The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochemistry approaches. In vitro enzymatic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymerization, and lignin structure.

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Recent advances in nuclear magnetic resonance (NMR) technology have made it possible to rapidly screen plant material and discern whole cell wall information without the need to deconstruct and fractionate the plant cell wall. This approach can be used to improve our understanding of the biology of cell wall structure and biosynthesis, and as a tool to select plant material for the most appropriate industrial applications. This is particularly true in an era when renewable materials are vital to the emerging bio-based economies. This protocol describes procedures for (i) the preparation and extraction of a biological plant tissue, (ii) solubilization strategies for plant material of varying composition and (iii) 2D NMR acquisition (for typically 15 min-5 h) and integration methods used to elucidate lignin subunit composition and lignin interunit linkage distribution, as well as cell wall polysaccharide profiling. Furthermore, we present data that demonstrate the utility of this new NMR whole cell wall characterization procedure with a variety of degradative methods traditionally used for cell wall compositional analysis.

NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D 13C–1H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4:1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d6-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

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 crosslinking between lignins and polysaccharides. Monolignol-hydroxycinnamate (primarily monolignol-pcoumarate) 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.

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.

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.

Pyrolysis-GC-MS pyrograms from a series of alfalfa preparations, a grass, an angiosperm wood, a cellulose, and an arabinoxylan were obtained under pyrolytic conditions optimal for aromatic components of plant cell walls. Approximately 130 pyrolytic fragments have been identified by a combination of mass spectral interpretation, comparison with literature data, and, where possible, confirmation with authentic compounds. Several new fragments not previously noted in pyrograms have been conclusively assigned including both guaiacyl-and syringylpropyne and a range of alcohols. Diagnostic peaks from arabinose and xylose components of forages were found, along with markers for protein. Pyrolysis-GC-MS was particularly valuable for screening and differentiating p-coumaric acid and ferulic acid derivatives in tissues.