Lignins and lignification: Selected issues

Lignin deposition in plant cell walls is one of the mechanisms which allowed the development of upright plants adapted to a terrestrial habitat. At the present time, lignins and lignification are the subject of very active research which has recently moved from chemical and biochemical aspects to more biological and developmental problems. In this review, three different topics will be addressed. (a) A first section will deal with recent advances related to the biosynthesis of lignins. It will be shown that a complex array of O-methyltransferases may control the production of differentially methylated monolignols, the precursors of lignins, but that the downstream enzymes in the synthesis of monolignols are probably not encoded by multigene families which would provide additional possibilities for fine-tuning the monomeric composition of lignins. In addition, recent results obtained on laccases will illustrate the difficulty in identifying the true nature of oxidases involved in the production of phenoxy radicals, the oxidation products of monolignols. (b) A second set of data will highlight the potential interest of Arabidopsis mutants for understanding lignin synthesis, deposition and function. Indeed, different classes of lignification mutants with modifications in lignin content or composition and alterations of vascular differentiation or global vascular pattern have already been characterized. The identification of the corresponding genes will undoubtedly give rise to new insights on key steps and regulation mechanisms in the lignification process. (c) The last section is dedicated to the future of lignin genetic engineering. It will be emphasized that, after a first period which has demonstrated the potential of the approach, it is necessary to consider in greater detail the unexpected side effects and compensation mechanisms associated with induced lignin modifications. New targets for future lignin genetic engineering experiments will be identified and the extension of the technology to new woody species, the advantages for the pulp industry and the problems associated with public perception of these new products will be envisaged. Lignification is a tightly regulated and dynamic process subject to modulation at different levels during normal development and in response to different stresses. Understanding these subtle mechanisms which also involve the other polymers of the cell wall is an important challenge facing plant biology as we enter the next century.     

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight (~1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter⁻¹, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter⁻¹ within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter⁻¹ could also completely degrade 1 g of lignin liter⁻¹ during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter⁻¹ increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day⁻¹. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day⁻¹. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.     

On the Role of Long-Chain Aldehydes in Mammalian Plasmalogen Biosynthesis

Abstract: [1-³H, 1-¹⁴C]Palmitaldehyde(³H:¹⁴C= 15) was injected intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was followed over a 24-h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower ³H:¹⁴C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium-free alcohols derived from ¹⁴C-labeled fatty acids. Virtually no ³H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.     

Modification of Lignins by Growing Cells of the Sulfate-Reducing Anaerobe Desulfovibrio desulfuricans

The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans was grown on medium supplemented with either Kraft lignin or lignosulfonate. Only lignosulfonate contributed to the growth of D. desulfuricans cells, by replacing sulfate, a natural electron acceptor for this microorganism. Kraft lignin added to the culture medium could not substitute for lactate or sulfate, both necessary culture medium components. However, it was found to enhance the viability of D. desulfuricans cells. When changes occurring in lignin during growth of Desulfovibrio cultures were monitored, it was found that both lignin preparations could be partially depolymerized. Spectrophotometric and elemental analysis of biologically treated lignins suggested that both the polyphenolic backbone and lignin functional groups were affected by D. desulfuricans. After treatment, a twofold increase in the sulfur content of Kraft lignin and a minor decrease (14%) in the sulfur content of lignosulfonate were observed. After biological treatment, Kraft lignin and lignosulfonate both bound larger quantities of heavy metals.     

