Guaiacyl-syringyl Lignin Research Articles

Cork cellular and chemical features underlying bark environmental protection in the miombo species Parinari curatellifolia

Parinari curatellifolia is an important evergreen tree from the Miombo woodland of south-central and eastern Africa. The bark is corky, suggesting an increased protection against the ecosystem high temperatures and drought conditions as well as against wild fires. The cork in the bark rhytidome of P. curatellifolia was analyzed here for the first time with a focus on chemical and cellular features. P. curatellifolia cork has the cellular characteristics of cork tissues, with typical honeycomb structure in the tangential section and a brick-wall layer in the transverse and radial sections, without intercellular voids. Chemically P. curatellifolia cork has 8.4 % extractives, 33.9 % suberin, 31.9 % lignin and 25.2 % polysaccharides of the cork. The hemicelluloses are mostly xylans, with a substantial proportion of arabinose and galactose. Suberin showed a proportion of long chain lipids to glycerol (LCLip:Gly, mass ratio) of 8.5, and the long chain monomeric composition included a similar proportion of α,ω-diacids and ω-hydroxy acids (35.4 % and 31.5 % of long chain monomers) with a substantial proportion of monoacids (19.4 % of long chain monomers). Lignin is a guaiacyl-syringyl lignin with S/G of 0.32 and H:G:S of 1:14.1:4.5. The rhytidome composition and the cellular and chemical features of its cork are in line with environment-targeted protective features namely as a transpiration and insulation barrier, and as an increased fire protection.

A tunable deep eutectic solvent-based processing for valorization of chestnut wood fiber as a source of ellagic acid and lignin

Three different deep eutectic solvent (DES)-based extraction procedures were rationally designed and optimized for the recovery of antioxidants from chestnut wood fiber (CWF), a clean and largely available solid waste of the tannin industry. First, a mild protocol was developed using a choline chloride (ChCl)/tartaric acid DES at 50 °C, for 90 min. Ellagic acid (EA) was identified as the only low molecular weight phenolic component of the extract. In other experiments, harsher conditions were explored involving treatment of CWF with ChCl-based DESs at 120 °C for 8 h, which afforded a solid sample characterized by high phenolic content (up to 1.0 mg of gallic acid equivalents/mg of sample) and antioxidant properties (EC50 <0.025 mg/mL in the 2,2-diphenyl-1-picrylhydrazyl assay), and containing guaiacyl-syringyl lignin along with EA. Based on these results, a sequential two-step DES-based treatment of CWF was eventually designed, allowing to selectively obtain both an EA-enriched and an EA-free, lignin-enriched sample, with an overall 50% w/w of the starting CWF dissolved. In particular, a 2.3% w/w yield of EA was achieved, which is significantly higher than those reported in the case of DES-based processing of other agricultural wastes. The proposed tunable, straightforward, and eco-friendly approach may allow to fully exploit CWF as a green, cheap, and easily accessible source of high-value products.

The regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube

Fruit lignification is due to lignin deposition in the cell wall during cell development. However, there are few studies on the regulation of cell wall lignification and lignin biosynthesis during fruit pigmentation. In this study, we investigated the regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube. The cellulose content decreased, while the lignin content increased in the winter jujube pericarp during pigmentation. Safranin O-fast green staining showed that the cellulose content was higher in the cell wall of winter jujube prior to pigmentation, whereas the lignin in the cell wall increased after pigmentation. The thickness of the epidermal cells decreased with pericarp pigmentation. A combined metabolomics and transcriptomics analysis showed that guaiacyl-syringyl (G-S) lignin was the main lignin type in the pericarp of winter jujube, and F5H (LOC107424406) and CCR (LOC107420974) were preliminarily identified as the key genes modulating lignin biosynthesis in winter jujube. Seventeen MYB and six NAC transcription factors (TFs) with potential regulation of lignin biosynthesis were screened out based on phylogenetic analysis. Three MYB and two NAC TFs were selected as candidate genes and further studied in detail. Arabidopsis ectopic expression and winter jujube pericarp injection of the candidate genes indicated that the MYB activator (LOC107425254) and the MYB repressor (LOC107415078) control lignin biosynthesis by regulating CCR and F5H, while the NAC (LOC107435239) TF promotes F5H expression and positively regulates lignin biosynthesis. These findings revealed the lignin biosynthetic pathway and associated genes during pigmentation of winter jujube pericarp and provide a basis for further research on lignin regulation.

