Apparent inhibition to digestion by lignin in normal and brown midrib stems

Lignin, a cell wall component, limits digestibility of plant cell walls. Brown midrib (bmr) mutants of forages have lignin with altered chemical composition compared with their normal counterparts. The objectives of this study were to determine if bmr lignin is more inhibitory to digestion than is normal lignin and if bmr has a consistent effect on rate of digestion across species and environments. Extent and rate of in-vitro cell wall digestion of normal and bmr stems of sorghum (Sorghum bicolor (L) Moench, two comparisons), millet (Pennisetum americanum (L) Leeke) and maize (Zea mays L, two comparisons) were determined. Samples were incubated in rumen fluid, and data were fitted with a first-order, nonlinear model to estimate concentrations of potentially digestible neutral detergent fibre (PDNDF), digestion rate of PDNDF, concentration of indigestible residue (IR), and lag time before digestion. The NDF, acid-detergent fibre (ADF), and acid-detergent lignin (ADL) analyses were conducted sequentially on undigested samples. The IR: ADL ratio was 37% greater for bmr than for normal plants, which indicates that bmr lignin inhibits digestion more than normal lignin per unit of lignin. Digestion rate of PDNDF was faster in bmr than in normal counterparts in one of the two sorghum comparisons (difference of 59%) and in the millet comparison (difference of 27%), but in neither maize comparison. The bmr mutants were lower than normal genotypes in NDF (9%) and ADL (47%) concentrations. The PDNDF concentration was 19% greater for bmr than for normal lines. Thus, decreased lignin concentration in bmr mutants increased the extent of NDF digestion but did not consistently increase the rate of digestion.     

Cell wall composition and degradability of forage stems following chemical and biological delignification

Chemical and biological delignification methods were used to investigate the relationship between the concentration and composition of lignin and degradation of forage cell walls. Stem material from lucerne (Medicago sativa L), smooth bromegrass (Bromus inermis Leyss) and maize (Zea mays L) stalks was treated with alkaline hydrogen peroxide, potassium permanganate, sodium chlorite, sodium hydroxide, nitrobenzene, and the lignolytic fungus Phanerochaete chrysosporium. Klason lignin and esterified and etherified phenolic acids were delermined. Cell wall neutral sugar and uronic acid composition and the extent of in-vitro degradability were measured. Chemical delignification generally removed lignin. but the fungal treatment resulted in the removal of more polysaccharide than lignin. The concentrations of esterfied and etherified p-coumaric and ferulic acids were generally reduced in treated cell walls; chlorite treatment preferentially removing p-coumaric acid whereas nitrobenzene treatment removed more ferulic acid. Syringyl moieties were completely removed from the core lignin polymer by nitrobenzene treatment of forage stems. Alkaline hydrogen peroxide and nitrobenzene were generally the most effective delignification treatments for improving polysaccharide degradability, with the grass species responding similarly to delignification whereas lucerne was somewhat less responsive. Fungal delignification, under these experimental conditions, did not improve cell wall degradability of these forages. Multiple regression and covariate analyses indicated that the lignin components measured were not powerful predictors of cell wall degradability. Neither the concentration nor the composition of the lignin fractions was consistently correlated with degradation. This lack of effect was attributed to the more generalised disruption of the cell wall matrix structure by delignification treatments.     

Phenolic constituents of cell wall types of normal and brown midrib mutants of pearl millet (Pennisetum glaucum (L) R Br) in relation to wall biodegradability

