Regulation of Lignin Biosynthesis and Its Role in Growth-Defense Tradeoffs

1Lignin is a heterogeneous polymer of monolignols and is polymerized at the surface of the cell walls. The three essential monolignols of plant lignin are p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Lignin is important for terrestrial plants by providing structural support for the upward growth of plants and enabling the long-distance water transportation, which are essential for the evolutionary adaptation of plants from the aquatic to terrestrial environment. Lignin also provides physical and chemical protection for plants against pathogen invasion.

2The biosynthesis process of lignin has attracted much attention as lignin is the major contributor to the recalcitrance of biomass feedstocks, dramatically increasing the cost of biomass deconstruction and biofuels production. To increase the digestibility of biomass, genetic engineering of lignin has been an active research area in the past decade and efforts have been guided toward reducing the cost of biofuel production. On the other hand, the lignin residues after biomass saccharification can be used to produce biodegradable plastic and lignin-derived value-added solvents and chemicals.

3Given the extensive biological and industrial importance of lignin, the understanding of lignin biosynthesis in plants is beneficial for both agricultural and industrial purposes. In this review, we summarize the current understanding of the lignin biosynthesis process and its transcriptional regulation. We then offer a working hypothesis on the transcriptional coordination of energy flux among plant growth, defense, and lignin biosynthesis.

4In plants, there are two major steps to produce lignin: monolignol biosynthesis and monolignol polymerization via free radical coupling. Enzymes catalyzing monolignol biosynthesis have been well defined in the model plant Arabidopsis. Genetic modulation of these enzymes has been shown to dramatically alter the accumulation and/or composition of lignin.

5In Arabidopsis, monolignols are synthesized from phenylalanine via the phenylpropanoid pathway. Therefore, most key phenylpropanoid biosynthetic enzymes are also critical for lignin biosynthesis. A recent study of 221 independent transgenic Populus lines has demonstrated the importance of phenylpropanoid biosynthetic enzymes for lignin biosynthesis in Populus. The phenylpropanoid pathway is essential in plants, providing precursors for numerous secondary metabolites, including monolignols, flavonoids, and coumarins. In the phenylpropanoid pathway, three enzymes: phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL), catalyze the first three steps in sequence to provide precursors for all of the downstream metabolites. In Arabidopsis and Populus, genetic inhibition of PAL, C4H, and 4CL genes has been shown to significantly decrease lignin content. In addition to these three enzymes, other phenylpropanoid biosynthetic enzymes, such as quinate/shikimate p-hydroxycinnamoyltransferase (HCT), p-coumaroylshikimate 3′-hydroxylase (C3′H), caffeoyl shikimate esterase (CSE), caffeic acid O-methyltransferase (COMT), and caffeoyl-CoA O-methyltransferase (CCoAOMT), which work downstream of 4CL, are also indispensable for normal lignin biosynthesis (Figure 1). In Arabidopsis, the down-regulation of HCT and C3′H has been shown to induce the enrichment of H units in lignin polymers. Similarly, the down-regulation of HCT gene families dramatically increased the accumulation of H units in Populus. In Arabidopsis, Populus, and Medicago truncatula, CSE mutants were shown to deposit less lignin. COMT has been found to be critical for the synthesis of S units. The Arabidopsis null allele of CCoAOMT (ccomt1) also exhibits reduced lignin content, as well as reduced G units. It is notable that these early steps of monolignol biosynthesis may have variations in monocots. A systematic study of lignin biosynthesis in switchgrass has demonstrated that the conversion of p-coumaroyl CoA to caffeoyl CoA in switchgrass may have an alternative route, which involves the formation of quinate esters catalyzed by HCT-like enzymes. In addition, the direct involvement of CCoAOMT in lignin biosynthesis was questioned because the knockdown of CCoAOMT genes (CCoAOMT1 and CCoAOMT2) did not change lignin content in switchgrass.

