Modeling Lignin Polymerization. I. Simulation Model of Dehydrogenation Polymers

Lignin is a heteropolymer that is thought to form in the cell wall by combinatorial radical coupling of monolignols. Here, we present a simulation model of in vitro lignin polymerization, based on the combinatorial coupling theory, which allows us to predict the reaction conditions controlling the primary structure of lignin polymers. Our model predicts two controlling factors for the β-O-4 content of syringyl-guaiacyl lignins: the supply rate of monolignols and the relative amount of supplied sinapyl alcohol monomers. We have analyzed the in silico degradability of the resulting lignin polymers by cutting the resulting lignin polymers at β-O-4 bonds. These are cleaved in analytical methods used to study lignin composition, namely thioacidolysis and derivatization followed by reductive cleavage, under pulping conditions, and in some lignocellulosic biomass pretreatments.Lignins are aromatic polymers that are predominantly present in secondarily thickened cell walls. These polymers make the cell wall rigid and impervious, allowing transport of water and nutrients through the vascular system and protecting plants against microbial invasion. Lignins are heterogeneous polymers derived from phenylpropanoid monomers, mainly the hydroxycinnamyl alcohols coniferyl alcohol (G-monomer) and sinapyl alcohol (S-monomer) and minor amounts of p-coumaryl alcohol (H-monomer). These monolignols differ in their degree of aromatic methoxylation (-OCH₃ group; Fig. 1). The resulting units in the lignin polymer are the guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units. They are linked by a variety of chemical bonds (Fig. 2) that have different chemical properties (Boerjan et al., 2003; Ralph et al., 2004; Vanholme et al., 2008).Open in a separate windowFigure 1.Chemical structures of three monolignols. A, H-monomer (p-coumaryl alcohol). B, G-monomer (coniferyl alcohol). C, S-monomer (sinapyl alcohol). G- and S-monomers are considered in our simulations. The G-monomer is methoxylated (-OCH₃ group) on position 3, and the S-monomer is methoxylated on positions 3 and 5.Open in a separate windowFigure 2.Chemical structures resulting from the possible bonding between two monomers (A) or a monomer and the bindable end of an oligomer (B). X and Y in the monomers denote the absence (for a G-unit) or presence (for an S-unit) of a methoxyl group at position 5 (see Fig. 1). The red line indicates the bonds generated by couplings of the B position and B, 4, or 5 position.Lignification is the process by which monomers and/or oligomers are polymerized via radical coupling reactions and typically occurs after the polysaccharides have been laid down in the cell wall. Lignin composition varies among cell types and can even be different in individual cell wall layers (Ruel et al., 2009). Lignin composition is also influenced by environmental conditions; for example, lignin in compression wood is enriched in H-units (Timell, 1986). Hence, both developmental and environmental parameters influence the composition and thus the structure of the lignin polymer (Boerjan et al., 2003; Ralph et al., 2004).Lignin is one of the main negative factors in the conversion of lignocellulosic plant biomass into pulp and bioethanol (Lynd et al., 1991; Hill et al., 2006). In these processes, lignin needs to be degraded by chemical or mechanical processes that are expensive and often environmentally polluting. Hence, major research efforts are devoted toward understanding lignin biosynthesis and structure. It has already been shown that reducing lignin content and modifying its composition in transgenic plants can result in dramatic improvements in pulping efficiency (Pilate et al., 2002; Baucher et al., 2003; Huntley et al., 2003; Leplé et al., 2007) and in the conversion of biomass into bioethanol (Stewart et al., 2006; Chen and Dixon, 2007; Custers, 2009). These altered biomass properties are related to the alterations in lignin composition and structure in terms of the frequencies of the lignin units and the bond types connecting them and possibly also their interaction with hemicelluloses (Ralph et al., 2004; Ralph, 2006).To study the parameters that influence lignin structure, lignin polymerization has been mimicked in vitro by experiments with dehydrogenation polymers (DHPs; Terashima et al., 1995). Indeed, lignification can be mimicked by oxidizing monolignols using a peroxidase, such as horseradish peroxidase (HRP), and supplying its cofactor hydrogen peroxide, producing synthetic DHP lignins. Monolignol oxidation can also be achieved without enzymes (e.g. by using transition metal one-electron oxidants, such as copper acetate). Some of these biomimetic DHPs have been suggested to be better models for wood lignins than HRP-generated DHPs (Landucci, 2000).In DHP experiments, the monolignols are either added in bulk (Zulauf experiment) or dropwise (Zutropf experiment) to the reaction mixture, yielding lignin polymers with very different bond frequencies (Freudenberg, 1956). Zutropf experiments approach the in vivo formation of lignin, which depends on the slow introduction of monolignols into the wall matrix via diffusion to the site of incorporation (Hatfield and Vermerris, 2001). Because the exact reaction conditions are known, such in vitro experiments have provided insight into the lignification process in planta. In this way, numerous factors were shown to influence lignin structure, including the relative supply of the monolignols, the pH, the presence of polysaccharides, hydrogen peroxide concentrations, and cell wall matrix elements in general (Grabber et al., 2003; Vanholme et al., 2008).Computer simulations of lignin polymerization can help explain and predict lignin structure from low-level chemical kinetic factors, including subunit-coupling probabilities and monolignol synthesis rates. Such models are helpful in explaining the mechanism behind a range of controlling factors identified in the experimental work, including (1) the ratio of coniferyl versus sinapyl alcohol monolignols, (2) the monolignol supply rate, and (3) the abundance of alternative monomers present during lignin biosynthesis in mutants and transgenics. Thus, computer models will also help in suggesting new targets for controlled lignin biosynthesis.Here, we propose a simulation model of synthetic lignin polymerization that is based upon an emerging consensus from a variety of observations and derives from a series of previous models of lignin polymerization (Glasser and Glasser, 1974; Glasser et al., 1976; Jurasek, 1995; Roussel and Lim, 1995). Our model uses a symbolic grammar to describe a constructive dynamical system (Fontana, 1992) or a rule-based system (Feret et al., 2009) in which it is not necessary to define all possible products in advance. We assume that G- and S-monomers and newly formed oligomers couple in a well-mixed medium, depending on coupling rules and experimentally measured coupling probabilities. To develop the model, we have used information from DHP experiments rather than natural lignins, as they are formed in a well-mixed medium and their reaction conditions are well known (e.g. the influx rate of monomers). Using information from natural lignin would have further complicated our model, as the structures of natural lignin polymers are influenced by many factors, including the possible involvement of dirigent proteins (Davin and Lewis, 2005), steric hindrance by polysaccharides, spatiotemporal regulation, and modifications during isolation procedures (Boerjan et al., 2003; Ralph et al., 2004).Using our simulation models, we study how putative controlling factors of lignin primary structure, including the influx rate of monomers and the relative amount of S-monomers, affect in silico lignin synthesis, and we compare our predictions with in vitro experiments. To predict the degradability of lignins formed in our simulations, we apply an in silico thioacidolysis, which cleaves the polymers at their β-O-4 positions. This simulates the molecular action of two of the most used methods to analyze lignin composition, thioacidolysis (Lapierre, 1993; Baucher et al., 2003) and derivatization followed by reductive cleavage (Lu and Ralph, 1997). The G+S-monomer yield is often taken as a reflection of the fraction of units bound by β-O-4 bonds. Cleavage of β-O-4 bonds is also the most important reaction in kraft pulping of wood (Baucher et al., 2003). The model predicts from first principles (1) that DHP lignins formed under Zutropf conditions have a higher β-O-4 content than those formed under Zulauf conditions, (2) that DHP lignins formed with high S content have a higher β-O-4 content than those formed with high G content, and (3) that a higher β-O-4 content does not necessarily reduce the average length of lignin fragments generated during in silico thioacidolysis.     

