A Possible Mechanism for the Oxidation of Sinapyl Alcohol by Peroxidase-Dependent Reactions in the Apoplast: Enhancement of the Oxidation by Hydroxycinnamic Acids and Components of the Apoplast

Apoplastic peroxidase isoenzymes from stems of Nicotiana tabacumrapidly oxidized sinapic acid and sinapyl alcohol, in additionto 4-coumaric acid, ferulic acid and coniferyl alcohol. By contrast,the peroxidase isoenzymes from stems of Vigna angularis oxidizedsinapic acid and sinapyl alcohol quite slowly but rapidly oxidizedcompounds with a 4-hydroxyphenyl or a guaiacyl group. However,the oxidation of sinapyl alcohol was greatly enhanced by 4-coumaricacid, ferulic acid and an ester of ferulic acid. Intercellularwashing fluid of V. angularis, which contained apoplastic components,also enhanced the oxidation of sinapyl alcohol. Based on theseresults, a possible mechanism for the oxidation of sinapyl alcoholis discussed on the assumption that the biosynthesis of ligninproceeds mainly via peroxidases which cannot oxidize sinapylalcohol in V. angularis. (Received October 23, 1995; Accepted April 3, 1996)     

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.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

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.     

Catalytic Profile of Arabidopsis Peroxidases,AtPrx-2, 25 and 71, Contributing to Stem Lignification

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.     

Lipid Peroxidation Induced by Phenolics in Conjunction with Aluminum Ions

Using the whole plant and model systems, we demonstrate that the aluminum ions (Al³⁺) stimulate phenolic-dependent lipid peroxidation. Lipid peroxidation in barley (Hordeum vulgare L. cv. Donor) roots was 30 % higher under AlCl₃ treatment than without Al. Major decomposition product of lipid peroxidation was 4-hydroxynonenal (4-HNE) but not thiobarbituric acid reactive substances (TBARS), a widely used markers for lipid peroxidation. Similarly, AlCl₃ stimulated lipid peroxidation of soybean liposomes in the presence of chlorogenic acid (CGA) and H₂O₂/horseradish peroxidase system which can oxidize phenolics. Al³⁺ was found to enhance lipid peroxidation induced by oxidized CGA. Intermediates of lignin biosynthesis in plants, including p-coumaric acid, ferulic acid, sinapic acid and coniferyl alcohol, also showed similar effects. These results suggest that Al³⁺ has a potential to induce oxidative stress in plants by stimulating the prooxidant nature of endogenous phenolic compounds.     

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.     

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.     

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.     

Formation of coniferyl alcohol from ferulic acid by the white rot fungus Trametes

The white rot fungus, Trametes sp., was cultivated in a medium containing ferulic acid, glucose and ethanol under aerobic conditions in submerged culture. The ferulic acid was transformed into coniferyl alcohol, coniferylaldehyde, dihydroconiferyl alcohol, vanillic acid, vanillyl alcohol, 2-methoxyhydroquinone and 2-methoxyquinone during 48–120 hr of cultivation. The amount of coniferyl alcohol in the culture reached a maximum after 90 hr with ca 40% of the initial amount of ferulic acid. Cinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, p -coumaric acid and sinapic acid were also transformed into the corresponding alcohols, benzoic acids and benzyl alcohols in the fungus culture.     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

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.     

Hydroxycinnamates and their in vitro and in vivo antioxidant activities

Hydroxycinnamates are among the most widely distributed plant phenylpropanoids present in the free, conjugated-soluble and insoluble-bound forms. This review will focus on the occurrence, in vitro and in vivo antioxidant activities of ferulic, coumaric, caffeic and sinapic acids and their derivatives. Hydroxycinnamates are found in almost all food groups though they are abundant in cereals, legumes, oilseeds, fruits, vegetables and beverages and render antioxidant activity by scavenging hydroxyl radical, superoxide radical anion, several organic radicals, peroxyl radical, peroxinitrite and singlet oxygen, among others. Further, their antioxidant activity as chain breaking antioxidants and reducing agents is also notable. Ferulic acid and its derivatives such as ferulic acid ethyl ester, ferulic acid dehydrodimers, feruloyl glycosides and curcumin have demonstrated potent antioxidant activity in both in vitro and in vivo systems. Similarly, caffeic acid and some of its derivatives such as caffeic acid phenethyl ester, rosmarinic acid, and chlorogenic acid exhibit antioxidant activity. The highest antioxidant activity was observed for caffeic acid whereas p-coumaric acid had the least effect among major hydroxycinnamic acids. The importance of structural effects on the potency of antioxidant activity of hydroxycinnamates is discussed. While this review also shows the existence of substantial body of evidences for in vitro antioxidant activity of hydroxycinnamates, there is a clear gap for in vivo information, particularly for sinapic and p-coumaric acids and their derivatives. The role of grains, fruits, vegetables and red wine in disease risk reduction and health promotion could partly be attributed to their constituent hydroxycinnamates.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87?nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions

An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively.     

Multiform biosynthetic pathway of syringyl lignin in angiosperms

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.     

Relationship between hydroxycinnamic acid content,lignin composition and digestibility of maize silages in sheep

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R ² value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R ² value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.     

Anomalous substrate specificities among the algal peroxidases

Semipurified tissue preparations from 13 red and brown algae oxidized pyrogallol and p-coumaric acid but could not oxidize guaiacol and other ortho (methoxy)-substituted phenols, including common lignin precursors such as coniferaldehyde. They also failed to oxidize the aromatic amine, benzidine. In contrast, preparations from green algae were like horseradish peroxidase and vascular plant preparations in their ability to oxidize unsubstituted phenols, those substituted at one or both ortho-positions, and benzidine. One brown alga, Postelsia, was also unable to oxidize the commonplace peroxidase substrates, iodide and eugenol. These results suggest a phylogenetic limitation on the potential for lignification based upon enzyme stereospecificity.     

3, 4-Dihydroxyphenylalanine is oxidized by phenoxyl radicals of hydroxycinnamic acid esters in leaves ofVicia faba L

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.     

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.