Lignin is polymerized mainly from hydroxycinnamyl alcohols, typically p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, through radical coupling (Fig. 1) (Boerjan et al., 2003). Lignins derived from these hydroxycinnamyl alcohols are commonly referred to as hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin, respectively. Except for S lignin, which is specific to certain lineages (e.g. flowering plants and the lycophyte Selaginella) (Weng et al., 2008b), H and G lignins are fundamental to all the tracheophytes (Fig. 2). Bryophytes do not synthesize lignin, but are widely found to accumulate soluble phenylpropanoids, such as flavonoids and lignans (Basile et al., 1999; Umezawa, 2003). Further, the genome of the reference moss Physcomitrellapatens contains orthologs of all the eight core lignin biosynthetic enzymes required for the biosynthesis of p-coumaryl alcohol and coniferyl alcohol (Fig. 3), whereas the green algae Chlamydomonas reinhardtii contains none (Silber et al., 2008; Weng et al., 2008b; Xu et al., 2009). This evidence suggests that the metabolic scaffold for monolignol biosynthesis evolved earlier than the rise of tracheophytes, and was probably established in the earliest land plants where some or all of the pathway served in the generation of UV-protectant molecules.
1. Recruitment of enzymes of primary metabolism to a new secondary metabolic pathway
The novel activities of the enzymes required for lignin deposition did not simply appear, but were gradually acquired through the Darwinian process of mutation and selection (Tawfik, 2006). It has been widely accepted that novel enzyme functions usually arise after gene duplication events, where one copy maintains the ancestral function, and the other copy becomes less constrained and can evolve (Lynch & Conery, 2000). In rare cases, the newly derived gene provides certain selective advantages to the host, and becomes fixed in the population (Lynch & Conery, 2000). Presumably, the primitive forms of phenylpropanoid metabolism emerged from a much larger number of metabolic pathways accidentally assembled using the evolving duplicated genes encoding primary metabolic enzymes and/or enzymes acquired from symbiotic organisms via horizontal gene transfer (Richards et al., 2006). It seems likely that specific combinations of new activities that yielded compounds with UV-absorbent properties would have been selected for in early terrestrial plants. Under subsequent purifying selection, those initial combinations could have converged into one particular metabolic scaffold, which has been preserved and elaborated on in land plants today.
Examination of the eight enzymes that constitute the core monolignol biosynthetic scaffold suggests that their ancestry is deeply rooted in the primary metabolism (Table 1, Fig. 3). Phenylalanine ammonia-lyase (PAL), the enzyme that converts phenylalanine to trans-cinnamic acid at the entry point of the phenylpropanoid pathway, is homologous to histidine ammonia-lyase (HAL), an enzyme involved in histidine degradation in primary metabolism. PAL exhibits considerable structural conservation with HAL, and mirrors HAL in catalytic mechanism by adopting the electrophilic 4-methylidene-imidazole-5-one group derived from the conserved alanine–glycine–serine (Ala–Gly–Ser) tripeptide (Baedeker & Schulz, 2002; Ritter & Schulz, 2004). Cinnamate 4-hydroxylase (C4H) and p-coumaroyl shikimate 3′-hydroxylase (C3′H) are cytochrome P450 monooxygenases (P450) that catalyze the first two aromatic hydroxylation reactions in the monolignol biosynthetic pathway. Although C4H and C3′H have greatly deviated from their primary metabolic cousins, for example sterol 14-demethylase, in their substrate recognition sites, they maintain highly conserved motifs responsible for heme binding and a common catalytic mechanism (Werck-Reichhart & Feyereisen, 2000). 4-Hydroxycinnamoyl-CoA ligase (4CL) activates p-coumaric acid to the activated thioester form p-coumaroyl CoA, which can then be channeled either further downstream into monolignol biosynthesis or into flavonoid biosynthesis if acted on by chalcone synthase (CHS). 4CL is homologous to long-chain fatty acyl-CoA synthetase, the first committed enzyme in the β-oxidation pathway for breaking down long-chain fatty acids. Like that of fatty acyl-CoA synthetase, the 4CL catalytic mechanism involves the formation of an acyl-adenylate intermediate (Schneider et al., 2003; Hisanaga et al., 2004). Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT) catalyzes the formation of p-coumarate esters using shikimate or quinate as an acyl acceptor. These specific p-coumarate esters then serve as the substrates for the phenylpropanoid meta-hydroxylation catalyzed by C3′H. HCT belongs to the BAHD acyltransferase superfamily, which has extensively diversified in plants, and has been suggested to be involved in the biosynthesis of a large number of secondary metabolites (D’Auria, 2006). Despite low primary sequence identity, the structure of the plant BAHD enzyme vinorine synthase shows a surprising overall similarity to the structures of primary metabolic CoA-dependent acyltransferases, such as carnitine acetyltransferase, which is required for the transport of long-chain acyl groups from fatty acids into mitochondria for β-oxidation (Wu et al., 2003; Ma et al., 2005). Caffeoyl-CoA O-methyltransferase (CCoAOMT) catalyzes the first transmethylation reaction on the aromatic 3-hydroxyl using S-adenosyl methionine (SAM) as the methyl donor. CCoAOMT shares a common architecture with mammalian catechol-O-methyl transferase, and contains a highly conserved α/β Rossmann fold, which forms the catalytic divalent cation and SAM-binding sites (Vidgren et al., 1994; Ferrer et al., 2005). Two oxidoreductases, (hydroxy)cinnamoyl-CoA reductase (CCR) and (hydroxy)cinnamyl alcohol dehydrogenase (CAD), are employed in the core monolignol biosynthetic pathway that reduces hydroxycinnamoyl-CoA esters to their corresponding alcohols. Whereas CCR shares significant similarities with mammalian 3-β-hydroxysteroid dehydrogenase responsible for steroid biosynthesis (Lacombe et al., 1997), CAD is homologous to the ethanol-degrading alcohol dehydrogenase widely spread in bacteria and animals (Youn et al., 2006). Like many of the oxidoreductases, both CCR and CAD contain highly conserved Rossmann fold NAD(P)H/NAD(P)(+) binding domains.
Table 1. The eight core monolignol biosynthetic enzymes and their representative cousins in primary metabolism | Enzyme | Enzyme family | Representative cousins in primary metabolism |
|---|
| PAL | Lyase, class I-like | Histidine ammonia-lyase |
| C4H | Cytochrome P450 monooxygenase | Sterol 14-demethylase |
| 4CL | Acyl-CoA synthetase | Long-chain fatty acyl-CoA synthetase |
| HCT | BAHD acyl transferase | Carnitine acetyltransferase |
| C3′H | Cytochrome P450 monooxygenase | Sterol 14-demethylase |
| CCoAOMT | SAM-dependent methyltransferase | Catechol-O-methyl transferase |
| CCR | Dehydrogenase | 3-β-Hydroxysteroid dehydrogenase |
| CAD | Dehydrogenase | Alcohol dehydrogenase |
Although difficult to address, it is intriguing to consider the progressive evolutionary paths by which individual enzymes were recruited and integrated into such a complex metabolic system. Based on a large-scale phylogenetic analysis, it was recently suggested that land plants obtained PAL from symbiotic bacteria or fungi via horizontal gene transfer, which could have initiated the early development of phenylpropanoid metabolism (Emiliani et al., 2009). The newly acquired ability to synthesize cinnamic acid from phenylalanine might have occurred concomitantly with the development of the activity of a promiscuous P450 that was the progenitor of C4H. On the one hand, cinnamic acid has been shown to act as a potent anti-auxin that inhibits plant growth (Vanoverbeek et al., 1951; Wong et al., 2005). Thus, an enzyme that can further metabolize cinnamic acid might have become immediately necessary to avoid growth abberrations (Schilmiller et al., 2009). On the other hand, the addition of the para-hydroxy group to the phenylpropanoid backbone shifts its UV absorbance spectra to the UV-B range, which might have provided an obvious selective advantage to the emerging land plants. It has been shown that, at least in flowering plants, PAL and C4H have further evolved for the ability to physically interact with one another, resulting in the recruitment of soluble PAL tetramers to the cytosolic surface of endoplasmic reticulum (ER) membranes where C4H is localized (Rasmussen & Dixon, 1999; Achnine et al., 2004). Although it remains to be tested whether this is a general phenomenon in land plants, the interaction between PAL and C4H could sequester the potentially harmful intermediate cinnamic acid, which may have the potential to act as an ionophore (McLaughlin & Dilger, 1980). Alternatively, or in addition, by tethering PAL to the ER membrane through PAL–C4H interactions, phenylpropanoid pathway input could be restricted to subpools of phenylalanine in the vicinity of the ER, which could prevent direct competition for bulk cytosolic phenylalanine pools used in protein synthesis.