Zur Verbreitung des Lignins bei Gefässkryptogamen

Ohne Zusammenfassung     

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (MLs), p-coumaryl alcohol, coniferyl alcohol (CA), and sinapyl alcohol (SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (Sarkanen and Ludwig, 1971; Boerjan et al., 2003; Ralph et al., 2004). The contribution of various MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three MLs, the lignification process has a high degree of metabolic plasticity (Boerjan et al., 2003; Ralph et al., 2004; Ralph, 2007; Vanholme et al., 2012). Of particular interest are ML conjugates in which the ester group can be acetate (Ac; Sarkanen et al., 1967; Ralph, 1996; Ralph and Lu, 1998; Del Río et al., 2007; del Río et al., 2008; Martínez et al., 2008), p-hydroxybenzoate (pBz; Venverloo, 1971; Monties and Lapierre, 1981; Landucci et al., 1992; Tomimura, 1992a, 1992b; Hibino et al., 1994; Sun et al., 1999; Kuroda et al., 2001; Lu et al., 2004, 2015; Morreel et al., 2004; Rencoret et al., 2013), p-coumarate (pCA; Monties and Lapierre, 1981; Ralph et al., 1994; Crestini and Argyropoulos, 1997; del Río et al., 2008, 2012a, 2012b; Withers et al., 2012; Rencoret et al., 2013; Petrik et al., 2014), or ferulate (FA; Grabber et al., 2008; Ralph, 2010; Wilkerson et al., 2014). In all cases, the MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (Lu and Ralph, 2002, 2008; Del Río et al., 2007; Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in Angelica sinensis and transformed into poplar (Populus alba × grandidentata), which naturally incorporates other acylated MLs, namely ML-pBz conjugates, into its lignin (Wilkerson et al., 2014). Plants that incorporate ML-FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which ML-FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (DFRC), that can release diagnostic chemical marker compounds from lignins containing ML-FAs (Lu and Ralph, 2014; Wilkerson et al., 2014). The DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that ML-FA conjugates are fully incorporated into the lignin of the FMT poplar (Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of pCA in lignin has been highly speculative. It is hypothesized that the pCA moieties may function as a radical sensitizer (Takahama and Oniki, 1996, 1997; Takahama et al., 1996; Ralph et al., 2004; Hatfield et al., 2008; Ralph, 2010). Peroxidases and/or laccases readily oxidize pCA to a radical but are poor oxidizers for SA. Free radicals of pCA readily undergo radical transfer to SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating pCA to an ML, like SA, to form SA-pCA, the most prevalent ML-pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating MLs in monocots (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014).In rice (Oryza sativa), enzymes have been characterized that function specifically in the addition of pCA onto hemicelluloses (Bartley et al., 2013) or lignin (Withers et al., 2012; Petrik et al., 2014). The p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many ML biosynthetic enzymes (Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward p-coumaroyl-CoA and an ML, and has kinetic efficiency with p-coumaryl alcohol > SA > CA. In most grasses, the PMT enzyme predominantly produces SA-pCA conjugates that are then incorporated into the lignin polymer (Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of PMT genes has been performed in Brachypodium distachyon and maize (Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of MLs before polymerization and not in the acylation of hemicelluloses (Petrik et al., 2014). RNA interference-mediated suppression of BdPMT resulted in decreased incorporation of ML-pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the BdPMT gene, which resulted in a complete absence of pCA-acylating B. distachyon lignins, did not affect plant growth or development (Petrik et al., 2014). The arabinose-bound FA and pCA levels remained virtually unchanged in the PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the p-coumaroyl-CoA substrate and its ML acylation. The PMT enzyme identified in maize (pCAT = ZmPMT) also displayed the highest catalytic efficiency with p-coumaroyl-CoA and SA as substrates (Marita et al., 2014). RNAi-mediated suppression of ZmPMT also resulted in decreased production of the ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (Ralph et al., 1994; Ralph, 2010). As a result, the pCA itself can be completely quantified by simple saponification. The units to which the pCA is attached are, like their normal ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the pCA’s terminal location makes p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (Arabidopsis thaliana) and poplar, neither of which naturally produces ML-pCA conjugates, to express a PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of PMT has on the native levels of ML-pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.     

Characterization of Lignins Isolated with Alkaline Ethanol from the Hydrothermal Pretreated Tamarix ramosissima

The objective of this study was to characterize the changes in lignin structure during hydrothermal pretreatment of shrub Tamarix ramosissima. Lignins in residual wood meal were isolated with alkaline ethanol solution and recovered with acid precipitation. A comparison between the recovered lignin fractions with milled wood lignin has been made in terms of yield, purity, gel permeation chromatography, Fourier transform infrared spectroscopy, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), 1D ¹³C and 2D heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopic techniques. Semiquantitative HSQC NMR showed that the relative amounts of β-O-4′ (around 76 % side chains) and resinol type substructures (16 %) of lignins were significantly modified during hydrothermal pretreatment. Py-GC/MS analyses brought direct evidences of these lignin samples with high S/G ratios ranging from 1.7 to 2.6. Moreover, the results indicated that an increase in the severity of the hydrothermal pretreatment enhanced the degradation of lignin unit side chains and the condensation of lignin and decreased the molecular weight of the recovered lignin fractions. This study demonstrated that the combination of autohydrolysis and alkaline ethanol process could potentially turn the recovered lignin fractions into value added products being in accordance with the “biorefinery” concept.     

