John H. Grabber

Lignin-ferulate cross-links in grasses: active incorporation of ferulate polysaccharide esters into ryegrass lignins

Abstract Active incorporation of ferulate polysaccharide esters into ryegrass lignins has been demonstrated by NMR spectroscopy of uniformly 13 C-labeled ryegrass. Observation, in the HMBC spectrum, of products of ferulate at its 8-position coupling with hydroxycinnamyl alcohols at the β-position (producing 8-β′-linked structures) is proof that ferulate-lignin radical cross-coupling reactions occur in vivo. Correlations of H-α′ (hydroxycinnamyl alcohol moiety) with guaiacyl and syringyl 1-, 2-, and 6-aromatic carbons in 8-β′ structures indicates that ferulates couple with both coniferyl and sinapyl alcohol monomers. As notable as the presence of this and other ferulate products is the absence of coupling of ferulate at its 8-position with the 5- and O -4-positions of lignin units. Such structures were significant when ferulate was biomimetically incorporated into a synthetic lignin. Since hydroxycinnamyl alcohols couple almost exclusively at their β-position in cross-coupling reactions, the 8-5′ and 8- O -4′ structures would only be formed by coupling with higher lignin oligomers (with no side-chain conjugation). Exclusive reaction of ferulates with lignin monomers is the first real evidence that ferulate polysaccharide esters in grasses are acting as initiation or nucleation sites for lignification and are critical entities in directing cell-wall cross-linking during plant growth and development.

Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls

Seven isomeric dehydrodimers of ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been synthesized and identified in extracts of saponified cell walls of cocksfoot, switchgrass, and suspension-cultured corn. Dehydrodimers (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid, (Z)-β-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxycinnamic acid, (E)-3-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-5-methoxycinnamic acid, (E,E)-4,4′-dihydroxy-3,5′-dimethoxy-β,3′-bicinnamic acid, 4,4′-dihydroxy-3,3′-dimethoxy-β,β′-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1,2-dihydronaphthalene-2,3-dicarboxylic acid, all arise from oxidative coupling of ferulate esters in cell walls and represent products of 8–5, 8–8, 8–O–4, 4–O–5, and 5–5 radical coupling. Prior literature has acknowledged only the presence of the 5–5-coupled dehydrodimer (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid. Consequently, by measuring only a single dehydrodimer and assuming inappropriate response factors, ferulate dehydrodimers have been underestimated by factors of up to 20. Synthetic routes to all seven isomers have been developed to provide structural authentication and determination of GC response factors.

Ferulate cross-linking in cell walls isolated from maize cell suspensions

Abstract Cross-linking of arabinoxylans by ferulate dehydrodimers and incorporation of feruloylated arabinoxylans into lignin were modelled with maize walls (Zea mays cv Black Mexican) containing 5.3–18.0 mg g−1 of total ferulates. The proportion of dehydrodimers to total ferulates increased from ca 20 to 45% when dilute hydrogen peroxide was added to walls containing bound peroxidase. About 45% of the dehydrodimers were coupled by 8-5 linkages, with 8-8, 8-O-4 and 5-5 coupled dehydrodimers each comprising 10–25% of the total. The quantity of ferulates released by saponification were reduced by 83–95% when exogenously supplied hydroxycinnamyl alcohols were polymerized into walls by wall-bound peroxidase and in situ generated hydrogen peroxide. Only 40% of the ferulates incorporated into lignin were recovered following hydrolysis of ether linkages. These results indicate that primary walls in grasses become extensively cross-linked by ferulic and dehydrodiferulic acids during lignification, and that only a portion of ferulates in lignified tissues are measurable by current solvolytic methods.

Metabolic engineering of novel lignin in biomass crops.

Lignin, a phenolic polymer in the secondary wall, is the major cause of lignocellulosic biomass recalcitrance to efficient industrial processing. From an applications perspective, it is desirable that second-generation bioenergy crops have lignin that is readily degraded by chemical pretreatments but still fulfill its biological role in plants. Because plants can tolerate large variations in lignin composition, often without apparent adverse effects, substitution of some fraction of the traditional monolignols by alternative monomers through genetic engineering is a promising strategy to tailor lignin in bioenergy crops. However, successful engineering of lignin incorporating alternative monomers requires knowledge about phenolic metabolism in plants and about the coupling properties of these alternative monomers. Here, we review the current knowledge about lignin biosynthesis and the pathways towards the main phenolic classes. In addition, the minimal requirements are defined for molecules that, upon incorporation into the lignin polymer, make the latter more susceptible to biomass pretreatment. Numerous metabolites made by plants meet these requirements, and several have already been tested as monolignol substitutes in biomimetic systems. Finally, the status of detection and identification of compounds by phenolic profiling is discussed, as phenolic profiling serves in pathway elucidation and for the detection of incorporation of alternative lignin monomers.