C4H was probably the first P450 to acquire a dedicated role in the phenylpropanoid pathway, and C3′H was likely to have been subsequently derived from it (Nelson, 2006). However, unlike C4H, which uses a free acid as substrate, C3′H acts on p-coumaroyl shikimate esters, the synthesis of which requires the activities of 4CL and HCT. Schoch et al. (2006) proposed that the involvement of shikimate, a key intermediate in the synthesis of aromatic amino acids, provides a layer of biochemical regulation on phenylpropanoid metabolism. As the availability of cytosolic pools of shikimate fluctuates in response to environmental cues, C3′H activity could then be fine-tuned accordingly (Schoch et al., 2006). CCoAOMT activity may have evolved soon after the occurrence of C3′H, as the caffeoyl ortho-diphenol intermediates produced by C3′H are highly reactive, and are subjected to oxidation by various enzymes, such as polyphenol oxidases and catechol dioxygenases (Masai et al., 1999; Cheynier & Moutounet, 2002). CCoAOMT converts caffeoyl-substituted intermediates to much more stable guaiacyl-substituted compounds, resulting in a ring modification pattern suitable for lignin biosynthesis later during plant evolution. It remains to be determined when the two oxidoreductases, CCR and CAD, evolved relative to the other enzymes in the phenylpropanoid pathway, but the profound impact of the occurrence of such a reductive pathway from phenolic acids to their alcohols is unmistakable. The addition of these hydroxycinnamyl alcohols to the biochemical repertoire of early land plants portended the origin of lignification later in the natural history of land plant evolution.
2. Lignin-like compounds in nonvascular plants
Lignin has generally been considered to be a hallmark of tracheophytes, but there are sporadic reports in the literature describing the detection of lignin or ‘lignin-like’ compounds from nonvascular plants, including brown algae, charophytic algae and mosses (Siegel, 1969; Reznikov et al., 1978; Delwiche et al., 1989). Many of the early findings were based on relatively crude detection methods according to today’s standard, and were either highly controversial or failed to stand further scrutiny (reviewed by Lewis & Yamamoto, 1990). For example, the lignin-like compounds found in the brown algae Fucus were later confirmed to be a series of phloroglucinol-derived polyphenols, which are more likely to be synthesized from fatty acids via the activity of polyketide synthases rather than from phenylpropanoid metabolism (Ragan, 1984). Recently, Ligrone et al. (2008) revisited this issue by immunogold labeling approaches on several bryophyte species and the charophytic alga Nitella flexilis using polyclonal antibodies raised against synthetic lignin-like polymers. In this study, weakly reacting lignin-related epitopes could be detected in the cell wall of all the nonvascular plant species examined, among which the moss Sphagnum cuspidatum even exhibited a labeling intensity similar to that of the tracheophyte controls (Ligrone et al., 2008). Although the specificity of the antibodies used in this study needs to be further tested, these data suggest that nonvascular plants could have evolved the ability to transport and accumulate polymerized phenolic compounds in the wall. These wall-bound phenolics could possibly exist as polyphenols that resemble genuine lignin to some extent. The detection of lignin-related epitopes in Nitella, a genus representing the closest lineage sister to land plants, further raised an interesting possibility that a primitive form of phenylpropanoid metabolism could have emerged before land colonization by plants. Based on these earlier findings, it was even more surprising when Martone et al. (2009) reported the identification of lignin in the red alga Calliarthron cheilosporioides, the ancestor of which diverged from all the other green plants over 1.3 billion yr ago. Using the derivatization followed by reductive cleavage (DFRC) method, a lignin analysis technique specific to the β-O-4 linkages found in lignin (Lu & Ralph, 1997), these authors showed that Calliarthron lignin contains H, G and S units, a composition typically found in angiosperms (Martone et al., 2009). The authors explained the independent occurrence of the angiosperm-type lignin in red algae by convergent evolution (Martone et al., 2009). This hypothesis requires Calliarthron to have independently evolved not only the core monolignol biosynthetic pathway necessary for the synthesis of H and G lignin, but also the additional branch for S lignin synthesis that is only present in limited land plant lineages. It will be of great importance to interrogate the genome of this species to determine definitively whether it encodes the enzymes necessary for monolignol synthesis or whether the presence of low levels of lignin in this species (only c. 1% of the amount found in terrestrial plants) has another explanation.