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in the cell wall. Recently, various glycosylated lignin oligomers have been revealed in Arabidopsis thaliana. Given that monolignol oxidation and monolignol radical coupling are known to occur in the apoplast, and glycosylation in the cytoplasm, it raises questions about the subcellular localization of glycosylated lignin oligomer biosynthesis and their storage. By metabolite profiling of Arabidopsis leaf vacuoles, we show that the leaf vacuole stores a large number of these small glycosylated lignin oligomers. Their structural variety and the incorporation of alternative monomers, as observed in Arabidopsis mutants with altered monolignol biosynthesis, indicate that they are all formed by combinatorial radical coupling. In contrast to the common believe that combinatorial coupling is restricted to the apoplast, we hypothesized that the aglycones of these compounds are made within the cell. To investigate this, leaf protoplast cultures were cofed with ¹³C₆-labeled coniferyl alcohol and a ¹³C₄-labeled dimer of coniferyl alcohol. Metabolite profiling of the cofed protoplasts provided strong support for the occurrence of intracellular monolignol coupling. We therefore propose a metabolic pathway involving intracellular combinatorial coupling of monolignol radicals, followed by oligomer glycosylation and vacuolar import, which shares characteristics with both lignin and lignan biosynthesis.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes. Sequences of NtCAD1-1 and NtCAD1-7 were deposited in GenBank under accession numbers AY911854 and AY911855, respectively.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.     

Biosynthesis and Genetic Engineering of Lignin

Lignin, a complex heteropolymer of cinnamyl alcohols, is, second to cellulose, the most abundant biopolymer on Earth. Lignification has played a determining role in the adaptation of plants to terrestrial life. As all extracellular polymers, lignin confers rheological properties to plant tissues and participates probably in many other functions in cell and tissue physiology orin cell-to-cell communication. Economically, lignin is very important because it determines wood quality and it affects the pulp and paper-making processes as well as the digestibility of forage crops. For all these reasons the lignin biosynthesis pathway has been the subject of many studies. At present, most genes encoding the enzymes involved in the biosynthesis of lignin have been cloned and characterized. Various recent studies report on the alteration of the expression of these genes by genetic engineering, yielding plants with modified lignin. In addition, several mutants have been analyzed with changes in lignin content or lignin composition resulting in altered properties. Thanks to these studies, progress in the knowledge of the lignin biosynthesis pathway has been obtained. It is now clear that the pathway is more complex than initially thought and there is evidence for alternative pathways. A fine manipulation of the lignin content and/or composition in plants is now achievable and could have important economical and environmental benefits.     

Lignin engineering

Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls. Several decades of research have elucidated the main biosynthetic routes toward the monolignols and demonstrated that lignin amounts can be engineered and that plants can cope with large shifts in p-hydroxyphenyl/guaiacyl/syringyl (H/G/S) lignin compositional ratios. It has also become clear that lignins incorporate many more units than the three monolignols described in biochemistry textbooks. Together with the theory that lignin polymerization is under chemical control, observations hint at opportunities to design lignin structure to the needs of agriculture. An increasing number of examples illustrates that lignin engineering can improve the processing efficiency of plant biomass for pulping, forage digestibility and biofuels. Systems approaches, in which the plant's response to engineering of a single gene in the pathway is studied at the organismal level, are beginning to shed light on the interaction of lignin biosynthesis with other metabolic pathways and processes.     

Genetical metabolomics of flavonoid biosynthesis in Populus: a case study

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.