The cell walls of parenchyma, rind and vascular bundle fractions of pearl millet (Pennisetum glaucum (L) R Br) were isolated from two brown midrib mutants (bmr) 5753 and 5778 and from their normal (N) near-isogenic line. The cell wall content of parenchyma was lower than that of vascular bundle which, in turn, was lower than that of rind. The amounts of ferulic and p-coumaric acids released by NaOH treatment of the cell walls were in the ranges 3-7 mg g^?1 and 2-26 mg g^?1, respectively. Parenchyma cell walls of the N line had the highest content of p-coumaric acid (26 mg g^?1). This content of p-coumaric acid in the N line contrasts with that of bmr 5753 parenchyma (2 mg acid g^?1 walls) and bmr 5778 (7 mg acid g^?1 walls). The concentration of p-coumaric acid was highest in parenchyma cell walls that had been found to be the least digested. Parenchyma, rind and vascular bundle cells walls of the N line had much higher ratios of p-coumaric acid to ferulic acid than the mutants; rind and vascular bundle walls were less digestible than parenchyma. Small amounts of truxillic acid dimers were released by NaOH from the parenchyma walls of bmr 5778. Treatment of parenchyma, rind and vascular bundle cells walls with purified ‘driselase’ (containing xylanases and cellulases) released p-coumaroyl and feruloyl trisaccharides. Between 25 and 53% of the ferulic acid that was released by the NaOH treatment could be accounted for as feruloyl trisaccharide, but only 1-19% of the p-coumaric acid was accounted for as p-coumaroyl trisaccharide.     

Digestibility of silages in relation to their hydroxycinnamic acid content and lignin composition

BACKGROUND: The effectiveness of the analysis of cell wall‐bound hydroxycinnamic acids and the composition of lignin to evaluate the in vivo digestibility of a silage collection with unknown botanical composition was evaluated. RESULTS: Syringyl units content and total etherified phenols showed the highest correlation coefficients with in vivo dry matter digestibility (IVDMD) (r = ? 0.792 and r = ? 0.703, respectively), while guaiacyl units and total phenols showed the highest correlation coefficients with in vivo organic matter digestibility (IVOMD) (r = ? 0.871 and r = ? 0.817, respectively). Using the above‐mentioned chemical parameters, 10 equations were also developed to predict in vivo digestibility. The prediction of IVDMD produced a high adjusted R² value (0.710) using syringyl, total lignin, etherified total phenols, esterified ferulic acid and total phenol content as predictors. The prediction of IVOMD produced a higher adjusted R² value (0.821) using guaiacyl, total phenols, total ferulic acid and etherified p‐coumaric acid content as predictors. CONCLUSION: Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, syringyl and guaiacyl content and etherified phenols emerge as good predictors of digestibility. Copyright © 2010 Society of Chemical Industry     

Lignification of switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii) plant parts during maturation and its effect on fibre degradability

Five cultivars of switchgrass (Panicum virgatum L) and four cultivars of big bluestem (Andropogon gerardii Vitman) were harvested at vegetative, boot and heading stages of maturity. Leaf and stem fractions were analysed for detergent fibre composition and 48-h ruminal in-vitro degradability, ester- and ether-linked non-core lignin phenolic acids, and core lignin composition. Big bluestem leaves contained more neutral detergent fibre than switchgrass, but general composition of the fibre did not differ. Stem fibre of switchgrass had relatively lower levels of cellulose and lignin at the vegetative stage than observed in big bluestem. Esterified and etherified p-coumaric and ferulic acid concentrations were generally higher in switchgrass plant parts. Yield of nitrobenzene oxidation products from core lignin was greater for switchgrass leaves, but very little difference in composition was noted. Leaf tissue contained lower concentrations of all lignin components than stems. Maturation resulted in increased total lignification, but all components did not respond in the same manner. Variation for all measures of lignification seemed to be as great within species as between the grass species. Degradability of fibre declined with maturation. The only species difference was that switchgrass fibre was more degradable at the vegetative stage. Relationships between lignification and fibre degradability were in agreement with some, but not all, previously reported relationships. Concentration of core lignin was only a significant predictor of fibre degradability when the relationship was examined across maturity stages. Within a stage of maturity, lignin composition was more closely related to fibre degradability than was concentration. However, the best predictors of fibre digestibility differed among species, plant part and maturity.     