It has been suggested that the UV autofluorescent lignin-like material surrounding the zygotes of several charophytic algae species, such as Coleochaete, is sporopollenin, a biopolymer that constitutes the outer wall of noneuphyllophyte spores and euphyllophyte pollen (Delwiche et al., 1989; Kroken et al., 1996). Sporopollenin is a constituent of all known land plants, and is polymerized from hydroxylated fatty acids and phenolics (Ahlers et al., 2003). Its occurrence predated lignin (Morant et al., 2007; Dobritsa et al., 2009). The presence of sporopollenin in freshwater algae suggests that the initial selective advantage for phenylpropanoid metabolism could well have been that it provided algal zygotes with a UV-protecting outer layer that ultimately facilitated their movement onto the land (Morant et al., 2007). The biochemical apparatus that initially functioned in the transport of sporopollenin phenolic monomers to the cell wall and the enzymes involved in subsequent polymerization processes may have been the ancestral forms of those that were later recruited and optimized for lignification.
Another group of phenylpropanoids, lignans, are often compared with, and even confused with, lignin. Lignans are dimers derived from hydroxycinnamyl alcohols, allylphenols or hydroxycinnamic acids, which are oxidatively coupled from radicals produced by the actions of laccases or peroxidases (Davin & Lewis, 2003). Examination of the phylogenetic distributions of lignans across land plants reveals an interesting pattern. Whereas hydroxycinnamyl alcohols are widely involved in the biosynthesis of various classes of lignans in tracheophytes, the lignans reported from bryophytes to date are only derived from hydroxycinnamic acids (Tazaki et al., 1995; Cullmann et al., 1999; Scher et al., 2003; Umezawa, 2003). This observation is correlated with the occurrence of lignin biosynthesis in tracheophytes, which requires a major redirection of metabolic flux towards the reduction of hydroxycinnamic acids and aldehydes into their corresponding alcohols. The presence of CCR and CAD orthologs in the Physcomitrella genome suggests that bryophytes may contain the biochemical capacity to synthesize hydroxycinnamyl alcohols; however, the major offshoots from phenylpropanoid metabolism remain at the level of the acids and are directed to the biosynthesis of flavonoids and lignans. Although the evolution of lignan biosynthesis via radical coupling of phenylpropanoids in bryophytes may represent early steps on the evolutionary path towards lignification, several fundamental differences exist between lignan and lignin biosynthesis. First, in comparison with lignan linkages that are solely derived from monomer–monomer coupling reactions, lignin linkages are primarily formed through monomer–oligomer or oligomer–oligomer coupling reactions (Ralph et al., 2004). Second, the majority of the linkages found in lignans are based on β-β′ with no involvement of the 4-hydroxyl of monomers in linkage formation (Fig. 4a). By contrast, lignin contains a much richer variety of interunit linkages, including β-O-4′, β-5′, 5-5′, 4-O-5′, β-β′ and β-1′ (Fig. 4b). The prevalent β-β′ linkages found in lignans only account for a minor proportion of the total linkages in lignin (Ralph et al., 2004). Third, it has been shown that the stereospecificity of certain lignans, for example (+)-pinoresinol, is controlled by dirigent proteins during coupling reactions (Davin et al., 1997). By contrast, the various linkages in the lignin polymer are formed via a combinatorial process, controlled only by the chemical properties and concentrations of the reactants and the conditions in the surrounding matrix (Ralph et al., 2004). To date, no evidence has been found that suggests that any agent, such as dirigent proteins, is involved in lignin polymerization. Fourth, lignans and their glucosides are primarily stored in the vacuole, indicating that lignans could be synthesized either in that compartment or in the cytosol, and then transported into the vacuole by specific transporters localized at the vacuole membrane. Alternatively, the dimerization and subsequent glycosylation could take place on the vacuole membrane or inside the vacuole. During lignification, monolignols need to be transported to the apoplast, where they encounter the enzymes that lead to their polymerization.
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