Intensification of pre-treatment and fractionation of agricultural residues

Lignocellulose biomass is a unique renewable and sustainable source that can be used in energy generation, production of transport fuels and in chemical industries to replace petroleum-based resources. However, due to its recalcitrance to chemical and enzyme breakdown, it needs to be pre-treated prior to processing. In this study, ultrasound assisted ozone was used to pre-treat the agricultural processing residues corn cob and spelt husk. Following organosolv fractionation, about 90 % and 94 % of lignin with high purity (95 %) were recovered for corn cob and spelt husk with guaiacyl-syringyl lignin the major fraction from corn cob and guaiacyl lignin from spelt husk respectively. In addition, cellulose pulp of 83.6 % and 85.2 % with 78 % purity were recovered for corn cob and spelt husk respectively with a 19 % decrease in cellulose crystallinity and 17 % decrease in degree of polymerization (DP). Thus, the improved quality separation of biomass components will enhance downstream conversion to value added products thereby improving the sustainability and cost effectiveness of the process.

Structural characteristics of lignin extracted from Jordanian olive cake using different fractionation conditions

ABSTRACT The chemical structure and properties of lignin-derived from lignocellulosic olive mill waste (Jeft) were investigated. Alkali and dioxane lignins were evaluated regarding the achieved high delignification and high purity of the extracted lignins. The results declared that the major constituents of olive cake are extractives, cellulose, hemicelluloses, and lignin. Furthermore, the olive cake has low relative crystallinity and the ash content does not exceed 2%. The yields of Alkali and Dioxane lignins were 15.3 ± 0.34, and 1.4 ± 0.43%, respectively. For the elucidation of structural features of lignin molecules, lignins samples were also subjected to UV spectroscopy and 1HNMR analysis. The analysis showed that the treatment under acidic condition did not significantly attack the β-aryl ether structure (β-O-4), while the alkali treatment shows an extensive cleavage of the β-aryl-ether linkage with the degradation of ester resulting in a structural alteration of lignin. High Delignification is achieved by dioxane process through progressive enrichment of carboxylic acid group in isolated lignin; also dioxane isolation has little influence on the structure of isolated lignin. Moreover, the results confirmed that Alkali and Dioxane lignin were guaiacyl-syringyl lignin, typical of hardwood lignins.

Characteristics of guaiacyl-syringyl lignin in reaction wood in the gymnosperm Gnetum gnemon L.

Abstract Gnetum gnemon L. is a unique gymnosperm species showing angiosperm-like features in terms of its morphology and chemical composition of the cell wall. Xylan is the main hemicellulose component, and its lignin is primarily composed of syringyl (S) and guaiacyl (G) units and small amounts of p-hydroxyphenyl (H) units. In the present study, in addition to branch, root, bark, and leaf samples, the reaction wood (RW) taken from the leaning stem of G. gnemon, was investigated mainly by alkaline nitrobenzene oxidation, ozonation and NMR spectroscopy. The leaning stem was wider on the lower side of the wood stem (lsW) than on the upper side (usW), similar to the case for compression wood (CW) in gymnosperms. The usW contained lignin with a higher S/G ratio, and β-O-4 structure had a higher erythro/threo ratio, while both ratios decreased around the periphery of the stem towards the lsW. The lignin content was higher towards the lsW. Overall, the lignin composition in the RW of this tree was similar to that in the tension wood of angiosperms. The H-units were minor components in the lignin, but the content was higher towards the lsW, which resembles the distribution of the H-units in a gymnosperm CW.

Structural characterization of lignin: A potential source of antioxidants guaiacol and 4-vinylguaiacol