Impact of lignin composition on cell‐wall degradability in an Arabidopsis mutant

An Arabidopsis mutant that does not deposit syringyl‐type lignin was used to test the hypothesis that lignin composition impacts cell‐wall degradability. Two lines of the ferulate‐5‐hydroxylase‐deficient fah1 mutant and the wild‐type control line were grown in the greenhouse. In Experiment 1, the plants were harvested at the mature seed stage. For Experiment 2, plants were harvested 5, 6, 7 and 8 weeks after sowing. In both experiments stems were collected and analysed for cell‐wall concentration and composition, and in vitro degradability of cell‐wall polysaccharide components by rumen micro‐organisms. The absence of syringyl‐type lignin was confirmed for the mutant lines by nitrobenzene oxidation and pyrolysis‐GC‐MS. Lignin concentration was the same for all three Arabidopsis lines, at all stages of maturity. The Arabidopsis stems were similar to forage legumes in that the potentially degradable cell‐wall fraction was very quickly degraded. Cell‐wall polysaccharide degradability did not differ among the Arabidopsis lines in the first experiment after 24‐h fermentations, but the cell‐wall polysaccharides of the fah1‐2 mutant line were less degradable after 96‐h than either the wild‐type or the fah1‐5 mutant. In contrast, in Experiment 2 no differences among lines were found for cell‐wall polysaccharide degradability after either 24‐ or 96‐h fermentations; however, signficantly higher levels of ester‐bound ferulic acid were found in the walls of the fah1 mutant lines. As expected, increasing stem maturity was correlated with reduced degradation of cell‐wall polysaccharides. These experiments indicate that either lignin composition, as measured by syringyl‐to‐guaiacyl ratio, does not alter cell‐wall degradability in Arabidopsis, or that the fah1 mutation has other effects on the cell walls of these mutants such that the impact of the change in syringyl‐to‐guaiacyl ratio is masked. © 1999 Society of Chemical Industry     

Phenolic acids released from bermudagrass (Cynodon dactylon) by sequential sodium hydroxide treatment in relation to biodegradation of cell types

Sections of solvent-extracted bermudagrass (Cynodon dactylon L Pers) leaf blades were treated sequentially with increasing concentrations of sodium hydroxide. The amounts of saponifiable phenolic acid monomers and cyclobutane dimers released and the digestibility of the treated blades (ie % dry weight loss) were determined. Leaf sections were examined by scanning electron microscopy for biodegradation of cell types and histochemically (light microscopy) for lignin after treatment with sodium hydroxide. Treatment with 0.1 m sodium hydroxide for 1 h resulted in only minor changes from untreated sections. However, this treatment for 24 h released 86% of the ferulic acid, 65% of the dimers, and c 50% of the p-coumaric acid. Digestibility was increased from 6.5% in the untreated control to 56.6%. Substantial loss of the slowly biodegradable tissues (ie epidermis and parenchyma bundle sheath) and partial biodegradation and disruption of the refractory tissues (ie sclerenchyma, xylem and mestome sheath) occurred; histochemical reactions for lignin were less intense after NaOH treatment. Treatment with 1 m sodium hydroxide for 24 h released 50% of the p-coumaric acid and the remainder of the alkali-extractable ferulic acid and dimers, increased digestibility to 72%, and increased biodegradation of mesophyll and phloem. Mestome sheath cell walls only gave a histochemical reaction for phenolics and the reaction was weak after 1 m NaOH treatment. Alkali treatment increased the biodegradation of all cell types, with lignified tissues reduced to single-cell fibres after 1 and 2 m treatments.     

Relationship of cell wall composition to in vitro cell wall digestibility of maize inbred line stems

The phenolic equipment of maize stem tissues was investigated in relation to the feeding value of the detergent fibre components. Sixteen maize inbred lines, including three brown‐midrib 3 mutants and their normal counterparts, were selected for highly divergent in vitro cell wall digestibility. These lines were grown during two years. Maize stems were analysed for detergent fibre concentration, esterified and etherified p‐hydroxycinnamic acids, lignin content and structure and in vitro digestibility. A large genotypic variation was found for neutral detergent fibre, cell wall phenolic composition and cell wall digestibility. Within the normal maize lines the in vitro neutral detergent fibre digestibility (IVNDFD) of stem fractions was negatively correlated with their Klason lignin content. A multiple regression model based on esterified p‐coumaric acid and lignin composition as two explanatory variates accounted for 58% of the IVNDFD variation. In this study, three normal maize inbred lines displaying a lignin content and a cell wall digestibility level close to those observed in the three bm3 lines could be detected, which opens up new breeding avenues. © 2000 Society of Chemical Industry     

