Paul F. Schatz

Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem levels of up to ∼65% P (from wild-type levels of ∼1%) resulting from down-regulation of C3H were measured by traditional degradative analyses as well as two-dimensional13C-1H correlative NMR methods. Such levels put these transgenics well beyond the P:G:S compositional bounds of normal plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in gymnosperm severe compression wood zones but are limited to a few percent in dicots. NMR also revealed structural differences in the interunit linkage distribution that characterizes a lignin polymer. Lower levels of key β-aryl ether units were relatively augmented by higher levels of phenylcoumarans and resinols. The C3H-deficient alfalfa lignins were devoid of β-1 coupling products, highlighting the significant differences in the reaction course for p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in conjunction with an approximate doubling of their proportion. The nature of each of the structural units was revealed by long range13C-1H correlation experiments. For example, although β-ethers resulted from the coupling of all three monolignols with the growing polymer, phenylcoumarans were formed almost solely from coupling reactions involving p-coumaryl alcohol; they resulted from both coniferyl and sinapyl alcohol in the wild-type plants. Such structural differences form a basis for explaining differences in digestibility and pulping performance of C3H-deficient plants.

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).

Identification of Lignin and Polysaccharide Modifications in Populus Wood by Chemometric Analysis of 2D NMR Spectra from Dissolved Cell Walls

2D 13C-(1)H HSQC NMR spectroscopy of acetylated cell walls in solution gives a detailed fingerprint that can be used to assess the chemical composition of the complete wall without extensive degradation. We demonstrate how multivariate analysis of such spectra can be used to visualize cell wall changes between sample types as high-resolution 2D NMR loading spectra. Changes in composition and structure for both lignin and polysaccharides can subsequently be interpreted on a molecular level. The multivariate approach alleviates problems associated with peak picking of overlapping peaks, and it allows the deduction of the relative importance of each peak for sample discrimination. As a first proof of concept, we compare Populus tension wood to normal wood. All well established differences in cellulose, hemicellulose, and lignin compositions between these wood types were readily detected, confirming the reliability of the multivariate approach. In a second example, wood from transgenic Populus modified in their degree of pectin methylesterification was compared to that of wild-type trees. We show that differences in both lignin and polysaccharide composition that are difficult to detect with traditional spectral analysis and that could not be a priori predicted were revealed by the multivariate approach. 2D NMR of dissolved cell wall samples combined with multivariate analysis constitutes a novel approach in cell wall analysis and provides a new tool that will benefit cell wall research.

Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

BackgroundRecent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production.ResultsIn the absence of anatomical constraints to digestion, lignification with normal monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation.ConclusionOverall, monolignol substitutes improved the inherent degradability of non-pretreated cell walls by restricting lignification or possibly by reducing lignin hydrophobicity or cross-linking to structural polysaccharides. Furthermore some monolignol substitutes, chiefly readily cleaved bi-phenolic conjugates like epigallocatechin gallate or diferuloyl polyol esters, are expected to greatly boost the enzymatic degradability of cell walls following chemical pretreatment. In ongoing work, we are characterizing the enzymatic saccharification of intact and chemically pretreated cell walls lignified by these and other monolignol substitutes to identify promising genetic engineering targets for improving plant fiber utilization.

Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8–8-coupling product acylating cereal plant cell walls

A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically derives from 8-8-coupling of ferulate followed by water addition to one of the incipient quinone methide moieties and internal trapping of the other. The finding adds complexity to the analysis of plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways occurring in planta.

The U.S. Dairy Forage Research Center (USDFRC) Condensed Tannin NMR Database

This Perspective describes a solution-state NMR database for flavan-3-ol monomers and condensed tannin dimers through tetramers obtained from the literature to 2015, containing data searchable by structure, molecular formula, degrees of polymerization, and 1H and 13C chemical shifts of the condensed tannins. Citations for all literature references are provided and should serve as valuable resource for scientists working in the field of condensed tannin research. The database will be periodically updated as additional information becomes available, typically on a yearly basis and is available for use, free of charge, from the U.S. Dairy Forage Research Center (USDFRC) Website.

Reports from Other Journals: Gleanings from Scientific American

Scientific American provides a rich resource of background and general interest material for topics of chemical interest that can be used to supplement and enhance chemistry lecture and laboratory courses.