John Ralph

Lignin Biosynthesis and Structure

Lignin is the generic term for a large group of aromatic polymers resulting from the oxidative combinatorial coupling of 4-hydroxyphenylpropanoids ([Boerjan et al., 2003][1]; [Ralph et al., 2004][2]). These polymers are deposited predominantly in the walls of secondarily thickened cells, making them

Repression of Lignin Biosynthesis Promotes Cellulose Accumulation and Growth in Transgenic Trees

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin–cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.

Combinatorial modification of multiple lignin traits in trees through multigene cotransformation

Lignin quantity and reactivity [which is associated with its syringyl/guaiacyl (S/G) constituent ratio] are two major barriers to wood-pulp production. To verify our contention that these traits are regulated by distinct monolignol biosynthesis genes, encoding 4-coumarate–CoA ligase (4CL) and coniferaldehyde 5-hydroxylase (CAld5H), we used Agrobacterium to cotransfer antisense 4CL and sense CAld5H genes into aspen (Populus tremuloides). Trees expressing each one and both of the transgenes were produced with high efficiency. Lignin reduction by as much as 40% with 14% cellulose augmentation was achieved in antisense 4CL plants; S/G-ratio increases as much as 3-fold were observed without lignin quantity change in sense CAld5H plants. Consistent with our contention, these effects were independent but additive, with plants expressing both transgenes having up to 52% less lignin, a 64% higher S/G ratio, and 30% more cellulose. An S/G-ratio increase also accelerated cell maturation in stem secondary xylem, pointing to a role for syringyl lignin moieties in coordinating xylem secondary wall biosynthesis. The results suggest that this multigene cotransfer system should be broadly useful for plant genetic engineering and functional genomics.

Lignin-ferulate cross-links in grasses: active incorporation of ferulate polysaccharide esters into ryegrass lignins

Abstract Active incorporation of ferulate polysaccharide esters into ryegrass lignins has been demonstrated by NMR spectroscopy of uniformly 13 C-labeled ryegrass. Observation, in the HMBC spectrum, of products of ferulate at its 8-position coupling with hydroxycinnamyl alcohols at the β-position (producing 8-β′-linked structures) is proof that ferulate-lignin radical cross-coupling reactions occur in vivo. Correlations of H-α′ (hydroxycinnamyl alcohol moiety) with guaiacyl and syringyl 1-, 2-, and 6-aromatic carbons in 8-β′ structures indicates that ferulates couple with both coniferyl and sinapyl alcohol monomers. As notable as the presence of this and other ferulate products is the absence of coupling of ferulate at its 8-position with the 5- and O -4-positions of lignin units. Such structures were significant when ferulate was biomimetically incorporated into a synthetic lignin. Since hydroxycinnamyl alcohols couple almost exclusively at their β-position in cross-coupling reactions, the 8-5′ and 8- O -4′ structures would only be formed by coupling with higher lignin oligomers (with no side-chain conjugation). Exclusive reaction of ferulates with lignin monomers is the first real evidence that ferulate polysaccharide esters in grasses are acting as initiation or nucleation sites for lignification and are critical entities in directing cell-wall cross-linking during plant growth and development.

Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis

Abstract Lignin is an abundant biopolymer with a high carbon content and high aromaticity. Despite its potential as a raw material for the fuel and chemical industries, lignin remains the most poorly utilised of the lignocellulosic biopolymers. Effective valorisation of lignin requires careful fine‐tuning of multiple “upstream” (i.e., lignin bioengineering, lignin isolation and “early‐stage catalytic conversion of lignin”) and “downstream” (i.e., lignin depolymerisation and upgrading) process stages, demanding input and understanding from a broad array of scientific disciplines. This review provides a “beginning‐to‐end” analysis of the recent advances reported in lignin valorisation. Particular emphasis is placed on the improved understanding of lignins biosynthesis and structure, differences in structure and chemical bonding between native and technical lignins, emerging catalytic valorisation strategies, and the relationships between lignin structure and catalyst performance.

Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Hydroxycinnamates in lignification

Hydroxycinnamates incorporate into lignins by various mechanisms. The polysaccharide esters of ferulate, in particular, and the range of dehydrodiferulates and higher oligomers in grasses, participate in free-radical (cross-)coupling reactions during lignification to become integrally bound into the lignin polymer, resulting in extensive cross-linking between lignins and polysaccharides. Monolignol-hydroxycinnamate (primarily monolignol-p-coumarate) conjugates are primary building blocks for lignins, again in grasses (but analogously with monolignol acetates and p-hydroxybenzoates in other plants); radical coupling reactions of the monolignol moiety of the conjugate result in lignins with pendant p-coumarate units acylating a variety of lignin structures. Recent evidence suggests that even the hydroxycinnamic acids themselves can be monomers in lignification in wild-type and transgenic plants, undergoing radical cross-coupling reactions to incorporate into the polymer with interesting consequences. The compatibility of ferulate, in particular, with lignification suggests that plants able to utilize monolignol-ferulate conjugates in their primary monomer supply will be particularly well suited for subsequent chemical delignification, potentially improving processes for biomass conversion to biofuels, and for chemical pulping.

Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls

Seven isomeric dehydrodimers of ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been synthesized and identified in extracts of saponified cell walls of cocksfoot, switchgrass, and suspension-cultured corn. Dehydrodimers (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid, (Z)-β-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxycinnamic acid, (E)-3-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-5-methoxycinnamic acid, (E,E)-4,4′-dihydroxy-3,5′-dimethoxy-β,3′-bicinnamic acid, 4,4′-dihydroxy-3,3′-dimethoxy-β,β′-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1,2-dihydronaphthalene-2,3-dicarboxylic acid, all arise from oxidative coupling of ferulate esters in cell walls and represent products of 8–5, 8–8, 8–O–4, 4–O–5, and 5–5 radical coupling. Prior literature has acknowledged only the presence of the 5–5-coupled dehydrodimer (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid. Consequently, by measuring only a single dehydrodimer and assuming inappropriate response factors, ferulate dehydrodimers have been underestimated by factors of up to 20. Synthetic routes to all seven isomers have been developed to provide structural authentication and determination of GC response factors.

Chemoselective Metal-Free Aerobic Alcohol Oxidation in Lignin

An efficient organocatalytic method for chemoselective aerobic oxidation of secondary benzylic alcohols within lignin model compounds has been identified. Extension to selective oxidation in natural lignins has also been demonstrated. The optimal catalyst system consists of 4-acetamido-TEMPO (5 mol %; TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl) in combination with HNO3 and HCl (10 mol % each). Preliminary studies highlight the prospect of combining this method with a subsequent oxidation step to achieve C-C bond cleavage.