The structure of lignin obtained from the ozone and soaking aqueous ammonia pretreatment of wheat straw has been characterized utilizing chemical analytical methods in order to reveal its antioxidant characteristics, including attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), pyrolysis/tetramethylammonium hydroxide-gas chromatography/mass spectrometry (Py/TMAH-GC/MS), gel permeation chromatography (GPC), ultra violet–visible spectroscopy (UV–vis), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant evaluation assay. The results demonstrated that the isolated lignin is a ρ-hydroxyphenyl- guaiacyl-syringyl (H-G-S) lignin, with S/G ratio of 0.35 and significant amounts of phenol 2-methoxy (guaiacol) and phenol 2-methoxy-4-vinyl (4-vinylguaiacol). The Py-GC/MS and Py/TMAH-GC/MS pyrograms indicated that the major units in this lignin are derived from hydroxycinnamic acids. The GPC results revealed the molecular weight of the lignin was considerably low and also the FTIR analysis showed that the lignin possessed hydroxyl and methoxy functional groups; the factors led to the extracted lignin having a comparable antioxidant activity to that of currently used commercial antioxidants. The UV–vis and DPPH antioxidant assay results suggested a percentage of inhibition of the DPPH radicals in the following order: guaiacol (103.6±1.36)>butylated hydroxytoluene (103.3±1)>ferulic acid (102.6±0.79)>pretreated lignin (86.9±0.34).

Extraction of flax shive using sodium ethoxide catalyst in anhydrous ethanol

This work investigated the yield and nature of solvent-soluble organic compounds extracted from flax shive using a room temperature reaction (20 °C) with sodium ethoxide catalyst at four different concentrations (0.2, 0.5, 0.7, and 1.0 M) in anhydrous ethanol. Results were compared with the use of aqueous sodium hydroxide (1.0 M) at two different reaction temperatures (20 °C and 100 °C). Quantitative yield from flax shive varied linearly with sodium ethoxide concentration and averaged 54.5 mg/g on a dry-mass basis (db) at 1.0 M. In contrast, the quantitative yield using 1.0 M sodium hydroxide was much lower, averaging 2.2 mg/g (db). Yield did not differ significantly due to changes of particle size in either case, or due to changes of temperature over the range considered in the case of sodium hydroxide. Analyses using proton nuclear magnetic resonance ( 1H NMR) confirmed all extracts to contain aromatic compounds, thus likely lignin derived, but found differences in chemical characteristics between the two extraction methods. One key difference was the presence of compounds with methyl ether groups in sodium hydroxide extracts that were absent in the case of sodium ethoxide extracts. Given that flax contains a mixed guaiacyl–syringyl lignin, methyl ether groups would be expected to be present. Control reactions on three model compounds were carried out to confirm that transesterification occurred with sodium ethoxide. These control reactions also demonstrated that methyl ether groups would be expected to remain intact under the extraction conditions reported here. In light of the higher yield of solvent soluble compounds recovered by extraction with basic ethanol, flax shive may represent a source of value-added phenolic constituents. This processing method may also represent a useful pre-treatment prior to the production of biofuels by cellulose degrading organisms.

Separation and Structural Characterization of Lignin from Hybrid Poplar Based on Complete Dissolution in DMSO/LiCl

In this paper, the physicochemical properties and structural features of six lignin preparations separated with a total dissolution of ball-milled wood in dimethyl sulfoxide and lithium chloride (DMSO/LiCl) followed by extraction with ethanol/water were investigated. These isolated lignin fractions were characterized using wet chemical analysis, FT-IR, and 1H and13C-NMR techniques. Experimental results showed that separated lignin preparations were relatively free of associated polysaccharides. These lignin fractions were classified as guaiacyl-syringyl lignin type: mainly composed of guaiacyl units with noticeable amounts of syringyl units and fewer p-hydroxyphenyl units. The molar ratio of non-condensed guaiacyl units to syringyl units (G/S) decreased as the ratio of LiCl to poplar weight increased. The results also showed that these lignin preparations consisted mainly of β-O-4 ether bonds combined with small quantities of β-β′ and β-5 carbon-carbon linkages. Furthermore, considerable amounts of esterified p-hydroxybenzoic acids and minor amounts of esterified p-coumaric acid were also detected in these lignin fractions.

Lignin characteristics of peculiar vascular plants

Trochodendron aralioides Sieb. et Zucc. and Chloranthus glaber (Thunb.) Makino [Sarcandra globra (Thunb.) Nakai] belong to primitive angiosperms, and are characterized by the absence of vessels. Ephedra sinica Stapf, which is classified in the gymnosperms, contains vessel elements and fiber together with tracheids. Podocarpus macrophyllus (Thunb. Ex Murray) D. Don var. macrophyllus is reported to give a weak positive Maule test, although it is classified as a gymnosperm. Podocarpus macrophyllus gave only vanillin on alkaline nitrobenzene oxidation, while E. sinica, T. aralioides, and C. glaber gave both vanillin and syringaldehyde. The molar ratio of syringyl to guaiacyl nuclei (S/V ratio) of C. glaber was significantly low (S/V = 0.08) in comparison with the value for T. aralioides (S/V = 1.56). The erythro form is the predominant diasteromeric form of β-O-4 intermonomer linkages of T. aralioides and E. sinica [molar ratio of erythro to threo forms (E/T ratio): 1.93 and 1.67, respectively]; however, P. macrophyllus and C. glaber lignins, of which S/V ratios were 0 and 0.08, gave approximately equal amounts of erythro and threo forms (E/T ratio: 1.01 and 1.14). Results from 1H NMR spectroscopy agree well with S/V ratios of alkaline nitrobenzene oxidation and E/T ratios of ozonation products. In conclusion differences in guaiacyl lignin and guaiacyl-syringyl lignin do not exactly reflect taxonomical differences, as reported in previous articles. It was found that guaiacyl-syringyl lignin is not necessarily linked to the presence of vessels.

Monolignol oxidation by xylem peroxidase isoforms of Norway spruce (Picea abies) and silver birch (Betula pendula)

We partially purified peroxidase isoform fractions from xylem extracts of a gymnosperm, Norway spruce (Picea abies (L.) Karst.), and an angiosperm, silver birch (Betula pendula Roth.), to determine the participation of xylem-localized peroxidases in polymerization of different types of lignin in vivo. Several peroxidase fractions varying in isoelectric point values from acidic to basic were tested for their ability to catalyze the oxidation of the monolignols coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol in vitro. All of the xylem peroxidases extracted from Norway spruce and most of those from silver birch showed the highest rate of oxidation with coniferyl alcohol in the presence of hydrogen peroxide. The exception was an acidic peroxidase fraction (pI 3.60-3.65) from silver birch that exhibited higher oxidation activity for sinapyl alcohol than for coniferyl alcohol. For the xylem enzyme fractions extracted from silver birch, the ability to oxidize the artificial phenolic substrate syringaldazine coincided with high specific activity for sinapyl alcohol. Therefore, we conclude that the acidic, neutral and basic xylem peroxidases of Norway spruce all function in the synthesis of guaiacyl-type lignin, whereas in silver birch the acidic peroxidases preferentially oxidize sinapyl subunits. The latter provides a mechanism for synthesis of guaiacyl-syringyl lignin typical of tracheid cell walls in angiosperm trees.

Pathways for monolignol biosynthesis via metabolic grids: coniferyl aldehyde 5-hydroxylase, a possible key enzyme in angiosperm syringyl lignin biosynthesis

Lignins are aromatic polymers of methoxylated phenylpropanoids connected by both ether and carbon-carbon linkages, and classified into three major groups, guaiacyl lignin in softwoods (gymnosperms), guaiacyl-syringyl lignin in hardwoods (angiosperms) and guaiacyl-syringyl-p-hydroxyphenyl lignin in grasses (gramineae) on their monomeric units. In grass lignins p-coumaric acid (5∼10%) is ester linked mostly at γ-position of the propyl side chain. Biochemical studies of lignification have elucidated that 1) lignins are formed by dehydrogenative polymerization of monolignols catalyzed by peroxidase and laccase, 2) biosynthesis of monolignols is via shikimate-cinnamate pathway, 3) syringyl lignin in hardwoods is formed from the syringyl monolignol, sinapyl alcohol, derived from 5-hydroxyguaiacyl precursors, and that 4) ferulate 5-hydroxylase, coniferyl aldehyde 5-hydroxylase and probably coniferyl alcohol 5-hydroxylase are involved in the biosynthesis of the syringyl monolignol from guaiacyl monolignol precursors in respective hardwoods.(Communicated by Saburo TAMURA, M. J. A., Oct. 14, 2003)

Caffeoyl-coenzyme A 3-O-methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development.

