Lignin Biosynthesis and Structure
Lignin is the generic term for a large group of aromatic polymers resulting from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids (Boerjan et al., 2003; Ralph et al., 2004). These polymers are deposited predominantly in the walls of secondarily thickened cells, making them rigid and impervious. In addition to developmentally programmed deposition of lignin, its biosynthesis can also be induced upon various biotic and abiotic stress conditions, such as wounding, pathogen infection, metabolic stress, and perturbations in cell wall structure (Caño-Delgado et al., 2003; Tronchet et al., 2010). Because lignin protects cell wall polysaccharides from microbial degradation, thus imparting decay resistance, it is also one of the most important limiting factors in the conversion of plant biomass to pulp or biofuels. The removal of lignin from plant biomass is a costly process; hence, research efforts are now aimed at designing plants that either deposit less lignin or produce lignins that are more amenable to chemical degradation (Sticklen, 2008; Weng et al., 2008a; Mansfield, 2009). The main building blocks of lignin are the hydroxycinnamyl alcohols (or monolignols) coniferyl alcohol and sinapyl alcohol, with typically minor amounts of p-coumaryl alcohol (Fig. 1). The monolignols are synthesized from Phe through the general phenylpropanoid and monolignol-specific pathways. Phe is derived from the shikimate biosynthetic pathway in the plastid (Rippert et al., 2009). Certain enzymes of the lignin biosynthetic pathway, namely the cytochrome P450 enzymes CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE (C3H), and FERULATE 5-HYDROXYLASE (F5H), are membrane proteins thought to be active at the cytosolic side of the endoplasmic reticulum (Chapple, 1998; Ro et al., 2001). Although metabolic channeling has been shown between PHENYLALANINE AMMONIA-LYASE (PAL) and C4H (Rasmussen and Dixon, 1999; Achnine et al., 2004), it remains unknown whether the other pathway enzymes are also part of metabolic complexes at the endoplasmic reticulum. The main biosynthetic route toward the monolignols p-coumaryl, coniferyl, and sinapyl alcohol (Boerjan et al., 2003). PAL, PHENYLALANINE AMMONIA-LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE:CoA LIGASE; C3H, p-COUMARATE 3-HYDROXYLASE; HCT, p-HYDROXYCINNAMOYL-CoA:QUINATE/SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE; CCoAOMT, CAFFEOYL-CoA O-METHYLTRANSFERASE; CCR, CINNAMOYL-CoA REDUCTASE; F5H, FERULATE 5-HYDROXYLASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; CAD, CINNAMYL ALCOHOL DEHYDROGENASE. The units resulting from the monolignols, when incorporated into the lignin polymer, are called guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units (Figs. 1 and 2). With some notable exceptions (Novo Uzal et al., 2009), lignins from gymnosperms are composed of G-units only (with minor amounts of H-units), whereas angiosperm dicot lignins are composed of G- and S-units. H-units are elevated in softwood compression wood and may be slightly higher in grasses (Boerjan et al., 2003). A variety of less abundant units have been identified from diverse species, and these may be incorporated into the polymer at varying levels (Ralph et al., 2004). Some units, such as those derived from the monomer sinapyl acetate, can make up to 85% of all S-units in the polymer (Lu and Ralph, 2008; Martínez et al., 2008). In addition to differences in lignin composition among taxa and species, lignin composition can also differ among cell types, as readily visualized by the histochemical Mäule staining, which is indicative of S-units, or when chemically analyzed by laser capture microdissection followed by microanalysis of lignin (Nakashima et al., 2008; Ruel et al., 2009). Lignins can even be dissimilar at the level of individual cell wall layers, as revealed by Raman, IR, and UV microspectroscopy, or by immunolabeling of the secondary wall with antibodies cross-reacting with specific lignin substructures (Shi et al., 2006; Gierlinger and Schwanninger, 2007; Gou et al., 2008; Ruel et al., 2009). Representation of a lignin polymer from poplar, as predicted from NMR-based lignin analysis (adapted from Stewart et al., 2009). It is commonly accepted that lignin evolved together with the adaptation of plants to a terrestrial life to provide them with the structural support needed for an erect growth habit (Fig. 3). Comparative genomics studies based on available plant genomes indicate that the complete biosynthetic pathway, except for F5H, first appeared in moss (based on the Physcomitrella genome sequence; Xu et al., 2009) but was absent from green algae. However, recent studies have also detected secondary walls and apparent lignin in the marine red alga Calliarthron, which diverged from vascular plants more than 1 billion years ago (Martone et al., 2009), indicating either a very strongly conserved evolutionary history of the relevant pathways or convergent evolution. In support of the latter hypothesis, parallels between Calliarthron and angiosperms are obvious; lignin in the secondary cell walls of Calliarthron may have evolved to resist the bending stresses imposed by breaking waves, similar to lignin in the walls of vascular plants that provides biomechanical support (Martone et al., 2009). In the lycophyte species Selaginella, convergent evolution has been demonstrated with the identification of a new F5H that is capable of functioning in S-lignin biosynthesis but that is structurally unrelated to F5H characterized