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The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Lignin, a polymer found in the plant secondary cell wall, is a major contributor to biomass' recalcitrance toward saccharification. Because of this negative impact toward the value of lignocellulosic crops, there is a special interest in modifying the content and composition of this important plant biopolymer. For many years this endeavor has been hindered by the plant growth inhibition that is often associated with manipulations to phenylpropanoid metabolism. Although the actual mechanism by which dwarfism arises remains unknown, recent advances in tissue-specific lignin complementation and better understanding of phenylpropanoid transcriptional regulation has made it possible to disentangle lignin modification from perturbations in plant development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled [ 13 C 6 ]-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Practical, high-yield lignin depolymerization methods could greatly increase biorefinery productivity and profitability. However, development of these methods is limited by the presence of interunit carbon-carbon bonds within native lignin, and further by formation of such linkages during lignin extraction. We report that adding formaldehyde during biomass pretreatment produces a soluble lignin fraction that can be converted to guaiacyl and syringyl monomers at near theoretical yields during subsequent hydrogenolysis (47 mole % of Klason lignin for beech and 78 mole % for a high-syringyl transgenic poplar). These yields were three to seven times those obtained without formaldehyde, which prevented lignin condensation by forming 1,3-dioxane structures with lignin side-chain hydroxyl groups. By depolymerizing cellulose, hemicelluloses, and lignin separately, monomer yields were between 76 and 90 mole % for these three major biomass fractions., (Copyright © 2016, American Association for the Advancement of Science.)

Lignocellulosic biomass represents an abundant and sustainable raw material for biofuel production. The recalcitrance of biomass to degradation increases the estimated cost of biofuel production and limits its competitiveness in the market. Genetic engineering of lignin, a major recalcitrance factor, improves saccharification and thus the potential yield of biofuels. Recently, our understanding of lignification and its regulation has been advanced by new studies in various systems, all of which further enhances our ability to manipulate the biosynthesis and deposition of lignin in energy crops for producing cost-effective second generation biofuels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Highly lignified vascular plant cell walls represent the majority of cellulosic biomass. Complete release of the biomass to deliver renewable energy by physical, chemical, and biological pretreatments is challenging due to the "protection" provided by polymerized lignin, and as such, additional tools to monitor lignin deposition and removal during plant growth and biomass deconstruction would be of great value. We developed a hyperspectral stimulated Raman scattering microscope with 9 cm(-1) spectral resolution and submicrometer spatial resolution. Using this platform, we mapped the aromatic ring of lignin, aldehyde, and alcohol groups in lignified plant cell walls. By multivariate curve resolution of the hyperspectral images, we uncovered a spatially distinct distribution of aldehyde and alcohol groups in the thickened secondary cell wall. These results collectively contribute to a deeper understanding of lignin chemical composition in the plant cell wall.

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content., (© 2015 American Society of Plant Biologists. All rights reserved.)

Key Message: The gene coding for F5H from Eucalyptus globulus was cloned and used to transform an f5h -mutant of Arabidopsis thaliana , which was complemented, thus verifying the identity of the cloned gene. Coniferaldehyde 5-hydroxylase (F5H; EC 1.14.13) is a cytochrome P450-dependent monooxygenase that catalyzes the 5-hydroxylation step required for the production of syringyl units in lignin biosynthesis. The Eucalyptus globulus enzyme was characterized in vitro, and results showed that the preferred substrates were coniferaldehyde and coniferyl alcohol. Complementation experiments demonstrated that both cDNA and genomic constructs derived from F5H from E. globulus under the control of the cinnamate 4-hydroxylase promoter from Arabidopsis thaliana, or a partial F5H promoter from E. globulus, can rescue the inability of the A. thaliana fah1-2 mutant to accumulate sinapate esters and syringyl lignin. E. globulus is a species widely used to obtain products that require lignin removal, and the results suggest that EglF5H is a good candidate for engineering efforts aimed at increasing the lignin syringyl unit content, either for kraft pulping or biofuel production.

