Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 α-helices, 6 and 7 β-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.     

Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine

Two types of structurally distinct O-methyltransferases mediate the methylation of hydroxylated monomeric lignin precursors in angiosperms. Caffeate 3-O-methyltransferase (COMT; EC 2.1.1.68) methylates the free acids and caffeoyl CoA 3-O-methyltransferase (CCoAOMT; EC 2.1.1.104) methylates coenzyme A esters. Recently, we reported a novel hydroxycinnamic acid/hydroxycinnamoyl CoA ester O-methyltransferase (AEOMT) from loblolly pine differentiating xylem that was capable of methylating both acid and ester precursors with similar efficiency. In order to determine the possible existence and role of CCoAOMT in lignin biosynthesis in gymnosperms, a 1.3 kb CCoAOMT cDNA was isolated from loblolly pine that showed 79–82% amino acid sequence identity with many angiosperm CCoAOMTs. The recombinant CCoAOMT expressed in Escherichia coli exhibited a significant methylating activity with hydroxycinnamoyl CoA esters whereas activity with hydroxycinnamic acids was insignificant. Moreover, 3.2 times higher catalytic efficiency for methylating caffeoyl CoA over 5-hydroxyferuloyl CoA was observed which could serve as a driving force towards synthesis of guaiacyl lignin. The secondary xylem-specific expression of CCoAOMT was demonstrated using RNA blot analysis, western blot analysis, and O-methyltransferase enzyme assays. In addition, Southern blot analysis indicated that CCoAOMT may exist as a single-copy gene in loblolly pine genome. The transgenic tobacco plants carrying loblolly pine CCoAOMT promoter-GUS fusion localized the site of GUS activity at the secondary xylem tissues. These data suggest that CCoAOMT, in addition to AEOMT, plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine.     

Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns.

The biosynthesis of lignin monomers involves two methylation steps catalyzed by orthodiphenol-O-methyltransferases: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferases (COMTs) and caffeoyl-coenzyme A (CoA)/5-hydroxyferuloyl-CoA 3/5-O-methyltransferases (CCoAOMTs). Two COMT classes (I and II) were already known to occur in tobacco (Nicotiana tabacum) and three distinct CCoAOMT classes have now been characterized. These three CCoAOMT classes displayed a maximum level of expression at different stages of stem development, in accordance with their involvement in the synthesis of lignin guaiacyl units. Expression profiles upon tobacco mosaic virus infection of tobacco leaves revealed a biphasic pattern of induction for COMT I, COMT II, and CCoAOMTs. The different isoforms were expressed in Escherichia coli and our results showed that CCoAOMTs and, more surprisingly, COMTs efficiently methylated hydroxycinnamoyl-CoA esters. COMT I was also active toward 5-hydroxyconiferyl alcohol, indicating that COMT I that catalyzes syringyl unit synthesis in planta may operate at the free acid, CoA ester, or alcohol levels. COMT II that is highly inducible by infection also accepted caffeoyl-CoA as a substrate, thus suggesting a role in ferulate derivative deposition in the walls of infected cells. Tobacco appears to possess an array of O-methyltransferase isoforms with variable efficiency toward the diverse plant o-diphenolic substrates.     

Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula x Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content.     

Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransferase

Caffeoyl coenzyme A 3-O-methyltransferases (CCoAOMTs) are S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) involved in lignin biosynthesis. Plant CCoAOMTs belong to a distinct family of OMTs, more closely related to the mammalian catechol OMTs than to other plant OMTs. The crystal structure of alfalfa (Medicago sativa) CCoAOMT in complex with the reaction products S-adenosine-l-homocysteine and feruloyl/sinapoyl CoAs presented here belong to a structurally and mechanistically distinct family of plant small molecule OMTs. These structures provide a new understanding of the substrate preferences and the catalytic mechanism accompanying CCoAOMT-mediated O-methylation of CoA-linked phenylpropanoid substrates.     

Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana

SUMMARY: Coumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its beta-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3'-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2OGD) family was designated as F6'H1. T-DNA insertion mutants of F6'H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6'H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6'H1 protein exhibited ortho-hydroxylase activity for feruloyl CoA (K(m) = 36.0 +/- 4.27 microM; k(cat) = 11.0 +/- 0.45 sec(-1)) to form 6'-hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)- and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.     