in angiosperms (Weng et al., 2008b, 2010). It is also noteworthy that cell cultures, but not woody cell walls of Ginkgo biloba, are able to synthesize S lignin; the S-lignin pathway in gymnosperms is generally thought to be absent (Novo Uzal et al., 2009). Clearly, the complex evolutionary puzzle regarding S lignin has yet to be resolved. Phylogenetic tree showing the distribution of lignin monomer composition across major lineages. *, S-units are only found in cell cultures of Ginkgo, not in wood (Novo Uzal et al., 2009). **, Lignin-like structures are reported in some mosses and green algae, but the presence of real lignin in these nonvascular species remains questionable; red algae have been barely studied (Weng et al., 2008b; Martone et al., 2009). Genes have been cloned for each of the steps of the lignin biosynthetic pathway and the impact on lignin amount and composition has been studied through mutants or reverse genetics in various species, mostly Arabidopsis (Arabidopsis thaliana), maize (Zea mays), alfalfa (Medicago sativa), poplar (Populus spp.), and tobacco (Nicotiana tabacum) but more recently also in flax (Linum usitatissimum; CCoAOMT; Day et al., 2009), the gymnosperms Pinus radiata (HCT and 4CL; Wagner et al., 2007, 2009) and Picea abies (CCR; Wadenbäck et al., 2008), and in rice (Oryza sativa; CAD; Zhang et al., 2006; Li et al., 2009). (For full gene/protein names not provided in the text, see legend of Fig. 1.) From these studies, it can be concluded that reducing the expression of each gene, except F5H, lowers lignin amount in the cell wall to varying extents, depending on the gene, the species, and the level of gene redundancy (Li et al., 2008; Nakashima et al., 2008; Vanholme et al., 2008). Secondary walls with decreased lignin amounts are often characterized by collapsed vessels. This “irregular xylem” phenotype, indicative of reduced mechanical cell wall strength, is not unique to reductions in lignin, as it is also noted in mutants with defects in the biosynthesis of other cell wall polymers (Brown et al., 2005). Strongly reduced lignin amounts result in altered plant development, but more importantly, modest reductions can lead to normal development, as nicely demonstrated by the allelic series of c4h (ref3) mutants in Arabidopsis; the strongest allele causes small and sterile plants, and the weaker alleles result in relatively normal plants with still less lignin (Ruegger and Chapple, 2001; Schilmiller et al., 2009). This implies that, for applications in crops, fine-tuning of the lignin level or targeting the transgene expression to only specific cell types will be an important endeavor. In addition to lignin content, lignin H/G/S composition also appears to be rather flexible. Silencing of HCT or C3H leads to lignin with H-unit levels as high as 100% of the total thioacidolysis lignin monomers (Franke et al., 2002; Abdulrazzak et al., 2006; Ralph et al., 2006; Besseau et al., 2007; Coleman et al., 2008). Down-regulation of F5H or COMT strongly reduces S-unit content; in contrast, up-regulation of F5H increases the S-unit content (Franke et al., 2000; Stewart et al., 2009). Furthermore, down-regulation of CAD increases the incorporation of cinnamaldehydes into the polymer (Baucher et al., 1998; Lapierre et al., 1999, 2004; Kim et al., 2003). The most remarkable shifts in lignin composition are seen when COMT is down-regulated; in these lignins, 5-hydroxyconiferyl alcohol, derived from the COMT substrate, is incorporated, although it is below the detection limit in wild-type lignin (Van Doorsselaere et al., 1995; Lapierre et al., 1999; Fig. 5). It is interesting that shifts in G and S levels, as well as shifts toward more aldehydes, generally have only minor effects on plant development. Obviously, because the relative ratio of the different monomers determines the frequency of the different bonds in the polymer, all these compositional shifts have repercussions on the structure of the polymer and may thus alter the cell wall properties. The effects of lignin pathway perturbations often go beyond alterations in lignin amount, composition, and cell wall structure. Indeed, several studies have now demonstrated that perturbing individual steps of the lignin biosynthetic pathway affects the expression level of other lignin pathway genes and also the expression of genes involved in a multitude of other, seemingly unrelated biological processes. These wider effects are also reflected at the metabolite level (Rohde et al., 2004; Sibout et al., 2005; Shi et al., 2006; Dauwe et al., 2007; Leplé et al., 2007). Uncovering their molecular basis might help mitigate the adverse effects on plant growth and development that often accompany lignin modifications. In the past few years, significant progress has been made in understanding the regulation of lignification. New data indicate a regulatory cascade of upstream transcription factors that control the formation of secondary walls by activating a range of other transcription factors. Some of these downstream transcription factors are then able to induce the expression of genes of the lignin biosynthetic pathway (Zhong and Ye, 2007). The regulatory cascade explains why several of the currently described transcription factors lead to enhanced or reduced lignification when misexpressed in plants while they do not directly regulate the lignin biosynthetic genes by binding to their promoters (Zhong et al., 2006, 2008). Therefore, yeast one-hybrid assays, protoplast transient expression assays, and electrophoretic