data of Rhodococcus sp. I24 suggested a coenzyme A-dependent, non-β-oxidative pathway for ferulic acid bioconversion, which involves feruloyl–CoA synthetase (Fcs), enoyl–CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh). This pathway was proven for Rhodococcus opacus PD630 by physiological characterization of knockout mutants. However, expression and functional characterization of corresponding structural genes from I24 suggested that degradation of ferulic acid in this strain proceeds via a β-oxidative pathway. The vanillin precursor eugenol facilitated growth of I24 but not of PD630. Coniferyl aldehyde was an intermediate of eugenol degradation by I24. Since the genome sequence of I24 is devoid of eugenol hydroxylase homologous genes (ehyAB), eugenol bioconversion is most probably initiated by a new step in this bacterium. To establish eugenol bioconversion in PD630, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was expressed in PD630 together with coniferyl alcohol dehydrogenase (calA) and coniferyl aldehyde dehydrogenase (calB) genes from Pseudomonas sp. HR199. The recombinant strain converted eugenol to ferulic acid. The obtained data suggest that genetically engineered strains of I24 and PD630 are suitable candidates for vanillin production from eugenol.     

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

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

Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C₆-C₁ aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants. Received: 24 January 1997 / Accepted: 14 May 1997     

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.     

Involvement of malate,monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Peroxidase associated with isolated horseradish cell walls catalyzes the formation of H₂O₂ in the presence of NADH. The reaction is stimulated by various monophenols, especially of coniferyl alcohol. NADH can be provided by a bound malate dehydrogenase. This system is capable of polymerizing coniferyl alcohol yielding an insoluble dehydrogenation polymer. NADH was found to be oxidized by two different mechanisms, one involving Mn²⁺, monophenol, and the superoxide radical O₂ ^·- in a reaction that is not affected by superoxide dismutase, and another one depending on the presence of free O₂ ^·- and probably of an enzyme-NADH complex. A scheme of these reaction chains, which are thought to be involved in the lignification process, is presented.Abbreviations DHP dehydrogenation polymer - GOT glutamate oxaloacetate transaminase (EC 2.6.1.1) - LDH lactate dehydrogenase (pig heart, EC 1.1.1.27) - MDH malate dehydrogenase (EC 1.1.1.37) - pCA p-coumaric acid - SOD superoxide dismutase (EC 1.15.1.1) - TLC thin-layer chromatography - XOD xanthine oxidase (EC 1.2.3.2)     

Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

A potential role for sinapyl p-coumarate as a radical transfer mechanism in grass lignin formation

Grass lignins are differentiated from other lignin types by containing relatively large amounts of p-coumaric acid (pCA) acylating the C-9 position of lignin subunits. In the case of a mature corn (Zea mays L.) stems, pCA constitutes 15–18% of a dioxane soluble enzyme lignin. The major portion of the pCA is specifically attached to syringyl residues. Studies with isolated corn wall peroxidases show that pCA readily undergoes radical coupling in the presence of hydrogen peroxide, whereas sinapyl alcohol radical coupling proceeds more slowly. Analysis of corn wall peroxidases did not reveal specific enzymes that would lead to the preferred incorporation of sinapyl alcohol as seen in other plants. The addition of ethyl ferulate, methyl p-coumarate, or sinapyl p-coumarate conjugates to a reaction mixture containing peroxidase, sinapyl alcohol, and hydrogen peroxide stimulated the rate of sinapyl alcohol radical coupling by 10–20-fold. Based on spectral analysis it appears that pCA and ferulate radicals form rapidly, but the radical is readily transferred to sinapyl alcohol. The newly formed sinapyl alcohol radicals undergo coupling and cross-coupling reactions. However, sinapyl alcohol radicals do not cross-couple with pCA radicals. As long as hydrogen peroxide is limiting pCA remains uncoupled. Ferulates have similar reaction patterns in terms of radical transfer though they appear to cross-couple in the reaction mixture more readily then pCA. The role of pCA may be to internally provide a radical transfer mechanism for optimizing radical coupling of sinapyl alcohol into the growing lignin polymer. Attachment of some pCA to sinapyl alcohol ensures localization of the radical transfer mechanism in areas where sinapyl alcohol is being incorporated into lignin.