Polymer properties of softwood organosolv lignins produced in two different reactor systems

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

Microbial degradation of lignin

The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.     

Changes in lignin content of leaf litters during mulching

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content. Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.     

Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis

Caffeic acid 3-O-methyltransferase (COMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, and is believed to occupy a pivotal position in the lignin biosynthetic pathway. A cDNA (TaCM) was identified from wheat and it was found to be expressed constitutively in stem, leaf and root tissues. The deduced amino acid sequence of TaCM showed a high degree of identity with COMT from other plants, particularly in SAM binding motif and the residues responsible for catalytic and substrate specificity. The predicted TaCM three-dimensional structure is very similar with a COMT from alfalfa (MsCOMT), and TaCM protein had high immunoreactive activity with MsCOMT antibody. Kinetic analysis indicated that the recombinant TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leads to S lignin via aldehyde precursors. Authority of TaCM encoding a COMT was confirmed by the expression of antisense TaCM gene in transgenic tobacco which specifically down-regulated the COMT enzyme activity. Lignin analysis showed that the reduction in COMT activity resulted in a marginal decrease in lignin content but sharp reduction in the syringl lignin. Furthermore, the TaCM protein exhibited a strong activity towards ester precursors including caffeoyl-CoA and 5-hydroxyferuloyl-CoA. Our results demonstrate that TaCM is a typical COMT involved in lignin biosynthesis. It also supports the notion, in agreement with a structural analysis, that COMT has a broad substrate preference.     

Universal fractionation of lignin–carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood

It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin–carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non‐wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan‐enriched LCC (glucomannan‐lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan‐enriched fraction (glucan‐lignin, GL), a mannan‐enriched fraction (GML) and a xylan‐enriched fraction (xylan‐lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in ¹H NMR, ¹³C CP‐MAS NMR and normal‐ or high‐sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis‐GC, thioacidolysis‐SEC and pyrolysis‐GC.     

Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

Fractal analysis of STM images of lignin polymer obtained by in vitro synthesis

Lignin, the structural polymer of the plant cell walls, is produced by free radical polymerization of phenolic alcohols, catalyzed by different peroxidases. The mechanism and the structural organization of lignin in the cell have not been completely understood. In this study we applied fractal analysis to images of lignin polymer obtained using scanning tunneling microscope. The analysis showed the regularity of the polymer at different levels of organization. According to the results obtained, at the 95% confidence level, there is no significant difference in the fractal dimension between images representing different organizational levels of lignin. In other words, lignin produced in in vitro conditions has fractal structural organization and, consequently the polymer can be expected to be regular in in vivo conditions. The value of the fractal dimension 1.929 +/- 0.021 is in good agreement with the theoretically predicted value for polyaddition and polycondensation mechanism of polymerization. The mechanism of in vivo lignin synthesis is discussed in terms of various experimental and theoretical evidences. In this paper, we could show that fractal analysis of the lignin polymer is a useful complementary approach to the experimental data collection in structural and phenomenological studies.     

Dual methylation pathways in lignin biosynthesis

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.     

Milled wood lignin: a linear oligomer

The degree of polymerization (DP) of softwood and hardwood milled wood lignin samples and their branching degrees were quantitatively evaluated by a novel end-group titration approach composed of QQ-HSQC, (31)P NMR, and DFRC coupled with (31)P NMR analysis techniques. The DP of lignin can be calculated when the C9 formula, the amounts of phenolic groups, pinoresinol (β-β), diphenylethane (β-1), and phenolic diphenyl (5-5') lignin subunits have been determined. Data on the degree of polymerization of lignin obtained by NMR techniques were not affected by supramolecular aggregation processes. (31)P NMR analysis coupled with DFRC and QQ-HSQC allowed a detailed evaluation of the occurrence of condensed units in lignin and showed the terminal nature of diphenyl ether and diphenyl subunits. The resulting data unequivocally show that milled wood lignin is a linear oligomer.     

Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation.

Practical utilization of the polysaccharides in the lignocellulosic complex is limited because of the high lignin content of the complex. In this study we focused on the effect of Mn on lignin and cellulose biodegradation during solid-state fermentation by the edible mushroom Pleurotus ostreatus. Preferential degradation of lignin was enhanced by the addition of Mn(II) to cotton stalks at concentrations ranging from 30 to 620 micrograms of Mn per g. This effect was most apparent when we compared mineralization rates of [14C] lignin with mineralization rates of [14C] cellulose. Enhanced selectivity was also observed when we analyzed residual organic matter at the end of the fermentation period by using crude fiber analysis. The cellulose fraction in the original material was 1.8 times larger than the cellulose fraction of lignin. The cellulose/lignin ratio increased during 32 days of solid-state fermentation from 2.5 in the control to 3.3 following the addition of Mn to the medium. The in vitro digestibility value for fermented cotton stalks was 53% of the dry matter. Addition of 600 micrograms of Mn per g to the cotton stalks resulted in a digestibility value of 65.4%. Enhancement of preferential lignin degradation could be result of either increased activity of the ligninolytic enzymes or production of Mn (III), which might preferentially degrade aromatic structures in the lignocellulosic complex.     

A new analysis of the depolymerized fragments of lignin polymer using ToF-SIMS

Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.     

Enzymatic synthesis of lignin-siloxane hybrid functional polymers

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by 2?Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.     