Determination of phenolic acids in plant cell walls by microwave digestion

Microwave digestion (750 W for 90 s) with 4 M NaOH was used to release esterified and etherified hydroxycinnamic acids from cell walls of maize (Zea mays L), wheat (Triticum aestivum L), barley (Hordeum vulgare L) and oilseed rape (Brassica napus L) stems. Subtraction of values for saponifiable phenolic acids obtained after treatment with I M NaOH at room temperature from digest results provided a measure of β-ether linked units. These were exclusively (E + Z)-ferulic acid in the cereal straws. Only trace amounts of ether- and ester-linked hydroxycinnamic acids were released from the dicotyledon, ripe straw. Microwave digestion was shown to be an order of magnitude more effective than dioxane-HCl at liberating β-ether bound phenolic acids and as effective, but substantially quicker, than previously described high-temperature alkaline digestions.     

Structure and digestibility of tissues in normal and brown midrib pearl millet (Pennisetum glaucum)

The tissues in leaf blades, midribs, sheaths and stems of normal (N), 5848, and two brown midrib (bmr) mutants, 5753 and 5778, of pearl millet (Pennisetum glaucum (L) R Br) were examined for structural characteristics related to digestibility. The anatomies of the various plant parts were not substantially different between N and bmr plants. The more rigid, lignified tissues such as vascular bundles in all plant parts and the rind of stems were not digested after incubation for 7 days in N or bmr plants, indicating that modifications in these tissues were not sufficient to affect biodegradation. Modifications in the digestible tissues resulted in faster and more extensive degradation in bmr plants, and these changes appear to be the most significant in relation to biodegradation. The parenchyma of midveins and stems, which occupies c 70 and 60% of the cross-sectional area of these respective parts, were the sites where modification in phenolics resulted in the greatest improvement in digestibility.     

Modification of in-vitro digestibility and cell-wall composition of cocksfoot,ryegrass and timothy by imazethapyr

Recent studies have shown that the compound imazethapyr (5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid) possesses growth regulatory activity and can be used to enhance nutritive value of grasses. However, little is known about possible effects of this material on fibre composition or potential fibre utilisation by ruminants. The objective of this research was to examine imazethapyr-treated cocksfoot (Dactylis glomerata L), ryegrass (Lolium perenne L × L multiflorum Lam), and timothy (Phleum pratense L) for possible treatment-induced changes in in-vitro digestibility and in concentrations of selected fibre constituents. Replicated field plots treated with imazethapyr at 0 (control) or 100 g ai ha^?1 during the vegetative (pre-elongation) stage of growth were harvested when controls were in the early stage of inflorescence emergence. Samples taken from treated plots 4 weeks post-treatment had higher in-vitro dry matter disappearance (IVDMD), lower neutral detergent fibre (NDF), lower acid detergent fibre (ADF), lower cellulose, lignin, and higher hemicellulose concentrations compared with controls. Imazethapyr treatment also reduced the concentration of p-coumaric acid in NDF, but had no effect on ferulic acid. The treatment effect on in-vitro NDF disappearance, however, was inconsistent among the grass species and was statistically non-significant (P = 0·10), implying that, under the conditions of this study, imazethapyr-related enhancements in IVDMD can be attributed primarily to a reduction in the amount of NDF and its associated constituents, as opposed to qualitative differences in NDF composition.     

The Use of an Extracellular Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids During Mashing