Degradation of C-Labeled Lignins and C-Labeled Aromatic Acids by Fusarium solani

Abilities of isolate AF-W1 of Fusarium solani to degrade the side chain and the ring structure of synthetic dehydrogenative polymerizates, aromatic acids, or lignin in sound wood were investigated under several conditions of growth substrate or basal medium and pH. Significant transformations of lignins occurred in 50 days in both unextracted and extracted sound wood substrates with 3% malt as the growth substrate and the pH buffered initially at 4.0 with 2,2-dimethylsuccinate. Degradation of lignin in such woods also occurred under unbuffered pH conditions when a basal medium of either 3% malt or powdered cellulose in deionized water was present. Decomposition of the lignin in these woods did not occur in cultures where d-glucose was present as a growth substrate. F. solani significantly transformed, as measured as evolved CO(2), both synthetic side chain (beta, gamma)-C- and U-ring-C-labeled lignins in 30 days under liquid culture conditions of only distilled deionized water and no pH adjustment. Degradation of dehydrogenative polymerizates by F. solani was reduced drastically when D(2) was the liquid medium. AF-W1 also cleaved the alpha-C from p-hydroxybenzoic acid and evolved CO(2) from the substrate, [3-C]cinnamic acid. Thus, the fungus cleaved side chain carbon from substrate that originally lacked hydroxyl substitution on the aromatic nucleus. Surprisingly, small amounts of C cleaved from aromatic acids by F. solani were incorporated into cell mass. Initial buffering of the culture medium to pH 4.0 or 5.0 with 0.1 M 2,2-dimethylsuccinate significantly increased F. solani degradation of all lignins or aromatic acids. Results indicated that AF-W1 used lignin as a sole carbon source.     

Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H₂O₂, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date. Abbreviations: 4CL – 4-coumarate:CoA ligase; C3H –p-coumarate 3-hydroxylase; HCT –p-hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyltransferase; CCoAOMT – caffeoyl-CoA O-methyltransferase; CCR – cinnamoyl-CoA reductase; F5H – ferulate 5-hydroxylase; CAld5H – coniferaldehyde 5-hydroxylase; COMT – caffeic acid O-methyltransferase; AldOMT – (5-hydroxyconifer)aldehyde O-methyltransferase; CAD – cinnamyl alcohol dehydrogenase; NMR – nuclear magnetic resonance (spectroscopy); DFRC – derivatization followed by reductive cleavage; TIZ – tosylation, iodination, zinc (a DFRC method); DHP – dehydrogenation polymer.     

Biosynthesis of the sesquiterpenoid ageratriol

Ageratriol is biosynthesized from agerol through a diepoxide derivative. Mevalonic acid incorporation revealed that the formation of the isopropenylic double bond is not stereospecific.     

Comparative Analysis of Lignins of Various Plant Forms by 31P NMR Spectroscopy

Russian Journal of Bioorganic Chemistry - Lignin is one of the most abundant biopolymers. Information about the functional composition and structure of various lignins may be useful in the study of...     

Elicitor-Induced Spruce Stress Lignin (Structural Similarity to Early Developmental Lignins)

Suspension cultures of Picea abies (L.) Karst released polymeric material into the culture medium when treated with an elicitor preparation from the spruce needle pathogen Rhizosphaera kalkhoffii. The presence of lignin (about 35%, w/w) was demonstrated by phloroglucinol/HCI reactivity and quantitation with thioglycolic acid. Carbohydrate (about 14%, w/w) and protein (about 32%, w/w) were also detected. Amino acid analysis revealed that hydroxyproline and proline predominated. Thioacidolysis and subsequent Raney nickel desulfurization allowed the analysis of lignin-building units and interunit bonds. Compared with spruce wood lignin, an approximately 20-fold higher relative amount of p-hydroxyphenyl units was determined. A high content of p-hydroxyphenyl units is typical for certain developmental lignins, such as conifer compression wood and middle lamella lignins, as well as all induced cell culture lignins so far analyzed. Cross-linkages of the pinoresinol type ([beta]-[beta]) in the excreted cell culture lignin were markedly increased, whereas [beta]-1 interunit linkages were decreased relative to spruce wood lignin. The amount and nature of cross-linkages were shown to be intermediate between those in wood lignin and in enzymatically prepared lignins. In summary, the elicitor-induced stress lignin was excreted as a lignin-extensin complex that closely resembled early developmental lignins.