mobility shift assays have been essential to prove the direct binding of a given transcription factor to the promoters of lignin genes (Table I). ↑, ↓, or =, Target gene up-regulated, down-regulated, or not affected, respectively. qPCR refers to quantitative reverse transcription-PCR results from overexpressing lines, what does not necessarily imply direct binding on the respective promoters, except for Myb58, for which the qPCR was done in an estradiol-inducible system in the presence of the protein synthesis inhibitor cycloheximide (Zhou et al., 2009). EMSA, Electrophoretic mobility shift assay; TEA, protoplast transient expression assay; Y1H, yeast one-hybrid assay. Direct promoter binding proven by TEA, EMSA, or Y1H. EMSA shows binding to the Pal box. EMSA shows binding to the AC-promoter element. ↑, ↓, or =, Target gene up-regulated, down-regulated, or not affected, respectively. qPCR refers to quantitative reverse transcription-PCR results from overexpressing lines, what does not necessarily imply direct binding on the respective promoters, except for Myb58, for which the qPCR was done in an estradiol-inducible system in the presence of the protein synthesis inhibitor cycloheximide (Zhou et al., 2009). EMSA, Electrophoretic mobility shift assay; TEA, protoplast transient expression assay; Y1H, yeast one-hybrid assay. Direct promoter binding proven by TEA, EMSA, or Y1H. EMSA shows binding to the Pal box. EMSA shows binding to the AC-promoter element. Microarray experiments on Arabidopsis cell suspension cultures that are induced to form tracheary elements have identified a set of transcription factors involved in this cell differentiation process (Kubo et al., 2005). Whereas VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 were shown to be key upstream regulators of the protoxylem and metaxylem formation, respectively (Kubo et al., 2005; Yamaguchi et al., 2008), SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) was described as an upstream regulator of interfascicular fiber development, and overexpression of SND1 led to ectopic formation of secondary cell walls (Zhong et al., 2006). Moreover, SND1 was able to regulate at least 10 other transcription factors (Zhong et al., 2007, 2008; Zhou et al., 2009), some of which activated the phenylpropanoid pathway (e.g. Myb46, Myb63, and Myb58; Table I). Engineering the expression of transcription factors has the to alter lignification with adverse effects on plant development for these transcription factors the promoters of the through the pathway in an some might be involved in the lignification process and not in other such as stress lignin formation, the plant able to to factors. their the monolignols are to the cell wall by a that remains a In one the monolignols are the membrane through their and that are upon their by in the cell However, Arabidopsis mutants in the have normal lignin levels et al., 2006; and Dixon, and In monolignols are to the membrane by However, et not for in they significant in the at is support for a for or in the of monolignols to the cell that monolignols are across the membrane by but on several by reverse genetics have not yet revealed on et al., 2005; studies are coniferyl and sinapyl alcohols might have the of through the membrane and 2006). Lignin oxidative of followed by combinatorial In the first the is The resulting is relatively to of the in the system (Fig. monomer may to form a a between coupling at their resulting in only the and (Fig. This coupling in a the ratio of each of the coupling on the chemical of each of the monomers and the in the cell wall (Ralph et al., 2004). the to be to a it can with monomer This of in which a monomer to the polymer, is the polymer one at a of lignin is in but relatively in coupling for of the et al., 2002; Wagner et al., 2009). each coupling are a as each to the making this of different from the that in the of several such as and The of a lignin in poplar is to be between and units et al., 2004; Stewart et al., 2009). of coniferyl alcohol of coniferyl alcohol with the at the or are not shown because coupling do not at these Whereas as a substrate, to their to types of enzymes to large gene of which the individual have making the process to in a may have to on lignification to gene redundancy et al., 2005; and 2006). may differ in their whereas some coniferyl alcohol, are specific toward sinapyl alcohol et al., 2006; et al., 2007). Because the structure of lignin on the of may in part the structure of the lignin for lignin structure by expression of specific Although monolignols might be direct with an the might be by Indeed, direct of the lignin polymer with enzymes may be given the of of in the cell The in sinapyl has been as a for lignification in sinapyl alcohol, is an for found in but the its to sinapyl alcohol also to lignin polymers et al., 2008). In the of lignification to provide the oxidative in the cell that this will have the to and into the lignin polymer, to chemical and This lignification also nicely explains why other can be into the lignin polymer and up the of lignins for applications by the and types of monolignols into the cell wall 2006; et al., 2008). to the molecular of lignin structure. lignin is a complex and of the generally at the frequency of the main units and the main types in the polymer, from a of the plant or in major and thioacidolysis and followed by are that the H/G/S composition of the lignin these only a of the polymer for and followed by monomers only from the of the polymer only those and S-units that