Flax (Linum usitatissimum) is a commercially important fiber crop in Europe. Lignification of its phloem fibers, although weak, causes a decrease in their commercial quality. In flax, fiber lignin mainly consists of guaiacyl (G) units in contrast to the mixed guaiacyl-syringyl (G-S) lignin type occurring in xylem fibers. G lignins are reported as more condensed polymers due to a higher frequency of 5-5 linkages, whereas the deposition of syringyl end groups in lignins increases the proportion of alky-aryl ether linkages as beta-O-4-bonds. The type of linkages within a lignin polymer depends on the methylation of either the 3-hydroxyl groups or both 3-OH and 5-OH groups, which is controlled by two enzymes: caffeate 3-O-methyltransferase (COMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT). First, we measured the in vitro activity of both OMTs in the flax stem tissues during stem development. CCoAOMT activity varied in the same way as COMT, i.e. increased gradually with stem maturity, from the top to the bottom of the stem, was maximum at the flowering stage and was lower, but still scorable, in the outer fiber-bearing tissues than in the xylem cells. In a second step, we focused our studies on the characterization of CCoAOMT in order to understand the implication of this enzyme in the lignification of flax fiber cells. CCoAOMT activity appeared to be associated with the accumulation of an acidic 33-kDa polypeptide identified as a CCoAOMT after immunological cross-reactivity with a poplar CCoAOMT and microsequencing. The differential accumulation of the CCoAOMT protein was confirmed by immunolocalization on tissue prints and correlated with that of the transcripts, suggesting a transcriptional regulation of CCoAOMT in the flax stem.

Characterization of the lignin obtained by alkaline delignification and of the cellulose residue from steam-exploded olive stones

The lignocellulosic by-products (whole stones and seed husks) obtained from pitted table olive and olive oil processing (respectively) were pretreated under various conditions of steam-explosion, with respect to pressure and time and with a dilute solution of sulfuric acid and without previous acid impregnation. The two materials exhibited considerable difference in behavior. The pre-impregnation of whole stones with low level of acid prior to steam treatment was necessary to improve the fractionation and the autohydrolysis of hemicellulose, while the results with seed husks were similar both with and without an acid catalyst. The lignins recovered from water-washed solid residue by alkaline-extraction followed by acidification were characterized. These lignin preparations from whole stones contained a very high amount of fat and almost no neutral sugars. The results also showed that these lignin preparations were guaiacyl-syringyl lignin, similar to exploded hardwood lignin, with an extensive cleavage of the β-aryl-ether linkage. The effect of treatment severity on the major physical properties of cellulose was also investigated. The cellulose fraction derived from steam-exploded whole stones was rapidly depolymerized as severity increased, leading to a degree of polymerization of 180, at a severity of log R 0 = 4.07. In seed husks, under similar severity conditions, depolymerization of cellulose was much slower, and only reached a value of 821 (at log R 0 = 4.07) or a value of 600 at log R 0 = 4.34 without acid impregnation. The cellulose derived from both steam-exploded materials was moderately crystalline, the relative crystallinity index values of seed husks being higher than those of whole stones. The results indicated that the cellulose from seed husks was more protected than that of the whole stones in the steam-explosion process, and could be used to produce a cellulose with different properties and applications depending on severity and on the different treated material.

Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides).

4-Coumarate:CoA ligases (4CLs, EC 6.2.1.12) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants. So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics. However, we have discovered two functionally and structurally distinct 4CL genes, Pt4CL1 and Pt4CL2 (63% protein sequence identity), that are differentially expressed in aspen (Populus tremuloides). The Escherichia coli-expressed and purified Pt4CL1 and Pt4CL2 proteins exhibited highly divergent substrate preference as well as specificity that reveal the association of Pt4CL1 with the biosynthesis of guaiacyl-syringyl lignin and the involvement of Pt4CL2 with other phenylpropanoid formation. Northern hybridization analysis demonstrated that Pt4CL1 mRNA is specifically expressed in lignifying xylem tissues and Pt4CL2 mRNA is specifically expressed in epidermal layers in the stem and the leaf, consistent with the promoter activities of Pt4CL1 and Pt4CL2 genes based on the heterologous promoter-beta-glucouronidase fusion analysis. Thus, the expression of Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen; Pt4CL1 is devoted to lignin biosynthesis in developing xylem tissues, whereas Pt4CL2 is involved in the biosynthesis of other phenolics, such as flavonoids, in epidermal cells.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Chemical properties of lignin from Aralia cordata

Lignin from Aralia cordata, was isolated and characterized spectrometrically and by chemical degradations. IR and 13C NMR spectra of the dioxan lignin showed a pattern of guaiacyl (G)-syringyl (S) lignin esterified with p-hydroxybenzoate. Alkaline hydrolysis of the lignin gave p-hydroxybenzoic acid. Molar ratios of the main syringyl to guaiacyl monomers recovered from alkaline nitrobenzene oxidation and from thioacidolysis are 1.3 and 1.2, respectively, indicating that A. cordata contains a guaiacyl-syringyl lignin, although the amount of syringyl lignin is a little lower than that in normal angiosperm wood.