O/N-alkyl and alkyl C are stabilised in fine particle size fractions of forest soils

Carbon stocks and organic matter composition in bulk soils and particle size fractions of Ah horizons from Luvisols, Leptosols and Phaeozems under European beech (Fagus silvatica L.) forest were investigated by elemental analysis, solid state ¹³C nuclear magnetic resonance (¹³C CPMAS NMR) spectroscopy and lignin analysis (CuO-oxidation). Radiocarbon age was used as an indicator for C turnover. The SOM of bulk soils and particle size fractions is dominated by O/N-alkyl C and alkyl C. Compared to sand and silt fractions, clay fractions had lower C/N ratios and ¹⁴C abundances. Aryl C and more specifically phenolic components (O-aryl C) decreased from sand to clay fractions. The concomitant decrease of lignin, determined by CuO oxidation, suggests that a major proportion of O-aryl C can be attributed to lignin. Positive nonlinear relations between the O-aryl C and the C/N ratio reveal the trend of decreasing O-aryl C proportions with increasing decomposition. Although lignin is believed to be highly recalcitrant, only low amounts of lignin are found in the stable clay fractions. In contrast to O-aryl C, the O/N-alkyl C contribution decreased from sand to silt fractions, but increased again in the clay fractions, whereas alkyl C contents exhibited lowest values in the sand fractions. These results are indicative of stabilisation processes operating specifically on polysaccharides and alkyl C, but not on aryl C, through association with the clay fraction.     

Lignin peroxidase: toward a clarification of its role in vivo

The extracellular lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium is thought to play an important role in lignin biodegradation. However, the majority of lignin-derived preparations actually experience overall polymerization at the hands of the enzyme in vitro. It has now been found that, in the presence of H2O2 at pH 4.0, the monomeric lignin precursor coniferyl alcohol is polymerized quantitatively by a lignin peroxidase preparation which is uncontaminated with MnII-dependent peroxidases. 13C NMR spectrometry of the resulting dehydropolymerisates from 13C-labeled monolignols confirms that the frequencies of different interunit linkages are very similar to those engendered through the action of horseradish peroxidase with H2O2. Indeed, lignin peroxidase does not ultimately seem to be a prerequisite for lignin degradation in vivo, yet its activity can still accelerate the conversion of lignin-derived preparations by P. chrysosporium to CO2. Consequently, lignin peroxidase can provisionally be expected to fulfill two important functions. On the one hand, the enzyme may detoxify lower molecular weight phenolic compounds released from lignins during their fungal decomposition. On the other hand, through the introduction of suitable functional groups, lignin peroxidase could indirectly enhance the susceptibility of macromolecular lignin structures toward depolymerization by another enzyme.     

Valorization of an industrial organosolv–sugarcane bagasse lignin: Characterization and use as a matrix in biobased composites reinforced with sisal fibers

In the present study, the main focus was the characterization and application of the by‐product lignin isolated through an industrial organosolv acid hydrolysis process from sugarcane bagasse, aiming at the production of bioethanol. The sugarcane lignin was characterized and used to prepare phenolic‐type resins. The analysis confirmed that the industrial sugarcane lignin is of HGS type, with a high proportion of the less substituted aromatic ring p‐hydroxyphenyl units, which favors further reaction with formaldehyde. The lignin–formaldehyde resins were used to produce biobased composites reinforced with different proportions of randomly distributed sisal fibers. The presence of lignin moieties in both the fiber and matrix increases their mutual affinity, as confirmed by SEM images, which showed good adhesion at the biocomposite fiber/matrix interface. This in turn allowed good load transference from the matrix to the fiber, leading to biobased composites with good impact strength (near 500 J m^?1 for a 40 wt% sisal fiber‐reinforced composite). The study demonstrates that sugarcane bagasse lignin obtained from a bioethanol plant can be used without excessive purification in the preparation of lignocellulosic fiber‐reinforced biobased composites displaying high mechanical properties. Biotechnol. Bioeng. 2010;107:612–621. © 2010 Wiley Periodicals, Inc.     

Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii

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.     

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.     

Comparison of different pretreatment methods for lignocellulosic materials. Part II: Influence of pretreatment on the properties of rye straw lignin

Several processes have been suggested to convert various types of lignocellulosic biomass into lignin products and saccharides. This paper evaluates the suitability of an organosolv process, a process using soda, a hydrothermal process and a process developed in this work, called the “Aquasolve process” for inclusion into a lignocellulosic biorefinery concept. Part II of this paper investigates the influence of the different pretreatment processes on the properties of rye straw lignin and evaluates their ability to produce high recoveries of high quality lignin.Specifications for high quality lignin products are defined and the isolated lignin fractions are analysed by Klason lignin, carbohydrate and ash content, elemental analysis, thermo-gravimetric analysis, ³¹P NMR, and size exclusion chromatography. The organosolv process shows the largest lignin recovery, followed by the soda and Aquasolve processes. Lignin products from the soda process, the Aquasolve process and with reservation the organosolv process show interesting properties for polymer applications.     

The role of of laccase in lignification

The enzymatic mechanism of monolignol polymerization in lignin biosynthesis is not known, although it has been the subject of significant interest for more than 60 years. Peroxidase had been considered to be the exclusive plant enzyme involved in the oxidative polymerization of lignin precursors. Recently, laccase and laccase-like oxidase activities have been associated with lignification. Laccase is bound to lignifying plant cell walls and can polymerize lignin precursors in vitro . Strong circumstantial evidence from different species implicates this enzyme in the polymerization of lignin precursors. Lignin has a complex structure and it has been difficult to analyze the heterogeneity of lignin by chemical and physical techniques. If lignin precursors are polymerized by enzymes that differ in their catalytic properties, then lignin heterogeneity could be produced by differential expression of multiple enzymes during plant development. When laccase genes are correctly identified, these ideas can be tested in genetic experiments where gain or loss of function can be predicted by the presence or absence of the functional gene.