| Abstract: | 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.Lignin, a complex phenylpropanoid polymer in the plant cell wall, is predominantly deposited in the cell walls of secondary-thickened cells (Vanholme et al., 2010). It is synthesized via oxidative radical coupling reactions from three prototypical monolignols, p-coumaryl, coniferyl, and sinapyl alcohols, differentiated by their degree of methoxylation ortho to the phenolic hydroxyl group. Considered within the context of the entire polymer, the main structural features of lignin can be defined in terms of its p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, derived respectively from these three monolignols (Ralph, 2010). Several novel monomers, all deriving from the monolignol biosynthetic pathway, have been found to incorporate into lignin in wild-type and transgenic plants. For example, monolignol acetate, p-hydroxybenzoate, and p-coumarate ester conjugates have all been shown to incorporate into lignin polymers and are the source of naturally acylated lignins (Ralph et al., 2004; Lu and Ralph, 2008); lignins derived solely from caffeyl alcohol were found in the seed coats of both monocot and dicot plants (Chen et al., 2012a, 2012b); lignins derived solely from 5-hydroxyconiferyl alcohol were found in a cactus (for example, in a member of the genera Astrophytum) seed coat (Chen et al., 2012a); a Medicago truncatula transgenic deficient in cinnamyl alcohol dehydrogenase exhibited a lignin that was overwhelmingly derived from hydroxycinnamaldehydes (instead of their usual hydroxycinnamyl alcohol analogs; Zhao et al., 2013); and iso-sinapyl alcohol was implicated as a monomer in caffeic acid O-methyltransferase down-regulated switchgrass (Panicum virgatum; Tschaplinski et al., 2012). These findings imply that plants are quite flexible in being able to use a variety of monomers during lignification to form the heterogenous lignin polymer. Most recently, and as addressed more fully here, the flavonoid tricin has been implicated as a monomer in monocot lignins (del Río et al., 2012). To our knowledge, tricin is the first monomer from outside the monolignol biosynthetic pathway to be implicated in lignification.Tricin 5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a member of the flavonoid family, is recognized as a valuable human health compound due to its antioxidant, antiaging, anticancer, and cardioprotective potentials (Ogo et al., 2013). Tricin and its derivatives can be solvent extracted from monocot samples such as wheat (Triticum aestivum), oat bran (Avena sativa), bamboo (Leleba oldhami), sugarcane (Saccharum officinarum), and maize (Zea mays). Extracted compounds can take the form of tricin itself, 7-O-glycosylated tricin, or the flavonolignan in which tricin is 4′-O-etherified by putative coupling with coniferyl alcohol (Ju et al., 1998; Bouaziz et al., 2002; Wenzig et al., 2005; Duarte-Almeida et al., 2007; Van Hoyweghen et al., 2010; Nakano et al., 2011; Bottcher et al., 2013; Moheb et al., 2013).In 2012, we reported, to our knowledge, the first evidence that tricin was incorporated into lignin, as implicated by two previously unassigned correlation peaks at δC/δH 94.1/6.56 and 98.8/6.20 in a heteronuclear single-quantum coherence (HSQC) NMR spectrum from the whole cell wall and an isolated milled wood lignin of (unacetylated) wheat straw (del Río et al., 2012). The same evidence has now been found in the HSQC spectrum of wheat straw lignin isolated via different methods (Yelle et al., 2013; Zeng et al., 2013). Additional studies have verified the presence of tricin in lignin fractions from a variety of monocots, including bamboo (You et al., 2013), coconut coir (Cocos nucifera; Rencoret et al., 2013), maize, and others examined in our laboratories. The implication that tricin is the first phenolic from outside the monolignol biosynthetic pathway found to be integrated into the polymer has prompted further study with the aim of identifying and mechanistically delineating the role of tricin in lignin and its biosynthetic incorporation pathway.Tricin, unlike the monolignols that derive from the shikimate biosynthetic pathway (Sarkanen and Ludwig, 1971), is derived from a combination of the shikimate and acetate/malonate-derived polyketide pathways (Winkel-Shirley, 2001), as shown in Supplemental Figure S1. After p-coumaroyl-CoA is synthesized from p-coumaric acid by 4-coumarate:CoA ligase, it branches from the monolignol biosynthetic route to be transformed via chalcone synthase and chalcone isomerase into naringenin, the central precursor of most flavonoids. Naringenin is subsequently converted into apigenin by flavone synthase. Further hydroxylation at C-3′ and C-5′ followed by O-methylation furnishes tricin (Koes et al., 1994; Winkel-Shirley, 2001). The incorporation of tricin into lignin, therefore, suggests that an additional biosynthetic pathway, namely the polyketide pathway, may be associated with cell wall lignification in monocots.The revelation that tricin is incorporated into the lignin polymer was precipitated by closer study of signals found within the NMR spectra of various monocot samples. Before this discovery, tricin had not been noted in any lignin fractions, and although it is reasonable to anticipate compatibility based on its chemical structure, there is no direct and reliable evidence to date showing that tricin is able to react with monolignols through radical coupling; therefore, the efficiency and selectivity of the coupling reactions between tricin and various monolignols were also unknown. Synthetic model compounds that would facilitate the elucidation of the role of tricin within plant cell walls are desirable as aids to be used in a mechanistic study of flavonolignin generation. (We coin the term flavonolignin to describe the racemic oligomers and polymers of monolignols that start from tricin or 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 that are presumably made in the cytoplasm Begum et al., 2010; Niculaes et al., 2014; Dima et al., 2015]).The overall objective of this study is to demonstrate that tricin incorporates into the lignin polymer of monocots, with maize/corn stover as the representative experimental material. To this end, we have synthesized tricin and various model compounds in which tricin is conjugated to monolignols in the manner expected for the lignification process. Next, we verified whether these synthetic compounds could be made from their assumed precursors under the biomimetic radical conditions anticipated for lignification. Subsequently, NMR data generated from these synthetic and biomimetic coupling products were compared with NMR data from native maize stover lignin, including high-Mr fractions. We conclude that tricin is a monomer in monocot lignification and that, because little syringaresinol is found in maize lignin, tricin is functioning as a nucleation site that initiates lignin polymer chains. |