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The secondary cell wall polymer lignin impedes the extraction of fermentable sugars from biomass, and has been one of the major impediments in the development of cost-effective biofuel technologies. Unfortunately, attempts to genetically engineer lignin biosynthesis frequently result in dwarfing or developmental abnormalities of unknown cause, thus limiting the benefits of increased fermentable sugar yield. In this brief review, we explore some of the possible mechanisms that could underlie this poorly understood phenomenon, with the expectation that an understanding of the cause of dwarfing in lignin biosynthetic mutants and transgenic plants could lead to new strategies for the development of improved bioenergy feedstocks., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

• There is a pressing global need to reduce the increasing societal reliance on petroleum and to develop a bio-based economy. At the forefront is the need to establish a sustainable, renewable, alternative energy sector. This includes liquid transportation fuel derived from lignocellulosic plant materials. However, one of the current limiting factors restricting the effective and efficient conversion of lignocellulosic residues is the recalcitrance of the substrate to enzymatic conversion. • In an attempt to assess the impact of cell wall lignin on recalcitrance, we subjected poplar trees engineered with altered lignin content and composition to two potential industrial pretreatment regimes, and evaluated the overall efficacy of the bioconversion to ethanol process. • It was apparent that total lignin content has a greater impact than monomer ratio (syringyl : guaiacyl) on both pretreatments. More importantly, low lignin plants showed as much as a 15% improvement in the efficiency of conversion, with near complete hydrolysis of the cellulosic polymer. • Using genomic tools to breed or select for modifications in key cell wall chemical and/or ultrastructural traits can have a profound effect on bioenergy processing. These techniques may therefore offer means to overcome the current obstacles that underpin the recalcitrance of lignocellulosic substrates to bioconversion., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.

The presence of the phenylpropanoid polymer lignin in plant cell walls impedes breakdown of polysaccharides to the fermentable sugars that are used in biofuel production. Genetically modified plants with altered lignin properties hold great promise to improve biomass degradability. Here, we describe the generation of a new type of lignin enriched in 5-hydroxy-guaiacyl units by over-expressing ferulate 5-hydroxylase in a line of Arabidopsis lacking caffeic acid O-methyltransferase. The lignin modification strategy had a profound impact on plant growth and development and cell-wall properties, and resulted in male sterility due to complete disruption of formation of the pollen wall. The modified plants showed significantly improved cell-wall enzymatic saccharification efficiency without a reduction in post-harvest biomass yield despite the alterations in the overall growth morphology. This study demonstrated the plasticity of lignin polymerization in terms of incorporation of unusual monomers that chemically resemble conventional monomers, and also revealed the link between the biosynthetic pathways of lignin and the pollen wall-forming sporopollenin., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Summary: Lignin, a phenolic polymer derived mainly from hydroxycinnamyl alcohols, is ubiquitously present in tracheophytes. The development of lignin biosynthesis has been considered to be one of the key factors that allowed land plants to flourish in terrestrial ecosystems. Lignin provides structural rigidity for tracheophytes to stand upright, and strengthens the cell wall of their water-conducting tracheary elements to withstand the negative pressure generated during transpiration. In this review, we discuss a number of aspects regarding the origin and evolution of lignin biosynthesis during land plant evolution, including the establishment of its monomer biosynthetic scaffold, potential precursors to the lignin polymer, as well as the emergence of the polymerization machinery and regulatory system. The accumulated knowledge on the topic, as summarized here, provides us with an evolutionary view on how this complex metabolic system emerged and developed.

Defects in phenylpropanoid biosynthesis arising from deficiency in hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) or p-coumaroyl shikimate 3'-hydroxylase (C3'H) lead to reduced lignin, hyperaccumulation of flavonoids, and growth inhibition in Arabidopsis thaliana. It was previously reported that flavonoid-mediated inhibition of auxin transport is responsible for growth reduction in HCT-RNA interference (RNAi) plants. This conclusion was based on the observation that simultaneous RNAi silencing of HCT and chalcone synthase (CHS), an enzyme essential for flavonoid biosynthesis, resulted in less severe dwarfing than silencing of HCT alone. In an attempt to extend these results using a C3'H mutant (ref8) and a CHS null mutant (tt4-2), we found that the growth phenotype of the ref8 tt4-2 double mutant, which lacks flavonoids, is indistinguishable from that of ref8. Moreover, using RNAi, we found that the relationship between HCT silencing and growth inhibition is identical in both the wild type and tt4-2. We conclude from these results that the growth inhibition observed in HCT-RNAi plants and the ref8 mutant is independent of flavonoids. Finally, we show that expression of a newly characterized gene bypassing HCT and C3'H partially restores both lignin biosynthesis and growth in HCT-RNAi plants, demonstrating that a biochemical pathway downstream of coniferaldehyde, probably lignification, is essential for normal plant growth.