6In addition to enzymes involved in the general phenylpropanoid pathway, enzymes specific for lignin biosynthesis have been identified and characterized, including cinnamoyl-CoA reductase (CCR), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD). These lignin biosynthesis-specific enzymes act downstream of phenylpropanoid biosynthetic enzymes and catalyze the biosynthesis of specific monolignols (Figure 1). The down-regulation of F5H can result in significant increase of G units in lignin. Arabidopsis mutants of CAD exhibit reduced lignin content, but increased accumulation of G and S units. Besides CCR, F5H, and CAD, several lignin biosynthesis-specific steps are catalyzed by COMT, including the conversions of caffeoyl aldehyde to coniferaldehyde, 5-hydroxyl coniferaldehyde to sinapaldehyde, and 5-hydroxy conifer alcohol to sinapyl alcohol (Figure 1).

7After biosynthesis, monolignols are polymerized to form lignin. It has been suggested that peroxidases and laccases are key enzymes catalyzing monolignol polymerization though experimental evidence is incomplete. Genetic studies of genes encoding peroxidase and laccase in Arabidopsis have illustrated the close relationship between these enzymes and lignin accumulation in secondary cell walls. found that double mutants of Arabidopsis peroxidases (atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71) have 11–25% reduction in lignin content. Additionally, another peroxidase PRX17 has been found to be critical for lignin accumulation in a recent genetic study in Arabidopsis. Similarly, laccases have been shown to affect lignin accumulation. A 20–40% reduction in lignin content has been observed in knockout mutants of two laccase genes (LAC4 and LAC17) in Arabidopsis. Moreover, the triple mutant of LAC4, LAC11, and LAC17 completely lost lignin deposition in roots.

8A recent study of the model grass Brachypodium distachyon defined a monolignol biosynthesis process with fewer steps. In this process, monolignols are produced from tyrosine, which is directly converted into p-coumarate by a grass bifunctional phenylalanine and tyrosine ammonia-lyase (PTAL). Consequently, this tyrosine shortcut of monolignol biosynthesis in grasses does not contain the steps catalyzed by C4H (Figure 1). With fewer steps, the tyrosine shortcut pathway is energetically more efficient than the canonical lignin biosynthesis pathway. The tyrosine shortcut pathway is capable of guiding carbon and electrons into the biosynthesis of lignin via skipping the production of cinnamate, which is the essential precursor of benzenoid volatiles and salicylic acid. Therefore, the discovery of the tyrosine shortcut pathway provides an alternative approach to optimize the energy investment of lignin production. Although PTAL activity has been detected in some non-grass species, such as tobacco callus and castor bean endosperm, the involvement of tyrosine shortcut pathway in lignin biosynthesis of non-grass species remains unstudied.

9In addition to the H, G, and S units of lignin, a natural lignin (called C-lignin) solely containing an unusual Catechyl (C) unit was found in seed coats of vanilla orchid and most Cactoidae genera. In C-lignin, caffeyl alcohols are linearly connected by benzodioxane bonds via radical coupling reactions putatively catalyzed by a peroxidase (Figure 1; ). Although the detailed caffeyl alcohol biosynthesis and polymerization processes remain unclear, the study in dicot plants demonstrated that the biosynthesis of C-lignin and conventional lignin may be controlled differently. C-lignin and conventional lignin were found to be synthesized in a spatially and/or temporally separated manner in seed coats of several dicot plants. Without side chains, the linear lignin has less cross-linking with cellulose and hemicellulose, and is capable of enhancing the digestibility of biomass. Further understanding of the mechanism of C-lignin biosynthesis will provide an alternative bioengineering approach to generate better biomass for biofuel production. In addition, the linear C-lignin may be an ideal natural material to replace fossil-fuel based materials for the production of carbon fibers. Compared with fossil-fuel-based feedstocks, the renewable and degradable features of C-lignin make it more environmentally attractive.

10A key finding of lignin biosynthesis studies is that AC elements widely exist in the promoters of major phenylpropanoid and lignin biosynthetic genes. AC elements are DNA motifs rich in adenosine and cytosine. Such significant enrichment of AC elements suggests that the phenylpropanoid and lignin biosynthesis may be under the control of specific types of transcription factors. In the past two decades, key transcription factors regulating the carbon flux into phenylpropanoid and lignin biosynthesis pathways have been identified and a hierarchical transcriptional network connecting these transcription factors has been established (; Figure 2).

11Among transcription factors regulating phenylpropanoid and lignin biosynthesis, MYB46 and its close homolog MYB83 are well-studied in various plant species. In Arabidopsis, MYB46 and MYB83 have been found to activate the expression of PAL1, C4H, 4CL1, C3′H1, HCT, CCoAOMT, CCR1, F5H1, CAD6 genes via binding to AC elements in the promoters of these genes. The pine and Eucalyptus MYB46 homologs (PtMYB4 and EgMYB2) were also found to be functional in the regulation of lignin biosynthesis. Moreover, four MYB46 homologs in Populus (PtrMYB002, PtrMYB003, PtrMYB020, and PtrMYB021) were capable of triggering ectopic lignin deposition in Arabidopsis and Populus plants. In addition to lignin, the biosynthesis of other secondary cell wall components, cellulose and xylan, can also be activated by MYB46. Downstream of MYB46/MYB83, multiple MYB transcription factors have been identified as specific regulators of lignin biosynthesis in Arabidopsis, including MYB58, MYB63, MYB85, MYB4, MYB32, and MYB7. Among them, MYB58 and MYB63 are close homologs and specifically activate lignin biosynthesis via targeting AC elements. By inducing 4CL expression, MYB85 is also capable of activating monolignol biosynthesis. In contrast, MYB4, MYB32, and MYB7 were found to negatively regulate the expression of lignin biosynthetic genes.

12Upstream of MYB46/MYB83, the NAC transcription factor SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING PROMOTING FACTOR 3 (SND1/NST3) and its close homologs NST1, NST2, VASCULAR-RELATED NAC DOMAIN6 (VND6), and VND7 regulate lignin biosynthesis in Arabidopsis. Similar to MYB46/MYB83, these NAC transcription factors also regulate the biosynthesis of cellulose and xylan. In Arabidopsis, the regulation of secondary cell wall biosynthesis by these NAC transcription factors is cell-type specific. For example, VND6 and VND7 are specifically expressed in xylem vessel cells and regulate secondary cell wall thickening. Knockdown of VND6 or VND7 using a dominant chimeric repressor specifically inhibits the formation of metaxylem and protoxylem vessels. In contrast, NST transcription factors were found to preferentially regulate the secondary cell wall biosynthesis of fiber cells [SND1/NST3 and NST1,], silique cells [SND1/NST3 and NST1,], and anther endothecium [NST1 and NST2]. However, the cell-type specificity seems to be absent in the woody plant Populus and monocots, suggesting distinct regulatory mechanisms may exist. In Populus, both VND and NST homologs were found to be expressed in vessels and fibers, though only homologs of VND transcription factors were found to be expressed in primary xylem vessels which have no secondary growth. In monocots, such as rice and maize, homologs of VND and NST transcription factors are named SECONDARY WALL-ASSOCIATED NAC (SWN) due to their regulatory roles in the secondary cell wall biosynthesis of vessel and fiber cells.

13Because of the essential regulatory roles during secondary cell wall formation, the hierarchical network comprised of NAC and MYB transcription factors is thought to be the major transcriptional regulatory mechanism of lignin and secondary cell wall biosynthesis. In this network, feed-forward loops are prevalent. For example, NAC transcription factors directly activate MYB46/MYB83. Meanwhile, NAC transcription factors and MYB46/MYB83 directly activate the downstream MYB58, MYB63, MYB85, and lignin biosynthetic genes (Figure 2). Similarly, MYB46/MYB83 directly activates MYB58, MYB63, and MYB85, and they together directly activate lignin biosynthetic genes (Figure 2). These feed-forward loops are probably to ensure the robust regulation of lignin biosynthesis. Besides feed-forward loops, the NAC-MYB network also contains feed-back regulations to fine-tune lignin biosynthesis (Figure 2). For example, SND1 and VND7 were found to directly target and activate the expression of themselves in Arabidopsis, representing positive feed-back regulation. A negative feed-back regulatory loop, in which SND1 is targeted and down-regulated by downstream MYB transcription factors including MYB4, MYB7, and MYB32, was also defined in Arabidopsis.

14Recent discoveries of new transcription factors in Arabidopsis, and other plant species, suggest that the regulatory network of lignin biosynthesis in plants extends beyond the established NAC-MYB network. Among these new transcription factors, several regulate members of the NAC-MYB network. A transcriptional repressor E2Fc has been reported to directly target VND6 and VND7 in a large-scale yeast one hybrid screening. The knockdown of E2Fc using RNAi induced ectopic lignin deposition in Arabidopsis roots, further demonstrating that E2Fc is a negative regulator of lignin biosynthesis. Moreover, Arabidopsis WRKY12 was found to repress lignin biosynthesis via direct repression of the NST2 gene. Knockdown of WRKY12 in Medicago was found to enhance lignin deposition in cell walls. The positive regulators of SND1 and its close homologs have also been identified in Arabidopsis. ASYMMETRIC LEAVES2-LIKE20/LATERAL ORGAN BOUNDARIES DOMAIN18 (ASL20/LBD18) and ASL19/LBD30 genes are activated by VND6 and VND7. Overexpression of ASL20/LBD18 and ASL19/LBD30, in turn, enhances the expression of VND7, suggesting a positive feedback loop amplifying VND7 expression. In addition, overexpression of MYB26 was found to increase lignin deposition and the expression of NST1 and NST2. The discovery of these positive and negative regulators demonstrates that the NAC-MYB network is regulated to spatially and/or temporally switch on or off lignin biosynthesis.

15Using genome-wide association studies (GWAS), a novel transcription factor was identified that regulates the expression of PtrMYB021 (homolog of MYB46) in Populus. This transcription factor (PtrEPSP-TF) is a Populus homolog of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, an enzyme catalyzing the shikimate pathway. Aside from the well-established enzyme function of EPSP synthase, PtrEPSP-TF can act as a transcriptional repressor. Genetic and molecular studies further revealed that PtrEPSP-TF activates PtrMYB021 expression and lignin biosynthesis by inhibiting the expression of a transcriptional repressor of PtrMYB021, which is named PtrhAT. Different from the ancestral EPSP synthase, the PtrEPSP-TF protein contains an additional helix-turn-helix (HTH) motif at its N-terminus, which is indispensable for the nuclear accumulation and transcriptional function of PtrEPSP-TF. However, the HTH motif is almost entirely missing in EPSP synthases of non-vascular plants, algae, and monocots. As opposed to herbaceous plants (e.g., Arabidopsis), woody perennial plants (e.g., Populus) have extensive secondary cell wall thickening over multiple growing seasons. The discovery of the additional regulatory loop of MYB46 in Populus supports the existence of woody plant-specific regulatory mechanisms in lignin biosynthesis. Moreover, the discovery of an activator (PtrEPSP-TF) and repressor (PtrhAT) of MYB46 provides alternative approaches to fine tune lignin biosynthesis.

16Crosstalk among biological processes is widespread. Genetic studies in Arabidopsis have demonstrated crosstalk among lignin biosynthesis, growth, and defense. Meanwhile, the complexity of the crosstalk is demonstrated by these studies. To date, the relationship of lignin content with growth and defense remains unpredictable due to the limited understanding of the underlying mechanisms.

17Lignin is thought to be indispensable for plant growth. The disruption of lignin biosynthesis by knocking down lignin biosynthetic genes, such as C4H and CCR1, was found to co-occur with the suppression of growth rate. However, recent studies suggest that lignin-growth crosstalk is more complex. A study of one C3′H mutant (ref8-1) illustrated that the growth suppression of ref8-1 might be due to the flavonoid hyperaccumulation, rather than the impaired lignin biosynthesis. On the other hand, increased lignin accumulation is also harmful for plant growth. The ectopic lignin deposition induced by the overexpression of NAC and MYB transcription factors, such as SND1, MYB46/83, and MYB58/63, was observed together with inhibited growth in Arabidopsis.