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene (in English)

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

Cloning of cDNA Encoding COMT from Chinese White Poplar (Populus tomentosa), Sequence Analysis and Specific Expression

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

Caffeoyl coenzyme A O-methyltransferase down-regulation is associated with modifications in lignin and cell-wall architecture in flax secondary xylem

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT, EC 2.1.1.104) down-regulated-flax (Linum usitatissimum) plants were generated using an antisense strategy and functionally characterized. Chemical analyses (acetyl bromide and thioacidolysis) revealed that the lignin quantity was reduced and that the Syringyl/Guaïacyl (S/G) lignin monomer ratio was modified in the non-condensed lignin fraction of two independent down-regulated lines. These modifications were associated with altered xylem organization (both lines), reduced cell-wall thickness (one line) and the appearance of an irregular xylem (irx) phenotype (both lines). In addition UV microspectroscopy also indicated that CCoAOMT down-regulation induced changes in xylem cell-wall structure and the lignin fractions. Microscopic examination also suggested that CCoAOMT down-regulation could influence individual xylem cell size and identity. As a first step towards investigating the cellular mechanisms responsible for the unusual structure of flax lignin (G-rich, condensed), recombinant flax CCoAOMT protein was produced and its affinity for different potential substrates evaluated. Results indicated that the preferred substrate was caffeoyl coenzyme A, followed by 5-hydroxyconiferaldehyde suggesting that flax CCoAOMT possesses a small, but probably significant 5′ methylating activity, in addition to a more usual 3′ methylating activity.     

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.     

Biochemical characterization of a cinnamoyl-CoA reductase from wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester-->aldehyde conversion in monolignol biosynthesis, which can divert phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis in wheat (Triticum aestivum L.), a cDNA encoding CCR was isolated and named Ta-CCR2. DNA hybridization analyses demonstrated that the Ta-CCR2 gene exists in three copies in the wheat genome. RNA blot hybridization indicated that Ta-CCR2 was expressed most abundantly in root and stem tissues that were in the process of lignification. The secondary and three-dimensional structures of Ta-CCR2 were analyzed by molecular modeling. Recombinant Ta-CCR2 protein purified from E. coli converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA and caffeoyl CoA with almost similar efficiency, suggesting that it is involved in both G and S lignin synthesis. Ta-CCR2 had a very low V max value for 4-coumaroyl CoA, which may serve as a mechanism to control metabolic flux to H lignin in vivo . Furthermore, the reaction mechanism of Ta-CCR2 was analyzed in relation to its possible three-dimensional structure. The activity of Ta-CCR2 in relation to lignin biosynthesis is discussed.     

Structural and docking studies of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> Cinnamoyl CoA reductase

Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

An alternative methylation pathway in lignin biosynthesis in Zinnia.

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.     

CCoAOMT suppression modifies lignin composition in Pinus radiata

A cDNA clone encoding the lignin‐related enzyme caffeoyl CoA 3‐O‐methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P. radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p‐hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide‐soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT‐deficient transgenic lines. Pyrolysis‐GC/MS and 2D‐NMR studies demonstrated that these reductions were due to depletion of G‐type lignin. Correspondingly, the proportion of H‐type lignin in PrCCoAOMT‐deficient transgenic lines increased, resulting in up to a 10‐fold increase in the H/G ratio relative to untransformed controls. 2D‐NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho‐diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D‐NMR. Comparison of the 2D‐NMR spectra from PrCCoAOMT‐RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT‐deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.     

The role of CCoAOMT1 and COMT1 in Arabidopsis anthers

Arabidopsis caffeoyl coenzyme A dependent O-methyltransferase 1 (CCoAOMT1) and caffeic acid O-methyltransferase 1 (COMT1) display a similar substrate profile although with distinct substrate preferences and are considered the key methyltransferases (OMTs) in the biosynthesis of lignin monomers, coniferyl and sinapoylalcohol. Whereas CCoAOMT1 displays a strong preference for caffeoyl coenzyme A, COMT1 preferentially methylates 5-hydroxyferuloyl CoA derivatives and also performs methylation of flavonols with vicinal aromatic dihydroxy groups, such as quercetin. Based on different knockout lines, phenolic profiling, and immunohistochemistry, we present evidence that both enzymes fulfil distinct, yet different tasks in Arabidopsis anthers. CCoAOMT1 besides its role in vascular tissues can be localized to the tapetum of young stamens, contributing to the biosynthesis of spermidine phenylpropanoid conjugates. COMT1, although present in the same organ, is not localized in the tapetum, but in two directly adjacent cells layers, the endothecium and the epidermal layer of stamens. In vivo localization and phenolic profiling of comt1 plants provide evidence that COMT1 neither contributes to the accumulation of spermidine phenylpropanoid conjugates nor to the flavonol glycoside pattern of pollen grains.     

Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase ofStellaria longipes

A cDNA clone encodingS-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyl-transferase (EC 2.1.1.104; CCoAOMT) fromStellana longipes Goldie (long-stalked chick-weed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme ofStellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in theStellana longipes genome and the absence of introns in the coding region of the cDNA-corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNA-encoded protein expressed inEschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.     

Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula x Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.