A purified extracellular ferulic acid esterase from Lactobacillus acidophilus K1 was successfully used during mashing for the release of free phenolic acids into sweet wort. The enzyme was produced in bioreactors and partially purified to obtain the monoenzyme preparation. Release of free ferulic and vanillic acid into the wort at 52°C (with the use of 4.09–14.60 enzyme activity units/L of the mash) and ferulic acid at 62°C (14.60 units/L) was observed. Free p‐OH‐benzoic and syringic acids were effectively released at 26°C at each enzyme concentration used. Free p‐OH‐benzoic acid was also released by the enzyme (14.60 U/L) at 52°C‐74°C. Free protocatechuic acid was effectively hydrolyzed by the enzyme preparation (8.75 U/L and 14.60 U/L) at 26°C‐52°C. Free caffeic acid (effectively released at 26°C‐62°C) originated from chlorogenic acid. No p‐coumaric acid was released due to the action of bacterial esterase during mashing. Ferulic acid esterase from L. acidophilus K1 exhibited no ability to release free phenolic acids during mashing at 62°C or at 74°C due to its low thermostability. In conclusion, L. acidophilus K1 is an attractive source for the production of ferulic acid esterase, which can be useful for the release of antioxidant phenolic acids in the early stages of mashing.     

粪产碱菌对阿魏酸厌氧降解的影响

粪产碱菌在以阿魏酸为唯一碳源的厌氧发酵中培养7 d,阿魏酸的降解率约为70%;研究秸秆厌氧发酵产气（以产气量反应菌群的活性）发现,体系接种5%粪产碱菌后,菌群的活性最强,生物气增量最大,达130 mL,比接种3%和7%粪产碱菌的体系提高85.71%和116.67%;同时体系的产酸效率和阿魏酸的降解率均显著提升,分别比接种3%和7%粪产碱菌的体系提高136%和110.71%以及25%和33.33%。傅里叶红外光谱检测表明：厌氧发酵体系接种5%粪产碱菌后秸秆中木质素、阿魏酸的特征官能团结构被有效破坏。秸秆厌氧发酵体系接入粪产碱菌可以有效降解阿魏酸等木质素降解衍生物、解除木质素及其降解产物对厌氧菌群的毒性同时提高产气效率,具有应用价值。     

Cell wall phenolics and polysaccharides in different tissues of quinoa (Chenopodium quinoa Willd)

Quinoa (Chenopodium quinoa Willd) is an Andean pseudo-cereal, of the Chenopodiaceae family, which is currently being studied for introduction in Northern Europe as an alternative to industrial crops. The aim of this work was to verify existence in quinoa of the distinctive cell wall features identified in other Chenopodiaceae, ie presence of pectin-bound ferulic acid and dehydrodiferulic acids. Alcohol-insoluble solids (AIS) were prepared from leaves, stems and roots of mature quinoa plants, representing 0.10, 0.20 and 0.47 g g⁻¹ respectively of the fresh weight. Ferulic acid and dehydrodiferulic acid derivatives were present in all the organs, with the highest concentrations in the leaves with 2.1 and 0.5 mg g⁻¹ AIS respectively. The ratio of dehydrodiferulic acid to ferulic acid was highest in the roots. Pectins extracted by hot HCl from AIS of leaves were rich in ferulic acid (3.4 mg g⁻¹), but also highly acetylated (DAc 20), and rich in rhamnose, two characteristics encountered in other Chenopodiaceae. © 1999 Society of Chemical Industry     

Investigation of the ester- and ether-linked phenolic constituents of cell wall types of normal and brown midrib pearl millet using chemical isolation,microspectrophotometry and 13C NMR spectroscopy

The cell walls of rind, parenchyma and vascular bundle fractions of pearl millet (Pennisetum glaucum (L) R Br) were isolated from two brown midrib mutants and their normal (N) near-isogeneic line. The walls were sequentially treated with 1 M NaOH at 25°C for 20 h to determine ester-linked phenolic acids and then with 4 M NaOH at 170°C for 2 h to determine ether-linked phenolic constituents. The untreated walls and their residues resulting from each treatment were analyzed by microspectrophotometry and ¹³C NMR spectroscopy. The amount of ester-linked p-coumaric acid was determined by chemical analysis and found to be two to six times higher in the N line with no difference among lines in ferulic acid content. Ether-linked ferulic acid was about 30% higher in the rind in the N line and ether-linked p-coumaric acid was only slightly higher with the greatest difference found in the rind tissue. Microspectrophotometry of the untreated tissues showed absorption maxima at 232–238 nm. 288–292 nm and 312–324 nm. Treatment with 1 M NaOH generally reduced or eliminated the 312–324 nm absorption, with 4 M NaOH removing the remainder of the 288–292 nm absorption. ¹³C NMR confirmed these reductions of aromatic functionalities by alkali treatments. The combination of techniques provides excellent correlation of two types of spectral data with chemical identification and quantitation and establishes that bmr mutants have less ester-linked p-coumaric acid and less ether-linked ferulic acid, thus providing a better understanding of the factors contributing to biodegradability.     