Diferulate cross-links impede the enzymatic degradation of non-lignified maize walls

We assessed the e†ect of ferulate substitution and diferulate cross- linking of xylans on the degradation of cell walls by two fungal enzyme mixtures, one of which contained feruloyl esterase and high xylanase activities. Non- ligni-ed cell suspensions of maize (Zea mays) were grown with 0 or 40 lM 2- aminoindan-2-phosphonic acid to produce walls with normal (17E 2m g g~1 )o r reduced (5E 1m g g~1) ferulate concentrations. Walls were incubated with mercap- toethanol to inhibit diferulate formation or with hydrogen peroxide to stimulate diferulate formation by wall bound peroxidases. Varying the ferulate substitution of xylans did not a†ect cell wall hydrolysis. In contrast, increasing ferulate dimer- isation from 18 to 40% reduced carbohydrate release by 94E122 mg g~1 after 3 h and by 0E48 mg g~1 after 54 h of enzymatic hydrolysis. Diferulate cross- links impeded the release of xylans, cellulose and pectins from walls. These results provide compelling evidence that diferulate cross-links reduce the rate and, to a lesser degree, the extent of wall hydrolysis by fungal enzymes. Our results also suggest that enzyme mixtures containing high xylanase activity but not feruloyl esterase activity can partially overcome the inhibitory e†ects of diferulate cross-linking on wall hydrolysis. 1998 SCI. (

Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls

AbstractPeroxidases are heavily implicated in plant cell wall cross-linking reactions, altering the properties of the wall and impacting its utilization. Polysaccharide-polysaccharide cross-linking in grasses is achieved by dehydrodimerization of hydroxycinnamate-polysaccharide esters; a complex array of hydroxycinnamic acid dehydrodimers are released by saponification. Ferulates are the major cross-linking agents, but sinapate-ferulate cross-products have been discovered implicating sinapates in a similar role. New dehydrodimers have been authenticated, expanding our knowledge of the chemistry, role, and extent of cross-linking reactions. Ferulate dehydrotrimers have been discovered; whether these trimers truly cross-link three independent polysaccharide chains or only two remains to be determined. Hydroxycinnamates and their dehydrodimers also undergo radical coupling reactions with lignin monomers and possibly oligomers, resulting in lignin-polysaccharide cross-linking in the wall. Both polysaccharide-polysaccharide and lignin-polysaccharide cross-links inhibit the enzymatic hydrolysis of cell walls. The cross-linking process has particular relevance to plant physiology, human and animal nutrition and health, and food technology. Abbreviations: CW – cell wall; DFA – dehydrodiferulic acid (or dehydrodiferulate in context); DSA – dehydrodisinapic acid; TFA – dehydrotriferulic acid; SA – sinapic acid (1S); TA – thomasidioic acid (5C3SS); IDF – insoluble dietary fiber; SDF – soluble dietary fiber; GC-MS – gas chromatography-mass spectrometry; NMR – nuclear magnetic resonance (spectroscopy).

p-Coumaroylated syringyl units in maize lignin : implications for β-ether cleavage by thioacidolysis

Abstract Recent NMR studies of lignin isolated from maize demonstrated that p -coumarate esters are attached exclusively to the γ-position of phenylpropane side chains. Thioacidolysis/desulphuration experiments have revealed that p -coumarate units are attached primarily ( ca 90%) to syringly moieties in maize lignin. In model studies with guaiacylglycerol and syringylglycerol-β-guaiacyl ethers, cleavage of β-ether linkages by thioacidolysis was reduced 40% by γ-acylation of phenylpropane side chains with p -coumarate. Our results indicate that γ - p -coumarate esters significantly reduce the yields of syringyl products recovered after thioacidolysis of grass lignins.

Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency)

A molecular marker compound, derived from lignin by the thioacidolysis degradative method, for structures produced when ferulic acid is incorporated into lignin in angiosperms (poplar, Arabidopsis, tobacco), has been structurally identified as 1,2,2-trithioethyl ethylguaiacol [1-(4-hydroxy-3-methoxyphenyl)-1,2,2-tris(ethylthio)ethane]. Its truncated side chain and distinctive oxidation state suggest that it derives from ferulic acid that has undergone bis-8-O-4 (cross) coupling during lignification, as validated by model studies. A diagnostic contour for such structures is found in two-dimensional (13)C-(1)H correlated (HSQC) NMR spectra of lignins isolated from cinnamoyl CoA reductase (CCR)-deficient poplar. As low levels of the marker are also released from normal (i.e. non-transgenic) plants in which ferulic acid may be present during lignification, notably in grasses, the marker is only an indicator for CCR deficiency in general, but is a reliable marker in woody angiosperms such as poplar. Its derivation, together with evidence for 4-O-etherified ferulic acid, strongly implies that ferulic acid is incorporated into angiosperm lignins. Its endwise radical coupling reactions suggest that ferulic acid should be considered an authentic lignin precursor. Moreover, ferulic acid incorporation provides a new mechanism for producing branch points in the polymer. The findings sharply contradict those reported in a recent study on CCR-deficient Arabidopsis.

Relationship of growth cessation with the formation of diferulate cross-links and p-coumaroylated lignins in tall fescue leaf blades

Abstract. We examined relationships among cell wall feruloylation, diferulate cross-linking, p-coumarate deposition, and apoplastic peroxidase (EC 1.11.1.7) activity with changes in the elongation rate of leaf blades of slow and rapid elongating genotypes of tall fescue (Festuca arundinacea Schreb.). Growth was not directly influenced by ferulic acid deposition but leaf elongation decelerated as 8–5-, 8–O–4-, 8–8-, and 5–5-coupled diferulic acids accumulated in cell walls. Growth rapidly slowed and stopped with the deposition of p-coumarate, which is primarily associated with lignification in grass cell walls. Accretion of ferulate, diferulates and p-coumarate continued after growth ended, into the later stages of secondary wall formation. The concentration of 8-coupled diferulates dwarfed that of the more commonly measured 5–5-coupled isomer, suggesting that the latter dimer is a poor indicator of diferulate cross-linking in cell walls. Further work is required to clearly demonstrate the role of diferulate cross-linking and p-coumaroylated lignins in the cessation of leaf growth in grasses.

Coniferyl Ferulate Incorporation into Lignin Enhances the Alkaline Delignification and Enzymatic Degradation of Cell Walls

Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production.