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3'H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.

The processes underlying lignification, which for many years have been the near-exclusive purview of chemists and biochemists, have more recently been approached using both classical forward genetic screens and targeted reverse genetic approaches such as antisense suppression, RNAi, and characterization of insertional mutants. In this review, we provide an overview of the current understanding of lignin biosynthesis and structure, with emphasis on mutant and transgenic plants that have contributed to this knowledge. We also discuss ongoing work aimed at elucidating the relationship between lignin structure and function in vivo, as well as the phenotypic consequences arising from genetic manipulation of the lignin biosynthetic pathway.

Poplar (Populus tremula x alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4HF5H transgenic's lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial beta-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (beta-beta) and spirodienone (beta-1) contents, but with virtually no phenylcoumarans (beta-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the gamma-positions of lignin side chains, were reduced by >50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes.

Lycophytes arose in the early Silurian ( approximately 400 Mya) and represent a major lineage of vascular plants that has evolved in parallel with the ferns, gymnosperms, and angiosperms. A hallmark of vascular plants is the presence of the phenolic lignin heteropolymer in xylem and other sclerified cell types. Although syringyl lignin is often considered to be restricted in angiosperms, it has been detected in lycophytes as well. Here we report the characterization of a cytochrome P450-dependent monooxygenase from the lycophyte Selaginella moellendorffii. Gene expression data, cross-species complementation experiments, and in vitro enzyme assays indicate that this P450 is a ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydroxylase (F5H), and is capable of diverting guaiacyl-substituted intermediates into syringyl lignin biosynthesis. Phylogenetic analysis indicates that the Selaginella F5H represents a new family of plant P450s and suggests that it has evolved independently of angiosperm F5Hs.

Lignin, a major component of the cell wall of vascular plants, has long been recognized for its negative impact on forage quality, paper manufacturing, and, more recently, cellulosic biofuel production. Over the last two decades, genetic and biochemical analyses of brown midrib mutants of maize, sorghum and related grasses have advanced our understanding of the relationship between lignification and forage digestibility. This work has also inspired genetic engineering efforts aimed at generating crops with altered lignin, with the expectation that these strategies would enhance forage digestibility and/or pulping efficiency. The knowledge gained from these bioengineering efforts has greatly improved our understanding of the optimal lignin characteristics required for various applications of lignocellulosic materials while also contributing to our understanding of the lignin biosynthetic pathway. The recent upswing of interest in cellulosic biofuel production has become the new focus of lignin engineering. Populus trichocarpa and Brachypodium distachyon are emerging as model systems for energy crops. Lignin research on these systems, as well as on a variety of proposed energy crop species, is expected to shed new light on lignin biosynthesis and its regulation in energy crops, and lead to rational genetic engineering approaches to modify lignin for improved biofuel production.

Ethanol and other biofuels produced from lignocellulosic biomass represent a renewable, more carbon-balanced alternative to both fossil fuels and corn-derived or sugarcane-derived ethanol. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides to simple sugars and the subsequent conversion of these sugars to usable fuel. Recent advances in the understanding of lignin composition, polymerization, and regulation have revealed new opportunities for the rational manipulation of lignin in future bioenergy crops, augmenting the previous successful approach of manipulating lignin monomer biosynthesis. Furthermore, recent studies on lignin degradation in nature may provide novel resources for the delignification of dedicated bioenergy crops and other sources of lignocellulosic biomass.