Lignin, suberin, phenolic acids and tyramine in the suberized, wound-induced potato periderm

Formation of standard guaiacyl-syringyl lignin has been shown, by thioacidolysis, during healing and suberization of wound potato tuber discs. Recovery of p-hydroxybenzaldehyde after nitrobenzene oxidation has been related to the presence of tyramine. Variations in the relative proportion of hydroxyphenyl, guaiacyl and syringyl degradation products obtained after nitrobenzene oxidation, thioacidolysis and alkaline hydrolysis suggest the formation, during suberization, of a complex phenolic network linked to the cell wall.

A microanalytical approach to plant tissue characterization: A comparative study of healthy and fungus-infected carnation by pyrolysis-mass spectrometry

Minute samples of xylem from healthy and Fusarium oxysporum f.sp. dianthi-infected stems of carnation cultivars differing in degree of resistance to F. oxysporum were investigated by in-source pyrolysis low voltage EI mass spectrometry and by pyrolysis under ammonia CI conditions. Healthy xylem of both cultivars showed PY-MS spectra with markers for cellulose, hemicelluloses, phenolic acids, and a mixed guaiacyl-syringyl lignin. At different stages of infection visibly infected, brown coloured, xylem was separated under the microscope from healthy-looking parts. The PY-MS spectra of the diseased xylem of susceptible ‘Lena’ were entirely different from those of healthy(-looking) areas, showing a drastic change in lignin pyrolysis product distributions. In most cases practically all syringyl constituents, both mono- and dimeric, had disappeared. In some cases also an overall decrease in relative intensity of the lignin pyrolysis products was observed. Invasion of the fungus thus induced a major change of cell wall composition as a result of lignin degradation along different pathways: demethoxylation of syringyl units, the major pathway in ‘Lena’, and propenyl side chain oxidation. The PY-MS spectra of infected ‘Novada’ generally showed the overall oxidation of lignin and less demethoxylation. The ‘Novada’ spectrum also showed m/z 239, 287, and an increase of 257 and 271, which are molecular ions indicative of the phytoalexins dianthalexin and the dianthramides methoxydianthramide S, hydroxydianthramide B and methoxydianthramide B. The compounds appeared comparatively volatile and therefore could be desorbed as a thermal extract before other pyrolysis fragments appear. The compounds were practically absent in adjacent, healthy looking, xylem. Low voltage EI MS of methanol extracts of about 2 mm 3 of infected ‘Novada’ xylem confirmed these data and in a single case also showed evidence for hydroxyanthranilic acid ( m/z 153) which is attributed to degradation of dianthramine. Spectra of thermal and methanolic extracts were quite similar.

Localization of phytoalexin accumulation and determination of changes in lignin and carbohydrate composition in carnation (Dianthus caryophyllus L.) xylem as a consequence of infection with Fusarium oxysporum f. sp. dianthi, by pyrolysis-mass spectrometry

Minute pieces of xylem and other tissues from stems of healthy and fungus-infected plants of two carnation cultivars Novada and Lena were investigated for lignification (lignin/polysaccharide ratios) and lignin composition by means of pyrolysis mass spectrometry and pyrolysis gas chromatography mass spectrometry. This technique proved also very effective for the localization of dianthramide phytoalexins which accumulate in carnation after infection withFusarium oxysporum f.sp.dianthi. The composition of healthy tissues from both cultivars was practically the same. In the resistant cultivar Novada, infection induced a change from guaiacyl-syringyl lignin into a mainly guaiacyl lignin in the gum-occluded parts of the xylem. Considerable amounts of the phenolic amide dianthalexin and of other dianthramide phytoalexins were present in occluded xylem, but not in adjacent phloem, medulla or unoccluded xylem. Xylem from susceptible ‘Lena’ suffering degradation was characterized by a loss of syringyl groups from the lignin and by demethylation of pectin in an early stage of infection. Small quantities of dianthalexin and other dianthramide phytoalexins were found in ‘Lena’ when local defense responses (particularly occlusion) had occurred. In both cultivars evidence for degradation of hemicellulose was found.