Characterisation of Lignin from CAD and OMT Deficient Bm Mutants of Maize

Internodes of the maize cell line W401 and bm₁ and bm₃ mutants expressed in W401 were harvested 5 days after anthesis (A5) and at silage (S) stage. The normal maize had a higher total phenolic (TP) content (80·5–90·5 g kg^-1 cell wall DM) than both bm₁ and bm₃ mutants (74·4–86·4 and 66·0– 84·2 g kg^-1 cell wall DM, respectively). TP were inversely related to cellulase digestibility with values of 85·4–91·5, 89·3–92·1 and 91·3–94·1% for normal, bm₁ and bm₃. Marked differences in p-coumaric acid concentrations were found ranging from 20·9 to 26·3 g kg^-1 cell wall DM for normal, 14·9 to 15·3 g kg^-1 for bm₁ to 10·1 to 14·4 g kg^-1 for bm₃. The ferulate pattern was entirely different with the bm₁ genotype providing the lowest total (9·1–10·7 g kg^-1) and etherified (1·9–2·3 g kg^-1) values. Although the bm₃ contained more total ferulate (11·5–13·1 vs 10·9–11·7 g kg^-1), the normal variety had a significantly greater amount of etherified ferulate (2·8–3·4 vs 3·2–4.1 g kg^-1) implying a greater extent of cross-linking between wall polymers. Recovery of guaiacyl and syringyl residues was greatest in the normal maize with the bm₁ occupying the middle position between the two extremes. Calculated S: G ratios from 4 M NaOH digestion and NMR were in good agreement with the normal line giving the highest ratio, bm₁ intermediate and bm₃ the lowest. Colorimetric analysis revealed a large increase in the aldehyde content of the in situ bm₁ lignin compared to normal and bm₃ genotypes although NMR failed to reveal significant numbers of aldehydic resonances. © 1997 SCI.     

Microspectrophotometric characterization of aromatic constituents in cell walls of hard and soft wheats

Cell walls of epidermis, hypodermis, nucellar epidermis, aleurone, and endosperm in hard and soft wheat (Triticum aestivum L) kernels were evaluated for aromatic constituents using ultraviolet (UV) absorption microspectrophotometry. Wheat varieties sampled were soft red winter Caldwell, hard red winter varieties Tam 200 and Karl, and hard red spring varieties Len and Yecora rojo. Spectra of cell walls of epidermis and hypodermis (beeswing cells) suggested larger amounts and a greater degree of polymerization of aromatic constituents in these walls compared to those of other cell types. Results also suggested the presence of ester-linked ferulic acid. Nucellar epidermal walls also had polymerized aromatics and ferulic acid, but amounts appeared to be much less than in the walls of the beeswing cells. UV spectra of aleurone walls suggested that the predominant aromatic was ester-linked ferulic acid; anticlinal walls had substantially more aromatic constituents than outer or inner periclinal walls. Endosperm walls lacked any indication of aromatics by this method. Grain hardness did not appear to be related to cell wall aromatics. UV absorption microspectrophotometry indicated significant variations in wall aromatics of different cell types of wheat grains.     

Fractionation and characterization of ball‐milled and enzyme lignins from abaca fibre

