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Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) straw and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4'-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4'-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4'-O-β)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds.