Review

Effects of altered lignin biosynthesis on metabolism and plant stress

Biofuels (2013) 4(6), 635–650

Holly L Baxter1,2 & C Neal Stewart Jr*1,2 Modification of lignin in bioenergy feedstocks has become a common strategy to increase saccharification and biofuel yield. The lignin biosynthetic pathway in several plant species has been dissected and key enzymes have been manipulated in transgenic plants. Recent analyses of lignin-modified plants have shown that decreasing lignin biosynthesis can alter carbon flow within the phenylpropanoid pathway and indirectly affect the synthesis of other secondary metabolites, many of which can play important roles in plant–environment interactions. In addition, lignin modifications have also been shown to induce the expression of various stress response-related genes. Examining and understanding these indirect effects of lignin modification on stress-related processes are essential, since they could ultimately impact the performance of low-lignin bioenergy feedstocks under agronomic field conditions. Recent efforts to characterize such effects will be discussed in this review.

While lignocellulosic feedstocks represent a promising alternative approach for improving the accessibility of renewable and sustainable alternative to petroleum- cell wall sugars and reducing the need for pretreatment based fuels, high production costs associated with con- is through genetic engineering of the lignin biosynthetic version processes currently prevent them from being pathway. Reducing lignin content or modifying its com- economically viable for large-scale implementation position can be achieved by downregulating or over- [1] . The production of biofuels from lignocellulosic expressing genes involved in either lignin biosynthesis feedstocks requires the depolymerization of cell wall or its regulation [3–5]. carbohydrates into simple sugars that can be utilized Early studies from a decade or more ago that contrib- during fermentation. However, the desired cellulose uted to our current understanding of lignin biosynthesis microfibrils are surrounded by a matrix of lignin and and its manipulation focused on poplar (Populus spp.) to hemicellulose, which greatly inhibits their accessibility improve pulping performance [6,7], as well as forage spe- to hydrolytic enzymes [1,2] . Lignin is a phenolic polymer cies, such as alfalfa (Medicago sativa L.) and tall fescue that reinforces the secondaryAuthor cell wall, confers struc- (Festuca arundinacea Proof), for improving digestibility [8–10]. tural integrity to the plant, aids in water transport and A great deal of insight into lignin engineering has also also plays an important role in plant responses to vari- been achieved through studies with the model species ous environmental stresses. The presence of lignin in Arabidopsis thaliana [11] and tobacco (Nicotiana tabacum) plant cells has been identified as a major contributor to [12] . Because the lignin biosynthetic pathway appears to the resistance of converting cell walls into fuel precur- be highly conserved among plant species, many of the sors [3]. Expensive and energy-intensive thermochemi- genetic engineering strategies that have proven to be cal pretreatments are generally required to disrupt the successful in model species can also be applied to lig- lignin–polysaccharide barrier and allow better access nocellulosic feedstocks [4]. Of particular importance to of the cellulose to hydrolysis prior to fermentation. An biofuel-related studies, A. thaliana has been identified as

1Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 2BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA *Author for correspondence: Tel.: +1 865 974 7324; E-mail: [email protected]

future science group 10.4155/BFS.13.56 © 2013 Future Science Ltd ISSN 1759-7269 635 Review Baxter & Stewart Jr

Key terms a relevant model system for study- to pathogen infection, wounding and mechanical dam- Recalcitrance: The resistance of ing the effects of lignin modifica- age is an important defense mechanism in plants [35], lignocellulosic biomass to enzymatic tion on cell wall recalcitrance. Cell and it has long been presumed that a reduction in lignin breakdown into fermentable sugars. wall phenotypes in Arabidopsis have content would render plants more susceptible to such Phenylpropanoid pathway: Metabolic been translatable to commercial stresses. However, recent studies suggest that changes pathway responsible for the synthesis of dicot species such as M. sativa and in stress susceptibility in lignin-modified plants are not diverse classes of plant secondary spp. [13] . Currently, con- necessarily direct results of reducing lignin content, but metabolites that play roles in Populus developmental and stress-related siderable research has focused on can be influenced by indirect effects of lignin pathway processes. developing strategies for the genetic perturbations on stress-related metabolism and gene Transcription factors: Proteins that improvement of bioenergy feed- expression. Some reports with low-lignin mutant and regulate gene expression by binding to stock candidate species including transgenic plants indicate that the accumulation or sup- specific genes and promoting or switchgrass (Panicum virgatum L.), pression of certain secondary metabolites resulting from repressing their transcription. The over-expression of transcription factors sorghum (Sorghum bicolor), willow the alterations to phenylpropanoid metabolism can affect that downregulate the expression of (Salix spp.), Miscanthus, sugarcane how plants interact with stresses [34,36–38]. Additionally, lignin biosynthetic genes is an effective (Saccharum spp.) and poplar [14] . recent studies have revealed an increase in the expression method for reducing lignin content. Transgenic modifications are likely of stress-related genes in lignin-modified plants, which to be the most direct way of improv- some reports hypothesize to be triggered by the altered ing the quality of lignocellulosic biomass for cost-effec- structural organization of the cell wall [31,39,40]. Evaluation tive conversion into biofuels [15], and manipulation of of these plants under stress in controlled greenhouse or the lignin biosynthetic pathway has thus far proven laboratory environments have indicated that these modi- to be an effective route for increasing saccharification fications to secondary metabolism and/or stress-related efficiency and/or ethanol yield in greenhouse-grown gene expression can interfere with or enhance the ability switchgrass, field-grown poplar, and greenhouse- and of the plant to tolerate stresses. field-grown sugarcane [16–23] . Additionally, opportuni- Because of the influence of lignin modification on ties for genetic engineering of lignin in other potential plant stress, it is possible that altering lignin biosyn- biofuel species might also exist. Sorghum mutants with thesis in second-generation bioenergy feedstocks could low lignin content from brown mid-rib mutations are impact their performance in the field. In order to be com- known for having enhanced digestibility and sacchari- mercially competitive, bioenergy crops should possess a fication[24,25] . The lignin biosynthetic genes underlying number of desirable agronomic traits, including high bio- some of these mutations have been identified, and may mass productivity under minimal agronomic inputs, high represent potential targets for downregulation [26–28]. water and nutrient use efficiency, a relative resistance to The majority of studies with lignin-modified plants pathogen and insect pests, and tolerance to a wide range have primarily focused on the effects of lignin pathway of abiotic conditions [41–43]. While modifying lignin con- modifications on lignin content and composition, and tent through transgenic approaches provides a promising the associated changes in pulping performance, digest- route for improving saccharification efficiency and etha- ibility or recalcitrance to saccharification. More in-depth nol production, information regarding the impact of such characterization of lignin-modified plants at the tran- modifications on the ability of these feedstocks to toler- script, protein and metabolite level have revealed that ate biotic and abiotic stresses when grown under natural these modifications can have unexpected effects on other environmental conditions is limited. This is an important metabolic processes beyond lignin biosynthesis. Monoli- consideration since the commercial viability of low-lignin gnols, the lignin monomers, are synthesized through the bioenergy feedstocks will depend on their growth and lignin-specificAuthor branch of the phenylpropanoid metabolic Prooffitness in an agronomic setting, which could be enhanced pathway. Phenylpropanoid metabolism encompasses a or debilitated from pleiotropic effects of lignin modifica- network of metabolic pathways responsible for synthesiz- tion. This review will provide an overview of efforts to ing a wide variety of secondary metabolites that play vari- characterize the effects of lignin pathway modifications ous roles in developmental and stress-related processes on phenylpropanoid metabolism and plant stress-related (Figure 1) [29,30], and several studies show that suppress- processes, including what has been learned from studies ing lignin biosynthesis can alter metabolic channeling in model plants, as well as recent knowledge gained from within the phenylpropanoid pathway and differentially studies with species that have been identified as potential affect the synthesis of other secondary metabolites [31–34]. bioenergy feedstocks. Since most of these studies have In addition to altering phenylpropanoid metabolism, been performed in controlled greenhouse environments, modifications to lignin biosynthesis can also be associ- the possible implications of altered stress-related metabo- ated with changes in how plants respond to biotic and lism for the field performance of low-lignin bioenergy abiotic stresses. The accumulation of lignin in response feedstocks will also be explored in this review.

636 Biofuels (2013) 4(6) future science group Effects of altered lignin biosynthesis on phenylpropanoid metabolism & plant stress Review

L- PAL BA2H Salicylic acid Benzoic acid Cinnamate COMT F5H COMT Caffeic acid Ferulate 5-OH ferulate Sinapate C4H C3H P-coumarate Lignin

4CL CAD CHI/CHR/CHS CCR 4-coumaroyl-CoA 3 malonyl-CoA IFS1 IFS2 CHS CHI FS1, FS2 5’OH eriodictyol F3’5’H Naringenin F3’H Eriodictyol IOMT Phytoalexins IFR1 F3H F3H F3H FLS IFR2 Dihydromyricetin F3’5’H Dihydrokaempferol F3’H Dihydroquercetin

Isoflavanones DFR DFRDFR Pterocarpan LAR Leucodelphinidin Leucopelargonidin Glyceollin II LDOX ANS LDOX ANS LDOXANS ANR Pelargonidin -3-ols UFGT UFGT epicatechin UFGT Condensing Delphinidin-3 glycoside Pelargonidin-3-glycoside Cyanidin-3-glycoside enzyme Tannins

Figure 1. The phenylpropanoid metabolic pathway. 4CL: 4-coumarate:CoA ligase; ANR: reductase; ANS: Anthocyanidin synthase (also called LDOX: dioxygenase); BA2H: Benzoic acid 2-hydroxylase; C3H: P-coumarate 3 hydroxylase; C4H: Cinnamate 4-hydroxylase; CAD: Cynnamyl alcohol dehydrogenase; CCR: Cynnamoyl CoA reductase; CHI: Chalcone isomerase; CHR: Chalcone reductase; CHS: ; COMT: Caffeic O-methyltransferase; DFR: Dihydroflavonol-4-reductase; F3’H: 3’-hydroxylase; F3’,5’H: Flavonoid 3’,5’-hydroxylase; F3H: 3-hydroxylase; F5H: Ferulic acid 5-hydroxylase; FLS: Flavonol synthase; FS: Flavone synthase; IFR: reductase; IFS: Isoflavone synthase; IOMT: Isoflavone O-methyltransferase; LAR: Leucoanthocyanidin reductase; PAL: Phenylalanine ammonia-lyase; UFGT: UDP-flavonoid glucosyltransferase. Reproduced from [30].

Alterations to phenylpropanoid metabolism abiotic stress. Initiation of the phenylpropanoid pathway Phenylpropanoid metabolismAuthor in plants is an intricate net- begins with theProof conversion of phenylalanine, an amino work of pathways responsible for the synthesis of a broad acid product of the shikimate pathway, to cinnamic acid range of secondary metabolites including , iso- via phenylalanine ammonia-lyase (Figure 1; [30]). The next flavonoids, sinapate esters and lignin. Intermediate and two steps are catalyzed by cinnamate 4-hydroxylase and end products of phenylpropanoid metabolism function 4-coumaroyl CoA ligase to produce p-coumaroyl CoA, as antimicrobial and antiherbivory compounds, antioxi- a metabolite that serves as a branch point from which dants, pigments and UV-protectants [29,44]. The com- other metabolic pathways in the phenylpropanoid net- pounds that specifically aid in plant responses to stress work diverge [44]. The enzymes and enzyme families that range from simple precursor metabolites such as hydroxy- control metabolic channeling among the major classes of cinnamic acids, to more complex compounds such as fla- phenylpropanoid products have been discussed in reviews vonoids, and stilbenes[45] . Phenylpropanoid [44,46], as well as the transcription factors and signaling metabolic pathways can be developmentally induced in networks that regulate gene expression among the various specific tissues, or activated in response to biotic and secondary metabolic pathways [47,48].

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Key term As the second most abundant branches by catalyzing the conversion of p-coumaroyl- RNAi-mediated gene silencing: RNAi natural polymer after cellulose, CoA into p-coumaroyl shikimate [56]. P-coumaroyl-CoA methods are used to block the lignin is a major end product of is a metabolite located at a branching point in the phenyl- translation of a specific target gene into phenylpropanoid metabolism. The propanoid pathway, where the monolignol and flavonoid its protein product, thus preventing it lignin-specific branch of the phen- biosynthetic pathways diverge. If catalyzed by HCT, from becoming functional. In lignin genetic engineering, this approach can ylpropanoid pathway is responsible p-coumaroyl-CoA is converted into shikimate and qui- be used to reduce the expression levels for the synthesis of p-coumaryl, nate esters, and carbon is directed toward the synthesis of lignin biosynthetic genes. coniferyl and sinapyl alcohol mono- of monolignols [56,57]. Alternatively, p-coumaroyl-CoA lignols. These monolignols serve as can also serve as a substrate for chalcone synthase (CHS), precursors for the production of p-hydroxyphenyl, guaia- an enzyme that catalyzes the initial step toward flavo- cyl and syringyl monomeric lignin subunits, respectively, noid biosynthesis. If catalyzed by CHS, the metabolic which undergo polymerization by peroxidases to form flux will instead be directed toward the production of the lignin polymer [49]. More recently, the discovery of a flavonoids, including flavonols, anthocyanins and tan- cachetyl lignin polymer (C-lignin), comprised solely of nins [58,59]. Thus, competition between HCT and CHS caffeyl alcohol monomers, has been observed in the seed for the same substrate makes these two enzymes highly coats of some species [50]. While many of the enzymes influential in controlling the metabolic flux toward either that participate in lignin biosynthesis can independently monolignol or flavonoid biosynthesis. utilize multiple compounds as substrates, mixed sub- Lignin downregulation by RNAi-mediated gene strate reactions reveal strong enzyme preferences for spe- silencing of HCT in A. thaliana resulted in a hyper- cific substrates and a high degree of competitive inter- accumulation of flavonoid products [32]. The observed activity among enzymes for particular substrates [51–53]. increase in flavonoid biosynthesis was suggested to be This gives rise to a main metabolic stream toward the a consequence of the metabolic flux being redirected production of monolignols, with each substrate being away from the lignin pathway in the absence of HCT, primarily catalyzed by a specific enzyme (Figure 2) [4,54]. and into the flavonoid pathway through CHS activ- Characterization of mutant and transgenic plants with ity. High performance liquid chromatography profiling altered lignin biosynthesis has helped elucidate the revealed that flavonoid products, most notably flavonols influence of individual enzymes on lignin content and and anthocyanins, accumulated in higher amounts in composition [49,55]. the HCT-downregulated plants compared with control While the effects of altered lignin biosynthesis on total plants. HCT-downregulated plants also exhibited a dark lignin content and lignin polymer composition have been purple coloration of the leaves relative to the controls studied in great detail, the consequences of lignin path- as a result of the accumulation. Similarly, way perturbations on other metabolic processes are not downregulation of HCT in alfalfa (M. sativa L.) also as well understood. Recent transcriptomic, proteomic resulted in an increase in flavonoid biosynthesis and an and metabolomic profiling of such plants have helped accumulation of anthocyanins, which were suggested reveal some of these broader effects. Several studies have to be a result of metabolic spillover from suppressed indicated that downregulating single or multiple lignin lignin biosynthesis into the flavonoid pathway[31] . In pathway genes can alter the metabolic flux through the addition to evidence supporting a metabolic spillover phenylpropanoid pathway, and differentially affect the into flavonoid metabolism, the expression of several biosynthesis of other secondary compounds (Table 1). flavonoid biosynthetic pathway genes were induced in Depending on the lignin biosynthetic gene(s) being sup- the HCT-silenced plants; the activation of these genes pressed, plants can undergo various metabolic changes could also be contributing to the observed flavonoid in responseAuthor to the reduced carbon flow into lignin bio - Proofaccumulation. A significant increase in coumaric acid synthesis. In the next sections, the known biochemical was also observed in HCT-silenced plants; researchers effects of altering the expression of characterized lignin hypothesized that the accumulating HCT substrate, biosynthetic genes will be examined. p-coumaroyl-CoA, may either undergo hydrolysis into coumaric acid or be redirected into the flavonoid ƒƒ Hydroxycinnamoyl CoA: shikimate biosynthetic pathway. hydroxycinnamoyl transferase Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl ƒƒ P-coumarate 3-hydroxylase transferase (HCT) is a member of a large gene family After being synthesized by HCT, p-coumaroyl shiki- encoding for acyltransferases, a group of enzymes that mate is converted into caffeoyl shikimate by p-couma- catalyze the acylation of secondary metabolites. HCT cat- rate 3-hydroxylase (C3H) [60,61]. Downregulation of alyzes an early step in the phenylpropanoid pathway and C3H in hybrid poplar (Populus grandidentata × Populus initiates the carbon flux toward the monolignol-specific alba) resulted in the accumulation of soluble secondary

638 Biofuels (2013) 4(6) future science group Effects of altered lignin biosynthesis on phenylpropanoid metabolism & plant stress Review

H N O 2 O O PAL C4H HO OH OH OH

p-coumaric acid phenylalanine cinnamic acid 4CL O O HCT HO O HO SCoA Shikimate p-coumaroyl shikimic acid p-coumaroyl-CoA C3H O

HO O Shikimate HO caffeoyl shikimic acid CCR O HCT CCoAOMT O HO SCoA HO SCoA MeO HO feruloyl-CoA caffeoyl-CoA

CCR O MeO O O HO O F5H COMT HO H HO H HO H HO H p-coumaraldehyde MeO MeO MeO sinapalaldehyde coniferaldehyde 5-hydroxy- CAD coniferaldehyde CAD CAD

CH OH 2 HO MeO HO CH OH COMT CH OH CH OH F5H 2 2 2 HO HO p-coumaroyl HO alcohol MeO MeO coniferyl MeO alcohol 5-hydroxy- sinapyl alcohol PER/LAC coniferyl alcohol PER/LAC PER/LAC H-lignin G-lignin S-lignin

Figure 2. One of the current views of the lignin biosynthetic pathway. The enzymes involved in the pathway are: 4CL: 4-coumarate- CoA ligase; C3H: Coumarate 3-hydroxylase; C4H: Cinnamate 4-hydroxylase; CAD: Cinnamyl alcohol dehydrogenase; CCoAOMT: Caffeoyl CoA 3-O-methyltransferase;Author CCR: Cinnamoyl-CoA reductase; COMT: Caffeic Proof acid 3-O-methyltransferase; F5H: Ferulate 5-hydroxylase; HCT: Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase; LAC: Laccase; PAL: Phenylalanine ammonia lyase; PER: Peroxidase. Reproduced with permission of the copyright owner from [4]. Copyright © 2009 by the Society for In Vitro Biology, formerly the Tissue Culture Association. metabolites, the majority of which were p-coumaric prevent a potentially toxic accumulation of this substrate acid-derived phenylglucosides, including O-p-coumar- by allowing it to be mobilized to the phloem [33]. oyl-b-D-glucoside and populoside, grandidentatin and trichocarposide. The researchers hypothesized that the ƒƒ Caffeoyl-CoA O-methyltransferase decreased C3H activity could cause p-coumarate to Caffeoyl-CoA O-methyltransferase (CCoAOMT) be diverted into ester-linked glucosides, which would methylates caffeoyl-CoA to feruloyl-CoA, as well

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Table 1. Summary of the effects of altered lignin biosynthesis in transgenic or mutant plants on the synthesis of phenylpropanoid-derived secondary metabolites. Gene Type of modification Plant Effect on phenylpropanoid metabolism Ref. HCT Downregulation Arabidopsis thaliana ↑ flavonoids [32] Downregulation Alfalfa ↑ flavonoids [31] ↑ coumaric acid [31] C3H Downregulation Poplar ↑ phenolic glucosides [33] CCoAOMT Downregulation Poplar ↑ phenolic glucosides [64] Downregulation Alfalfa ↑ caffeoyl glucosides [65] CCR Mutant A. thaliana ↑ feruloyl malate [69] ↓ sinapoyl malate [69] Downregulation Poplar ↑ ferulic acid [40] ↑ sinapic acid [40] Downregulation Tobacco ↑ ferulic acid [39,71] ↑ sinapic acid [39,71] COMT Mutant A. thaliana ↓ sinapoyl malate [36,73] ↑ hydroxyferuloyl malate [36,73] Downregulation Switchgrass ↑ ferulic acid [74] ↑ 5-hydroxyferulic acid [74] ↑ vanillin [74] ↑ 5-hydroxyconiferaldehyde [74] ↑ iso-sinapyl alcohol [74] CAD Downregulation Switchgrass ↑ chlorogenic acid [17] Downregulation Flax ↑ ferulic acid [75] ↑ p-coumaric acid [75] Downregulation Tobacco ↑ syringic acid [76] ↑ ferulic acid [76] ↑ p-coumaric acid [76] ↑ sinapic acid [76] ZmMYB42 Overexpression A. thaliana ↓ flavonoids [38] transcription factor ↓ sinapoyl malate [38] ZmMYB31 Overexpression A. thaliana ↑ flavonoids [34] transcription factor ↓ sinapoyl malate [34] ↑: Accumulation; ↓: Suppression. as 5-hydroxyferuloyl-CoA to sinapoyl-CoA. This monolignol-specific branches of the lignin biosynthetic enzyme, along with caffeic acid 3-O-methyltransferase pathway [66–68]. For most species, the predominant (COMT), is involved in catalyzing the methylation of role of CCR in lignin biosynthesis is the reduction of the monolignol precursors [62,63]. Downregulation of feruloyl-CoA into coniferaldehyde [49]. In A. thaliana, CCoAOMT in poplar resulted in a redirection of the poplar and tobacco, suppressed CCR activity has been metabolic flux away from monolignol biosynthesis, and shown to affect the soluble phenolics composition, most into the pathway leading from caffeic acid to sinapic notably by increasing the accumulation of various feru- acid. The Authorincreased flux into this pathway resulted in Prooflate derivatives. In studies with A. thaliana mutants an increased synthesis and accumulation of phenolic deficient in CCR, a decreased flow of metabolites into acid glucosides in transgenic plants relative to controls, lignin biosynthesis was accompanied by a reduction including O3-b-D-glucopyranosyl-caffeic acid, O in sinapoyl malate biosynthesis, and an increased flow 4-b-D-glucopyranosyl-vanillic acid and O 4-b-D-glu- toward the synthesis of feruloyl malate, as well as an copyranosyl-sinapic acid [64]. Similarly, downregulation incorporation of ferulic acid into the lignin polymer of CCoAOMT in alfalfa resulted in an accumulation of [69]. Two possible mechanisms employed by plants to caffeoyl glucosides [65]. avoid the accumulation of monolignol precursors is by sequestering them into storage metabolites or incor- ƒƒ Cinnamoyl-CoA reductase porating them into the cell wall structure [70]. It was Cinnamoyl-CoA reductase (CCR) catalyzes cinnam- hypothesized that the CCR-deficient mutants avoid a oyl-CoA esters to their corresponding cinnamalde- potentially toxic buildup of feruloyl-CoA, the preferred hydes, which is considered to be the first step in the substrate of CCR, via one or both the following

640 Biofuels (2013) 4(6) future science group Effects of altered lignin biosynthesis on phenylpropanoid metabolism & plant stress Review redirection mechanisms: by hydrolyzing feruloyl-CoA malate in COMT mutants compared with controls; into ferulic acid, which is further processed into feru- this compound is a derivative of the COMT substrate, loyl malate; or by transferring feruloyl-CoA-derived 5-hydroxyconiferaldehyde [36]. In switchgrass, RNAi- ferulic acids to the cell wall and incorporating them mediated gene silencing of COMT resulted in an accu- into the lignin structure [69]. Transgenic poplar and mulation of phenolic acids and aldehydes, most notably tobacco plants with CCR-downregulation exhibited in ferulic acid, 5-hydroxyferulic acid and ferulic acid- modifications to the soluble phenolics composition glycoside conjugates, vanillin and 5-hydroxyconiferal- similar to those observed in A. thaliana CCR mutants dehyde. Additionally, a novel monolignol-like metabo- [39,40,71] . In poplar, CCR downregulation was associ- lite was observed in the transgenic plants, identified ated with an overall increase in the soluble phenolics as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol content, most notably in the levels of ferulic and sinapic (iso-sinapyl alcohol) [74]. acid esters, relative to control plants [40]. In addition, the concentration of ferulic acid in transgenic tissues ƒƒ Cinnamyl alcohol dehydrogenase increased relative to coniferaldehyde. It was suggested Cinnamyl alcohol dehydrogenase (CAD) catalyzes the that the decreased conversion of feruloyl-CoA into last step in the monolignol-specific pathway by reducing coniferaldehyde in CCR-downregulated plants caused cinnamaldehydes into their corresponding cinnamyl a diversion of the metabolic flow away from lignin bio- alcohols [68]. In switchgrass, downregulation of CAD synthesis, which was compensated for by an increased resulted in a 40–170% increase in the level of chloro- flow toward the synthesis of ferulic acids. Similar to that genic acid (caffeoyl quinic acid) in most of the trans- observed in the CCR mutants of A. thaliana, NMR and genic lines relative to the control [17] . Downregulation of thioacidolysis analyses of CCR-downregulated poplar CAD in flax Linum( usitatissimum L.) led to an increase indicated that some of the accumulating ferulic acid in total phenolic content, including a twofold increase was transported to the cell wall and cross-coupled with in ferulic acid and 30% increase in p-coumaric acid lignin. Transgenic tobacco with suppressed CCR activ- compared with the control plants [75]. In CAD-down- ity also exhibited an increased metabolic flux toward regulated tobacco, an increased metabolic flux toward the production of ferulic acid and sinapic acid relative to the production of soluble phenolics was also observed, control plants [39,71] . In one of these studies, an increase particularly in levels of syringic, ferulic, p-coumaric and in glycosylated and quinylated derivatives of feruloyl- sinapic acids [76]. Additionally, ferulic acid increased by CoA was observed in transgenic plants relative to their 58-fold in the transgenic plants relative to the wild- controls [39]. It was proposed that the accumulating type controls. Another study with CAD-downregulated feruloyl-CoA could be quinylated or redirected toward tobacco reported an increase in the accumulation of the synthesis of ferulic acid. The ferulic acid could then the two substrates of CAD, coniferaldehyde and sina- be partially converted to sinapic acid and detoxification paldehyde, in the transgenic plants relative to controls of these acids could occur via glycosylation. Increased [39]. Additionally, it was suggested that CAD down- transcript levels of 3-deoxy-D-arabino-heptulosonate- regulation may also result in a partial flux toward the 7-phosphate, a regulator of the carbon flux into shi- shikimate pathway, as indicated by increased transcript kimate metabolism, indicated that the metabolic flux levels of 3-deoxy-D-arabino-heptulosonate-7-phosphate may also be partially redirected toward the shikimate synthase [39]. pathway. ƒƒ Myeloblastosis transcription factors ƒƒ Caffeic acid 3-O-methyltransferase Rather than silencing the expression of individual lignin COMT is a member ofAuthor the O-methyltransferases, a biosynthetic Proofgenes, an alternative approach for decreas - family of enzymes that catalyze the methylation of ing lignin in cell walls is to manipulate the activity of various secondary metabolites in the phenylpropanoid transcription factors that act as repressors of lignin genes. pathway [72]. In lignin biosynthesis, COMT is primar- Myeloblastosis (MYB) proteins comprise a diverse fam- ily responsible for catalyzing the O-methylation of ily of transcription factors that play important regula- the 5-hydroxyl group of 5-hydroxyconiferaldehyde to tory roles in various plant functions. The R2R3-MYB produce sinapaldehyde [37]. A suppression of COMT family of transcription factors has been described as enzyme activity has been shown to affect the pheno- playing an important role in the regulation of lignin lics profiles of A. thaliana, tobacco and switchgrass biosynthetic genes [5]. The activities of R2R3-MYB [36,37,73,74]. A. thaliana mutants deficient in COMT transcription factors are generally not restricted to lignin activity accumulate significantly less sinapoyl malate biosynthesis, but rather have an influence on gene expres- relative to controls [36,73]. One of these reports also sion in multiple pathways within the phenylpropanoid indicated a significant increase in hydroxyferuloyl network, including phenolics, anthocyanins, lignins

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Key term and flavonols[77] . A. thaliana plants clearly understood. The reduction in lignin content may Pathogenesis-related proteins: Play a with over-expression of ZmMYB42, trigger signaling pathways that cause the activation of general role in plant response and an R2R3-MYB transcription fac- stress genes [34]. One hypothesis is that the activation adaptation to biotic stresses, typically tor derived from maize (Zea mays), of stress-related genes could be triggered by changes to induced by the host plant in response exhibited a suppression of several the structural organization of the secondary cell wall, to viral, bacterial or fungal infections. lignin biosynthetic genes and a cor- either by mimicking cell wall wounding or pathogen responding decrease in total lignin damage [39,40], or by facilitating the release of primary content relative to control plants [38]. Additionally, these cell wall polysaccharide components that act as elicitors plants were reported to have a 66% reduction in total of defense responses [31]. In addition, there is accumulat- phenolic content, mainly from a significant reduction ing evidence that altering cell wall integrity can induce in flavonols. Suppression of flavonoid biosynthesis was long-term modifications to gene expression, including attributed to the negative regulation of ZmMYB42 on the constant activation of defense-related genes [79,80]. two major flavonoid pathway genes. In addition to flavo- noid and lignin suppression, these plants also exhibited ƒƒ Biotic stress-related effects lower levels of sinapoyl malate compared with controls. Several phenolic compounds play essential roles in This reduction in sinapoyl malate biosynthesis could mediating plant–microbe interactions [81], and modi- be related to the negative regulation of ZmMYB42 on fications to the phenolic profile resulting from altered the expression of cinnamate 4-hydroxylase, ferulate phenylpropanoid metabolism have been shown to 5-hydroxylase and COMT, as a reduced expression of influence such interactions in mutant and transgenic these genes in mutant plants has been associated with a plants with suppressed lignin biosynthesis. Hydroxy- decrease in sinapoyl malate synthesis [73,78]. Additionally, feruloyl malate, a derivative of the COMT substrate, this reduction could also be explained by the negative accumulated in A. thaliana mutants deficient in COMT regulation of ZmMYB42 on aldehyde dehydrogenase, activity [36]. Elevated levels of this compound were the enzyme catalyzing the initial step toward sinapoyl correlated with an enhanced resistance to the downy malate biosynthesis [38]. A. thaliana plants expressing mildew-causing oomycete pathogen, Hyaloperonospora another R2R3-MYB transcription factor, ZmMYB31, arabidopsidis, and an inhibition of asexual sporulation also exhibited a significant decrease in lignin content was observed in the COMT mutant plants compared and a reduction in sinapoyl malate biosynthesis rela- with wild-type controls. In vitro assays showed that tive to control plants [34]. However, in contrast to the application of hydroxyferuloyl malate to the oomycete ZmMYB42 plants, there was an increase in flavonoid pathogen enhanced sexual reproduction and weakened biosynthesis in ZmMYB31 over-expressing plants rela- mycelium vigor [36]. Additionally, transgenic tobacco tive to controls. This increase in flavonoid biosynthesis plants with downregulated COMT activity developed was accompanied by an accumulation of anthocya- significantly smaller tumors than control plants when nins in transgenic tissues. The researchers concluded inoculated with Agrobacterium tumefaciens, a soil bac- that the over-expression of this transcription factor in terium that causes crown gall disease. These plants A. thaliana results in a reduced flux of carbon into lignin had lower Agrobacterium virulence (vir) gene-inducing and sinapoyl malate biosynthesis, and a redirection of activities and lower levels of soluble phenolics includ- the flux into flavonoid biosynthesis[34] . ing acetosyringone, a known elicitor of vir expression in Agrobacterium [37]. Effects of lignin modification on plant stress An upregulation of genes encoding for pathogenesis- As discussed above, suppressing single or multiple lig- related proteins and other proteins involved in plant nin biosyntheticAuthor steps can lead to the accumulation or Proofdefense toward pathogens has been observed in pop- suppression of other secondary metabolites. Many of lar, A. thaliana and alfalfa plants with modified lignin these compounds play important roles in plant–envi- biosynthesis. In CCR-downregulated poplar, increased ronment interactions, and altering their biosynthesis transcript levels of a U-box domain protein resembling can influence the susceptibility of plants to stresses. the CMPG1 protein triggered by fungal elicitors in Additionally, transcriptomic and proteomic studies Petroselinum crispum were observed relative to control have indicated that lignin modifications can trigger plants [40]. In A. thaliana plants over-expressing the an increase in the expression of stress response-related ZmMYB31 transcription factor, a proteome ana­lysis genes [31,34,39,40], and in one report there was a broad showed increased levels of osmotin relative to control upregulation of defense-related genes that was associ- plants [34]; osmotin is a pathogenesis-related (PR) pro- ated with an enhanced tolerance to biotic and abiotic tein that primarily functions as a plant defense pro- stresses [31] . The exact mechanisms triggering changes tein by providing resistance against a variety of fungal in gene expression in lignin-modified plants are not yet pathogens [82]. Downregulation of HCT in alfalfa

642 Biofuels (2013) 4(6) future science group Effects of altered lignin biosynthesis on phenylpropanoid metabolism & plant stress Review resulted in a constitutive activation of defense responses, the transgenic plants exhibited elevated photorespira- and a transcriptome ana­lysis of stem tissue revealed that tion. This elevated photorespiration was suggested to the expression of various PR transcripts was 5- to 56-fold be caused by an enhanced efficiency of photosystem II higher in transgenic plants than in controls [31] . It was (PSII), the first protein complex in the light-dependent suggested that this constitutive PR gene expression could reactions of photosynthesis. Gas-exchange analyses be related to the altered cell wall integrity. Fragments indicated that the increased efficiency of PSII in the of pectin, a galacturonic acid-rich component of plant transgenic plants was not associated with a change in primary cell walls, were shown to be more easily released photosynthetic CO2 assimilation. Therefore, researchers from the secondary cell wall in transgenic alfalfa com- hypothesized that the transgenic plants absorbed more pared with controls [31] . Previous studies have shown that light energy than could be used for photosynthesis, and galacturonides can trigger defense responses in several the observed increase in photorespiration could be a plant species [83–85]. In addition, these plants had higher protective mechanism against light-induced damage to levels of salicylic acid (SA) relative to controls, a biotic the photosynthetic apparatus [39]. An accumulation of defense-related phytohormone that has previously been H2O2 was also observed in the leaves of the CCR-down- shown to stimulate the upregulation of PR proteins in regulated plants, and lesions developed that resembled response to infection and enhance resistance to a wide those observed in catalase-deficient tobacco grown under range of pathogens [31,86]. Levels of SA were correlated high-light conditions. It was hypothesized that photo- with the amount of extractable pectin in the cell wall. respiration, specifically the elevated levels of photores-

SA can be synthesized either from cinnamate by phe- piratory H2O2, could be triggering the oxidative stress nylalanine ammonia-lyase activity, or from chorismate phenotype. An alternative hypothesis for the observed by isochorismate synthase activity, and the production oxidative stress phenotype suggested that the altered cell of SA through the isochorismate pathway has been wall structure could be actively inducing the expression observed in A. thaliana plants during defense response of oxidative stress-related genes as part of a wound-like [87]. Therefore, it was hypothesized that the continuous response. In support of this hypothesis, metabolomic leaching of these pectic elicitors from the cell wall as profiling showed an increase in the levels of feruloyl tyra- a result of reduced lignification could trigger the pro- mine in CCR-downregulated plants compared with the duction of SA through the isochorismate pathway, and controls [39]. In solanaceous plants, feruloyl tyramine high levels of SA could then stimulate the production has been shown to accumulate at the site of wounding of PR proteins responsible for the observed constitutive or pathogen attack in order to reinforce the cell wall [93]. defense response [88]. Since constitutive expression of PR Similar to that observed in tobacco, CCR-downregula- proteins in plants has previously been shown to confer tion in poplar appeared to trigger a wound-like stress resistance to a wide range of pathogens [89,90], trans- response and a corresponding increase in the expression genic alfalfa plants were exposed to alfalfa anthracnose of oxidative stress-related transcripts, also hypothesized (Colletotrichum trifolii) to test whether elevated levels of to be induced by a defective cell wall [40]. Additionally, PR transcripts would translate into enhanced pathogen these plants had significantly higher levels of transcripts resistance. Incidence and severity of necrotic legions encoding for a PSII reaction center protein and a gluta- were significantly lower in HCT-downregulated plants mine synthetase compared with controls, indicating a compared with the controls [31] . possible connection between oxidative stress and photo- respiration similar to the one described in CCR-down- ƒƒ Abiotic stress-related effects regulated tobacco [40]. In A. thaliana, proteome analyses Oxidative stress, photorespiration & UV sensitivity indicated that plants with over-expression of ZmMYB31 Lignin modifications Authorhave been shown to be associ- had increased Proof levels of enzymes involved in protecting ated with the activation of genes involved in oxidative against oxidative stresses compared with control plants. stress responses in tobacco, poplar and A. thaliana. In ZmMYB31 also downregulates the expression of aconi- tobacco, transgenic plants had elevated transcript levels tase, an enzyme that plays a role in mediating oxidative of metallothionein and glutathione S-transferase, two stress and associated cell death, and it has been reported enzymes involved in detoxifying oxidative stress metab- that mutant A. thaliana plants deficient in this enzyme olites, along with heat shock transcription factors that are more tolerant to oxidative stress [34,94]. promote transcription of genes involved in protection The suppression of lignin biosynthesis has also been against oxidative stress damage [39,91,92]. In addition to shown to affect the production of secondary metabo- oxidative stress-related transcripts, an increase in the lites that function as UV protectants. UV light-induced abundance of photorespiratory-related transcripts and stress can induce the synthesis of flavonoids in epider- metabolites were observed in transgenic plants relative mal tissues of plants, where they function as a protective to controls, and gas-exchange analyses confirmed that screen against UV damage and subsequent cell death

future science group www.future-science.com 643 Review Baxter & Stewart Jr

by absorbing light in the UV-B range [59,95]. Sinapoyl Future perspective malate and other sinapate esters have also been recog- The use of systems biology-based approaches that nized as important UV protectants in A. thaliana [95]. incorporate information at the transcript, protein and In CCR-deficient mutant plantlets ofA. thaliana, a sub- metabolite level are continually improving our under- stantial reduction in sinapoyl malate biosynthesis was standing of how plants respond to altering lignin. It is observed relative to control plants [69]. Exposure to UV important to examine and understand these broader light resulted in slower growth of the mutant plantlets effects of lignin modification since the commercial relative to controls, and their leaves were yellow, indi- viability of altered-lignin bioenergy feedstocks will cating an increased sensitivity to UV light. Similarly, depend not only on improved biofuel traits, but also ZmMYB42 over-expression in A. thaliana resulted in on their ability to perform at least as well as their non- the downregulation of several phenylpropanoid path- transgenic counterparts in the field. This performance way genes including those involved in sinapoyl malate includes optimal growth potential in the absence of and flavonoid biosynthesis[38] . As a result, transgenic stress, the ability to tolerate abiotic stresses and the abil- plants were more sensitive to UV light, as indicated by ity to resist pests. Vulnerability on any of these fronts a yellowing of the leaves relative to control plants [38]. would likely make modified feedstocks an undesirable A. thaliana plants with over-expression of another MYB risk to farmers. Therefore, field-based experiments to transcription factor, ZmMYB31, also had lower levels characterize lignin-modified feedstocks in these crucial of sinapoyl malate compared with controls [34]. How- areas are absolutely required. ever, unlike the ZmMYB42 plants, ZmMYB31 over- To date, most of the research with transgenic low- expression resulted in an increase in levels of UV-pro- lignin feedstocks grown in the field has been done in tecting flavonoids. Despite this increase in flavonoids, trees and forage crops for evaluating traits related to transgenic A. thaliana plants were still highly sensitive improved pulping or digestibility, respectively [6,98]. to UV radiation compared with controls, displaying More recently, saccharification efficiency has been an upward leaf curling phenotype resembling those assessed in field-grown bioenergy crop species[21,23,99] . observed in mutant plants deficient in UV-protectant Such studies have provided valuable insight into the compounds [34,95]. The researchers concluded that these impacts of altered lignin content on susceptibility to results, in addition to previous studies involving mutant biotic and abiotic stresses in a field setting. In a 4-year plants, suggest that sinapate esters may play a more field evaluation of poplar plants with reduced lignin important role than flavonoids in protectingA. thaliana content for improved pulping performance, there was against UV radiation [34]. no effect of lignin modification on the susceptibil- ity to insect herbivory or rust (Melampsora sp.) [6]. Water stress Similarly, field-grown COMT-downregulated sugar- In HCT-downregulated alfalfa, transcriptome profiling cane was not more susceptible to orange rust (Puccinia revealed an upregulation of abiotic stress-related tran- kuehnii) compared with nontransgenic controls [21] . scripts, the majority of which were heat and drought On the other hand, COMT-downregulation in field- stress-related [31] . Metabolomic analyses of transgenic grown perennial ryegrass plants led to an increased plants also showed elevated levels of abscisic acid, a phy- susceptibility to rust (Puccinia species) relative to tohormone that plays a central role in sensing water defi- control plants [100] . In addition to examining biotic ciency and activating the expression of drought stress- stress interactions in lignin-modified crops, abiotic related genes [31,96]. To investigate whether the observed stress-related effects have also been assessed in the changes in drought stress-related gene expression were field. A 2-year field study of low-lignin poplar with associatedAuthor with an enhanced tolerance to drought, Proofdownregulation of 4-coumaroyl CoA ligase found plants were deprived of water for a period of 9 days. that the transgenic events with the strongest reduction Transgenic plants exhibited fewer symptoms of drought in lignin content were associated with significantly stress than controls and were able to recover completely impaired xylem transport efficiency, resulting in fre- after 5 days of rehydration, while control plants showed quent shoot dieback despite being watered regularly extensive damage and did not survive. Leaf water poten- [101] . Interestingly, further studies with these plants tial measurements showed that water potential in the found that the impaired xylem water conductivity in transgenics decreased at a slower rate than the controls the lignin-downregulated lines was a result of accumu- throughout the experiment. Additionally, changes that lating phenolics and tyloses that were being deposited are commonly associated with adaptation to drought in xylem vessels and impeding water transport [102] . stress, including reduced leaf transpiration rate and In addition to impairment of water transport, the increased number of stomatal cells, were observed in reduction in lignin content in transgenic poplar lines transgenic leaf tissue [31,97]. was also associated with decreased wood strength and