p-Coumaroyl-CoA 3'-hydroxylase (C3'H) is a cytochrome P450-dependent monooxygenase that catalyzes the 3'-hydroxylation of p-coumaroyl shikimate and p-coumaroyl quinate. We used RNA interference to generate transgenic hybrid poplar suppressed in C3'H expression and analyzed them with respect to transcript abundance, cell wall structure and chemical composition, and soluble metabolite levels. RT-PCR expression profiles confirmed the down-regulation of C3'H in a number of lines, which generally correlated very well with reduced total cell wall lignin content. The most strongly repressed line was chosen for further analysis and compared with the wild-type trees. In-depth characterization revealed that along with the significant decrease in total lignin content, a significant shift in lignin monomer composition was observed, favoring the generation of p-hydroxyphenyl units at the expense of guaiacyl units while the proportion of syringyl moieties remained constant. Suppression of C3'H also resulted in the accumulation of substantial pools of 1-O-p-coumaroyl-beta-d-glucoside and other phenylpropanoid glycosides, and p-coumaroyl shikimate, providing further insight into the role of C3'H in the lignin biosynthetic pathway. The data presented indicate that when down-regulated, C3'H becomes a rate-limiting step in lignin biosynthesis and further support the involvement of hydroxycinnamic acid shikimate esters in the lignin biosynthetic pathway.

Considerable interest in lignin biosynthesis has been fueled by the many roles that lignin plays in development and in resistance to biotic and abiotic stress, as well as its importance to industry and agriculture. Although the pathway leading to the lignin polymer has been studied for decades, new insights into the enzymes of the pathway have required a complete re-evaluation of how we think lignin precursors are synthesized. Although free hydroxycinnamic acids have long been thought to be key intermediates, it has become apparent that many of the hydroxylation and methylation steps in the pathway occur instead at the level of hydroxycinnamic acid esters, and their corresponding aldehydes and alcohols.

The end products of the phenylpropanoid pathway play important roles in plant structure and development, as well as in plant defense mechanisms against biotic and abiotic stresses. From a human perspective, phenylpropanoid pathway-derived metabolites influence both human health and the potential utility of plants in agricultural contexts. The last known enzyme of the phenylpropanoid pathway that has not been characterized is p-coumarate 3-hydroxylase (C3H). By screening for plants that fail to accumulate soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. We have now shown that the ref8 mutant is defective in the gene encoding C3H. Phenotypic characterization of the ref8 mutant has revealed that the lack of C3H activity in the mutant leads to diverse changes in phenylpropanoid metabolism. The ref8 mutant accumulates p-coumarate esters in place of the sinapoylmalate found in wild-type plants. The mutant also deposits a lignin formed primarily from p-coumaryl alcohol, a monomer that is at best a minor component in the lignin of other plants. Finally, the mutant displays developmental defects and is subject to fungal attack, suggesting that phenylpropanoid pathway products downstream of REF8 may be required for normal plant development and disease resistance.

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.

Nuclear magnetic resonance (NMR) of isolated lignins from an Arabidopsis mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the F5H gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type Arabidopsis has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent beta-aryl ether (beta-O-4), phenylcoumaran (beta-5), resinol (beta-beta), biphenyl/dibenzodioxocin (5-5), and cinnamyl alcohol end-group structures. The lignin isolated from the F5H-deficient fah1-2 mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In fah1-2 transgenics in which the F5H gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the fah1-2 transgenics in which F5H expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly beta-aryl ether units, mainly with erythro-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.

The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the K(m) values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [(14)C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) is considered necessary to activate the hydroxycinnamic acids for the biosynthesis of the coniferyl and sinapyl alcohols subsequently polymerized into lignin. To clarify the role played by 4CL in the biosynthesis of the guaiacyl (G) and syringyl (S) units characteristic of angiosperm lignin, we generated 4CL antisense Arabidopsis lines having as low as 8% residual 4CL activity. The plants had decreases in thioglycolic acid-extractable lignin correlating with decreases in 4CL activity. Nitrobenzene oxidation of cell walls from bolting stems revealed a significant decrease in G units in 4CL-suppressed plants; however, levels of S lignin units were unchanged in even the most severely 4CL-suppressed plants. These effects led to a large decrease in the G/S ratio in these plants. Our results suggest that an uncharacterized metabolic route to sinapyl alcohol, which is independent of 4CL, may exist in Arabidopsis. They also demonstrate that repression of 4CL activity may provide an avenue to manipulate angiosperm lignin subunit composition in a predictable manner.