18The crosstalk between lignin biosynthesis and defense is even more complicated. Lignin is a well-known defense polymer, which has antimicrobial activity, forms a physical barrier to block pathogen invasion, and prevents the ingress or diffusion of toxins from pathogens. It has been widely observed that lignin deposition and lignin biosynthetic genes are induced during the attack of various pathogens. However, the genetic suppression of multiple lignin biosynthesis-related genes was shown to enhance pathogen resistance. For example, Arabidopsis MYB46 mutants (with impaired lignin biosynthesis) displayed enhanced disease resistance, which was thought to be caused by cell wall integrity damage-triggered high immunity level. The repression of HCT in Arabidopsis and Medicago was found to trigger the accumulation of defense hormone salicylic acid (SA) and the expression of PATHOGENESIS-RELATED (PR) genes.

19Studies on the Arabidopsis HCT mutant further illustrated the crosstalk among lignin biosynthesis, growth, and defense. HCT mutant has reduced lignin content, stunt growth, and enhanced defense. The reduced lignin content was found to trigger the accumulation of SA and the expression of PR genes, which subsequently enhance the pathogen resistance. Impaired lignin biosynthesis is generally thought to cause growth deficiency. However, in HCT mutant, the stunt growth was found to be unrelated with the reduced lignin content, but caused by the increased SA level.

20Transcriptional co-regulation has been identified as a mechanism balancing growth and defense. In a recent study, found that transcriptional networks downstream of jasmonic acid (JA) and phytochrome B signaling form a hub to coordinate growth and defense. The down-regulation of both JA and phytochrome signaling (jazQ phyB double mutant) has been shown to uncouple growth and defense, resulting in plants displaying more growth and more defense.

21Besides the co-regulation of growth and defense, evidence has accumulated that indicates the possible existence of transcriptional co-regulatory mechanisms coordinating growth and lignin biosynthesis, as well as defense and lignin biosynthesis. For example, the semi-dominant mutant (ref4-3) of one subunit of the transcriptional co-regulatory complex Mediator (MED5b) was found to exhibit the dwarfism phenotype, as well as repressed phenylpropanoid and lignin production. Disruption of MED5b is capable of rescuing both the growth and lignin biosynthesis deficiencies of the C3′H mutant, further demonstrating the involvement of transcriptional mechanisms in the co-regulation of growth and lignin biosynthesis. On the other hand, several transcription factors have been found to affect lignin biosynthesis, as well as defense responses. Overexpression of one Medicago WRKY transcription factor gene WRKY W109669 in tobacco was shown to enhance the accumulation of lignin and the transcription of PATHOGENESIS-RELATED 2 (PR2) gene, which is a defense gene induced in response to virus infection. A recent genetic study of a Gossypium barbadense ethylene response-related factor gene (GbERF1-like) also demonstrated the transcriptional co-regulation of defense and lignin biosynthesis. Overexpression of GbERF1-like in Arabidopsis significantly enhanced the transcription of phenylpropanoid/lignin biosynthetic genes (e.g., PAL3, C4H, C3′H, HCT, CCoAOMT1, CCR1), as well as pathogenesis-related genes including PR3, PR4, and PLANT DEFENSIN1.2 (PDF1.2). Moreover, the Arabidopsis myb46 mutants were found to be resistant to the necrotrophic pathogen Botrytis cinerea and to increase PR3 and PDF1.2 expression after Botrytis cinerea infection.

22More importantly, genetic studies in Arabidopsis have demonstrated the possibility of the transcriptional co-regulation of growth, defense, and lignin biosynthesis. In mutants of C4H and CCR1, which display significantly reduced lignin accumulation, many defense-responsive genes and many growth-related genes (i.e., auxin response genes) are down-regulated. A MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) was found to regulate the expression of miRNA156, which negatively regulates inflorescence development and positively regulates tolerance to recurring environmental stress. A recent study further identified the regulatory role of AGL15 in lignin biosynthesis. In a study by, AGL15 was found to directly repress PRX17 expression to regulate lignin formation.