644 Biofuels (2013) 4(6) future science group Effects of altered lignin biosynthesis on phenylpropanoid metabolism & plant stress Review stiffness, and an increase in the formation of tension previously been shown to deter the activity of fungi wood [103] . Taken together, the outcomes of these field and insect pests of poplar, and it was suggested that trials suggest that some types of lignin modifications the elevated levels could potentially enhance defense may be more preferable than others for preserving the responses of transgenic poplar toward biotic pests [33]. growth and viability of low-lignin crops in the field, A constitutive expression of PR genes has also been and this will likely vary from species to species. More shown to influence stress interactions, and was shown extensive field studies are necessary in order to gain to increase the tolerance of transgenic alfalfa plants to a better idea of the types of lignin modifications that anthracnose infection [31]; thus, a constitutive activa- will be best tolerated by plants in their environment. tion of biotic defense-related genes could potentially Particularly, the indirect effects of modified lignin bio- render plants more prepared for defending themselves synthesis on secondary metabolism and stress-related against pathogens in a field setting. gene expression should be further explored under field Modifications to the lignin biosynthetic pathway conditions, as these factors could significantly impact have also been shown to suppress or enhance levels the susceptibility of plants to stresses in an agronomic of secondary compounds that could influence plant setting. acclimation to abiotic stresses. Plants have been shown In the absence of pertinent field data, we can spec- to accumulate anthocyanins and/or flavonoids as a ulate on the potential agronomic effects of altered protective mechanism against salinity stress, UV-B stress-related metabolism in low-lignin bioenergy damage, cold temperatures and water stress [111,112]. feedstocks. As previously discussed, modified lignin As previously discussed, downregulation of lignin biosynthesis can lead to the differential accumulation pathway genes can result in an increased or decreased or suppression of various phenylpropanoid pathway synthesis of these compounds. Altered levels of these intermediates, some of which have been shown to compounds could influence plant sensitivity to abi- influence plant–microbial interactions under con- otic stresses in the field. A field study comparing salt- trolled environmental conditions. Therefore, it is tolerant and salt-susceptible clones of sugarcane found possible that this altered biochemistry in feedstocks that the accumulation of soluble phenolics, anthocya- could influence interactions between the plant and nins and flavones were threefold higher in the toler- bacterial or fungal pests in the field. In particular, ant clone, and these compounds were suggested to levels of lignin precursors such as p-coumaric acid, promote salinity tolerance by protecting cytoplasmic ferulic acid and sinapic acid were shown to accumulate structures and chloroplasts from the damages of salin- in plants with suppressed lignin biosynthesis; in vitro, ity stress [111]. In addition to their roles in salinity tol- these metabolites have been shown to have antibac- erance, levels of anthocyanins and some phenolic acids terial and antifungal properties [104] . In switchgrass, might play an important role in the acclimation of the downregulation of CAD led to an accumulation plants to cold stress, as they have been shown to accu- of chlorogenic acid [17]; higher levels of chlorogenic mulate in the leaf mesophyll cells in plants exposed acid have been shown to correlate with increased plant to low temperatures [113] . A. thaliana with suppressed resistance to bacterial pathogens and insect herbivory CCR activity, or with over-expression of MYB tran- [105,106]. In addition to influencing plant–microbial scription factors, were shown to be more sensitive to interactions, biochemical changes in the plant tis- UV damage due to the consequences of suppressed sue as a result of altered secondary metabolism could lignin on sinapoyl malate and/or flavonoid biosyn- influence the palatability of plants to insects. While thesis, as previously discussed. A recent study found the relationship between total lignin content and sus- that UV-B radiation, in addition to inhibiting plant ceptibility to insect herbivoryAuthor is unclear [107], a recent growth, can Proofalter plant tissue biochemistry and result field study comparing switchgrass lines with naturally in a significant reduction in cell wall digestibility and varying lignin levels suggested that other factors, such the enzymatic release of sugars from the biomass [114] . as the accumulation of compounds that interfere with Although this study was performed in a greenhouse, nutrition, may be more important than overall lignin the results could have significant implications for etha- content in explaining resistance to insect herbivory nol production from field-grown low-lignin feedstocks [108] . In maize, higher levels of p-coumaric and feru- that have been made more susceptible to the effects lic acids were strongly associated with an increased of UV radiation. resistance to infestations by maize weevil (Sitophi- Given this knowledge, it is conceivable that altera- lus zeamais) and stem borer (Sesamia nonagrioides), tions to phenylpropanoid metabolism and/or the respectively, in the field [109,110]. In transgenic poplar upregulation of stress-related genes resulting from with downregulation of C3H, an accumulation of phe- lignin modification could positively or negatively nolic glucosides was observed. These compounds have influence the agronomic performance of low-lignin

future science group www.future-science.com 645 Review Baxter & Stewart Jr

transgenic feedstocks. It is also important to note that Acknowledgements there can be significant metabolic costs associated with The authors would like to acknowledge the anonymous reviews for an increased allocation of energy into the production their helpful comments and suggestions. of defense-related secondary metabolites and consti- tutive expression of defense-related genes, such that Financial & competing interests disclosure a higher resistance to stresses through either these This work was supported by funding from the Southeastern Sun Grant mechanisms could be associated with stunted growth Center (TN, USA) , the US Department of Agriculture-funded Integrated phenotypes and consequent reductions in biomass Biomass and Supply Systems center (TN, USA) and the BioEnergy Science yield [115,116 ]. This potential trade-off between stress Center (TN, USA). The BioEnergy Science Center is a US Department resistance and biomass yield should be considered, of Energy Bioenergy Research Center supported by the Office of Biological since high biomass production is also an important and Environmental Research in the US Department of Energy Office of trait for bioenergy feedstocks. In conclusion, exten- Science (Washington, DC, USA). The authors have no other relevant sive field evaluations and an improved understanding affiliations or financial involvement with any organization or entity with of the relationship between lignin and stress-related a financial interest in or financial conflict with the subject matter or metabolism are necessary to ensure the sustainable materials discussed in the manuscript apart from those disclosed. growth of transgenic low-lignin bioenergy feedstocks No writing assistance was utilized in the production of this in agronomic field environments. manuscript.

Executive summary Background ƒƒ Genetic engineering strategies to reduce total lignin content or modify lignin composition can significantly improve saccharification efficiency and ethanol yield in lignocellulosic feedstocks. ƒƒ Recent studies have revealed that lignin pathway modifications can affect various stress-related processes. This review examines the mechanisms underlying these changes and the influences they can have on how plants interact with stresses. Altered phenylpropanoid metabolism ƒƒ The lignin biosynthetic pathway constitutes one of the major branches of the phenylpropanoid pathway. Phenylpropanoid metabolism is responsible for the synthesis of secondary metabolites that play developmental and stress-related roles in plants. ƒƒ Suppressing steps along the lignin biosynthetic pathway can alter carbon flow within the phenylpropanoid pathway, leading to the accumulation or suppression of other secondary metabolites. ƒƒ The biochemical changes that result from lignin modifications vary depending on which step(s) along the lignin biosynthetic pathway are suppressed. Effects on plant stress ƒƒ Alterations to phenylpropanoid metabolism resulting from lignin downregulation can affect the biosynthesis of secondary compounds that play important roles in plant–environment interactions. ƒƒ Lignin modifications can trigger the upregulation of stress-response related genes. ƒƒ Experiments in controlled environments demonstrate that changes to these processes can positively or negatively influence the susceptibility of lignin-modified plants to stresses. Future perspective ƒƒ In order to be commercially viable, lignocellulosic feedstocks must perform at least as well as their nontransgenic counterparts in an agronomic field setting. Examining and understanding the broader impacts of altered lignin biosynthesis on stress-related metabolism will help to identify what types of lignin modifications will be best tolerated by the plant without compromising growth and fitness.

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