are with other units are from the polymer and by Lapierre et al., and Ralph, A thioacidolysis now some and Mansfield, 2009). which the of with the higher of to be the to the of the different lignin units and the (Fig. Ralph et al., 1999; Ralph and 2010). Although the are from lignins, the lignin can be analyzed when such as in which most of the polysaccharides are by with or when cell wall either or in the et al., 2008; Kim and Ralph, 2010). of the lignins in these most of the of the lignin structure to be The of walls now a of all of the various polysaccharides and in the cell A new development in data analysis is the of analysis that provides even hence, a of the cell as demonstrated by the differences between wood and normal wood in poplar et al., 2009). of an Arabidopsis lignin that the lignin from the cell wall but is in polysaccharides their removal with The provides a of of the lignin structure. The aromatic the ratio to be readily of the and The of the the individual structures the as their and the to be visualized and units are only seen at levels not the other from the units (H) from the incorporation of 5-hydroxyconiferyl alcohol are below the detection limit in wild-type Arabidopsis lignin but are seen in lignins from Although a of the data have been those in Fig. the molecular of The analysis of lignin from wild-type and or plants by with has in their A is that of and of revealed the of units derived from 5-hydroxyconiferyl the resulting units, when this new monomer into the polymer, were revealed as new in the (Ralph et al., 2001; et al., 2003; et al., of plants and a incorporation of into In addition to the and thioacidolysis was for a from coupling of into lignin et al., 2007; Ralph et al., 2008). These units are now indicative of incorporation into These new units not be with coupling from lignification of which are abundant in data also provide into other of the lignin polymer, such as polymer as nicely demonstrated by the analysis of lignin from poplar et al., 2009). Lignins from these have the S content, with an ratio of than for wild-type poplar readily seen by the lignin is composed only of and units (with small amounts of for types, see Fig. 5). the is and the level is of the of the lignin can only be 10 units, as it is a that a lignin can only have a in that factors high level and the and the high content of units with a to this remarkable and biomass conversion et al., 2003; Stewart et al., 2009). lignins have high molecular and are their structures the of and S-units in individual polymers and the bonds between is not that are typically based on lignin from cell In because lignin is of lignin is except for individual understanding monolignols the to a given provides into the factors that lignin structure. In an to the structures of have been from poplar and analyzed by et al., of these structures were and all were in with combinatorial oxidative the relative of these were analyzed from plants with lignin, they nicely with the lignin structural data as by of the total lignin, the of as a and more than or to in lignin structure. In has the to new lignin sinapyl was as a new monomer (or monomer by its with sinapyl alcohol (Lu et al., 2004; et al., the identification of such by a of their in the This has now the development of an that of individual lignin et al., 2010). the analysis of that still have an unknown Therefore, the structural and of the full of these by which to in as may lignin monomers and their biosynthetic new in lignin research is to biosynthetic pathways toward the biosynthesis of that, upon incorporation into the lignin polymer, will lignin This from the of lignin from the that lignin is able to readily units that from biosynthesis in plants with pathway and from in which plants with altered lignin structures are shown to be and more et al., 2002; et al., 2003; Ralph et al., 2006; Leplé et al., 2007). This of lignin monomers into the polymer has been demonstrated in a system by coniferyl together with normal monolignols into maize cell walls et al., 2008). The resulting lignin, now strongly in bonds in the lignin at and such can also be by it may result in less biomass to the into the factors that lignin structure and can be by to coupling of monolignols is on their of to the cell their chemical and the oxidative in the among other factors et al., 2008). A has now been that the frequency of the different types, the of different their and the relative of the different polymers varying This in can be to the that lignin degradation in such as or biomass in et al., 2010). The is such that it can the incorporation of lignin monomers to the on lignin more has revealed that lignin perturbations have on the regulation of the pathway and on other metabolic pathways and biological et al., 2008). this it to lignin and it can be to the effects of lignin perturbations while into and still has a to although studies have demonstrated the effects of lignin on such as and in the only a few studies have plants in the 2005; et al., 2006; et al., 2007; Leplé et al., 2007; Wadenbäck et al., 2008; et al., 2008; et al., 2010). However, in the plants are to various and stresses by and that are or even to in a are thus an essential toward to the effects of reduced lignin on the conversion of biomass to have recently been et al., 2009). In addition to plants, have been shown years ago for a of in grasses et al., and et al., 2001; et al., 2006). With the of the the of from the plant cell wall and their as a for that have been to a range of is more for help in the