An Erratum has been published for this article in Journal of the Science of Food and Agriculture 79(15) 1999, 2122. Ball‐milled and enzyme lignins were produced from abaca fibre via ball milling for 6 days followed by cellulase treatment for 3 days. The crude lignin preparations were fractionated into milled lignin (ML), enzyme lignin (EL), hemicellulose‐rich milled lignin (HRML), and lignin‐rich enzyme lignin (LREL) fractions using a two‐step precipitation method instead of a traditional ether precipitation procedure. The yield and chemical composition of the resulting lignin samples are reported. The ML and EL fractions contained low amounts of associated neutral sugars (2.0–3.3%) and uronic acids (1.4–1.5%), and showed relatively low average molecular weights (2500–2660), while the LREL and HRML fractions contained large amounts of bound polysaccharides (35.6–38.3%), and showed high molecular weights (8800–25000). The four lignin fractions are composed of a large proportion of syringyl units with fewer guaiacyl and p‐hydroxyphenyl units. The ML is mainly composed of β–O–4 ether bonds between the lignin structural units. The less common β–β, β–5 and 5–5′ carbon–carbon linkages are also present in the lignin molecules. It was found that uronic acids and 41–63% of p‐coumaric acids are esterified to lignin in the three lignin‐rich fractions of ML, EL and LREL. This level increased to over 90% in the hemicellulose‐rich fraction of HRML. For ferulic acids, 92–97% were found to be etherified to lignin in the three lignin‐rich fractions of ML, EL and LREL, while in the hemicellulose‐rich fraction of HRML this reduced to only 13%, suggesting that a majority of the ferulic acids are esterified to hemicelluloses or lignin in this fraction. © 1999 Society of Chemical Industry     

Biological variability in lignification of maize: Expression of the brown midrib bm3 mutation in three maize cultivars

Cell wall-linked phenolics were investigated in maize internodes located at three positions of the stem (top-middle-bottom). While the lignin content did not change drastically with position, the amount of cell wall-ester linked p-coumaric acid sharply increased from the top to the bottom internodes of the stem. Conversely, the saponified ferulic acid content remained relatively unchanged along the stem. Moreover, the highest syringyl content of the β-O-4-lignin structures was found in the basal (most mature) internode. Therefore, enhanced p-coumaric esterification of the cell wall and preferential deposition of syringyl units in the lignin polymer might indicate an extended maturity stage of the cell wall of maize internodes. The bm3 mutation in the three maize hybrids is expressed by lignin reduction and ester-bound p-coumaric decrease in the mutant lines. Furthermore, all bm3 hybrids synthesised lignin polymers which were characterised by a very low S/G molar ratio (0.16-0.43). This ratio originates from the substantial reduction of the syringyl unit content in β-O-4-lignin structures compared with the normal lignin. The occurrence in the same range of the 5-hydroxyguaiacyl unit in bm3 lignin from each type of hybrids was noteworthy, demonstrating the high heritability of the bm3 mutation at the molecular level. The alkaline solubility of lignin was greater for the three mutant lines compared to the normal cultivars. Furthermore, the alkali-labile fraction of lignin of both normal and mutant lines had a monomeric composition which was consistent with the non-condensed structures of in-situ lignin.     

Biological variability in lignification of maize: Expression of the brown midrib bm2 mutation

The cell wall phenolic components in the internodes of three maize genotypes, namely normal, bm2 and bm3 maize, were determined. The bm2 and bm3 brown midrib mutations lowered the lignin content of the bottom, middle and top internodes to a similar extent. However, unlike bm3, the bm2 trait did not induce a sharp reduction of the level of ester-bound p-coumaric acid in maize internodes. The other main alkali labile phenolic acid, ferulic acid, reached similar levels in the three genotypes. The main difference between bm2 and bm3 mutations occurred in the alkyl aryl ether linked structures of the lignin component. In contrast to bm3 lignins, which are characterised by a low syringyl content, the bm2 lignin had a lower content of guaiacyl units than lignin of normal maize internode. Consequently, the syringyl/guaiacyl molar ratio of bm2 lignin gave higher values (2.7–3.2) than those from either normal (0.9–1.5) or bm3 lignins (0.3). The alkali solubility of lignin was also compared between the three genotypes. Incorporation of the bm3 trait in maize led to a high recovery of alkali soluble lignin whereas the bm2 lignin had a similar solubility to the normal one in 2 M NaOH. The monomeric composition of the alkali soluble lignins was consistent with the non-condensed structures of the in-situ polymer. Although the bm3 and bm2 mutations had different effects on lignification, the modification of the cell wall phenolic level was also found in the bm2 maize stem as previously studied.