23Collectively, genetic studies have illustrated the existence of the transcriptional co-regulation mechanism coordinating growth, defense, and lignin biosynthesis. It is generally recognized that changes in lignin biosynthesis affect the structure and integration of cell walls, which in turn affects growth and defense. However, little is known about the genetic factors co-regulating growth, defense, and lignin biosynthesis or factors bridging the transcriptional co-regulations of growth-lignin biosynthesis and defense-lignin biosynthesis. This is a much-needed research area for future studies.

24Growing in a dynamic environment, plants have evolved sophisticated strategies to determine the energy allowance between growth (to compete for light) and defense (to fight against pathogens). The competition for energy suggests that growth and defense of plants may have a negative relationship, which means the activation of defense processes negatively affect the growth and reproduction of plants. Such tradeoffs between growth and defense have been observed in many studies. Although the tradeoff between growth and defense is fundamental for plant survival in a changing environment, it is detrimental for plant yield.

25Lignin biosynthesis is traditionally thought as one indivisible part of plant growth and defense, to provide structural support, transport water, and act as physical barrier. However, discoveries of transcriptional mechanisms underlying the crosstalk of lignin biosynthesis, growth, and defense suggest the possibility to unlock lignin biosynthesis from growth and defense. On the other hand, lignin accounts for approximately 30% of the organic carbon in the biosphere. This large amount of energy invested in lignin and its precursors has the potential to compensate the costly expenditure of defense, which consequently would mitigate the tradeoff between growth and defense. Collectively, the genetic modification of the co-regulatory mechanism represents a potential strategy to overcome growth-defense tradeoffs.

26To date, the lignin biosynthesis process and its regulatory mechanism in the model plant Arabidopsis are well established. Although the lignin biosynthesis process has been well-studied and monolignol biosynthetic enzymes have been systematically analyzed in woody plants, such as Populus, the regulation of lignin biosynthesis remains poorly understood. The regulatory mechanism in a woody perennial plant is much more complex than that in Arabidopsis, due to the complex genome and long life-cycle. With the application of genome-wide approaches, such as GWAS, expression quantitative trait loci (eQTL), and expression quantitative trait nucleotide (eQTN) mapping, in plant studies, it is plausible to identify causal genes affecting complex traits including lignin biosynthesis. Together with efficient post-GWAS characterization and validation, species-specific mechanisms of lignin biosynthesis can be defined.

27The concept of a transcriptional hub to coordinate carbon and energy flux into growth, defense, and lignin biosynthesis is just emerging based on studies in Arabidopsis. A comprehensive understanding of this transcriptional hub will have significant impacts on the field of bioengineering. Current studies of NAC-MYB network are restricted in secondary cell wall biosynthesis, although RNA-seq analyses have illustrated that NAC and MYB transcription factors can induce broader gene expression changes. By identifying genome-wide targets of secondary cell wall-related NAC and MYB transcription factors using ChIP-seq, molecular mechanisms linking lignin biosynthesis, growth, and defense are expected to be discovered. In the woody plant Populus, although data is relatively limited, our recent study demonstrated the transcriptional co-regulation of lignin biosynthesis and defense. A defense-responsive WRKY transcription factor was found to regulate the expression of a Populus HCT gene (PtHCT2). With the identification and characterization of transcription factors with multiple functions in the regulation of growth, defense, and lignin biosynthesis, a transcriptional network needs to be established to unveil how growth, defense, and lignin biosynthesis are co-regulated. With this knowledge, the resource flux in plants can be fine-tuned depending on human demand, which will greatly reduce the cost of agricultural and forestry biofuels production.

28MX drafted the manuscript. JZ, TT, GT, J-GC, and WM revised the manuscript.

29The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.