Effect of down-regulation of cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems

The effect of down-regulation of tobacco cinnamyl alcohol dehydrogenase (CAD) on cell wall composition and degradability has been assessed. CAD activity was only 20, 16, 14 and 7%, relative to the controls, in four populations of plants (designated 40-1, 40-2, 48 and 50, respectively) transformed with CAD antisense mRNA. Cell wall residues of stem samples were analysed for polysaccharide composition, gravimetric and acetyl bromide lignins and lignin nitrobenzene oxidation products. In situ disappearance and cellulase solubility of both initial dry matter and CWR were determined. The populations of plants with depressed CAD activity showed no change in lignin content but some consistent changes in cell wall composition and digestibility were identified. The syringyl content of lignins decreased and the syringaldehyde to vanillin ratio (S/V) was consequently reduced. Dry matter degradability, as measured by both methods, was significantly improved in all CAD-depressed samples except for population 40-1, which was the least CAD-depressed. Increased in situ disappearance of cell wall (ISCWD) was found in all plants exhibiting more than 80% CAD down-regulation and was maximal (7 percentage units) in population 50 which had the greatest CAD depression. The rates of ISCWD increased slightly in some populations (40-2 and 50). The relationship between S/V and ISCWD was significant (R = -0·68) only in the samples from a selected population of mature, most depleted plants. Other modifications may therefore also contribute to the improvement in degradability. However the changes in lignin composition that were observed in CAD-depressed tobacco are largely similar to those seen in some maize and sorghum mutants with altered lignification and improved digestibility. These data therefore suggest that depressing CAD activity may be an effective method for improving digestibility in forage crops. © 1998 SCI.     

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     

Variations in anatomy and ultraviolet microspectrometry between normal and brown midrib mutant maizes possessing different rumen degradabilities

Normal and brown midrib mutant (bmr) maize (Zea mays L) were examined for variations in their morphological composition. The degradability of the leaf blade, leaf sheath and stem, proportional area of specific tissues in leaf blade, and the ultraviolet (UV) absorption spectra of cell walls were measured and related to variations in cell wall degradability by rumen microorganisms. The UV and infrared (IR) absorption spectra of the lignins isolated from leaf blades of both types, before and after reduction with sodium borohydride, were recorded. The bmr3 maize had higher dry matter (DM) and neutral detergent fibre (NDF) degradabilities for leaf blade, leaf sheath, and stem than the normal counterpart. Approximately 35% and 26% of the observed difference in DM degradability was attributed to the difference in DM degradability of stem and leaf blade, respectively, and 39% to the difference in DM composition of stem. Distinct differences in tissue degradation of the leaf blades were observed for mesophyll cell walls in the midrib portion, which were thinner and of greater number in the bmr3 maize. Sclerenchyma cells were present only in the vascular bundles in the bmr3 leaf blade, while in the normal type those cells were underneath the epidermis tissue. The bmr3 plant also had large epidermal cells. UV microspectrometry of mesophyll cell walls of the bmr3 maize showed their lower UV absorbance around 320 nm compared to that of the normal, but not at 280 nm. Considerable increase in the UV absorbance at 280 nm was observed for the isolated lignins after reduction, suggesting a lesser degree of lignification in the bmr3 maize tissues. Lowered UV absorbance of the isolated lignin around 320 nm after reduction was associated with the removal of the IR bands at 1730, 1660, 1600, and 1250 cm^?1.     

14C labelling of lignins of normal and bm3 maizes for fermentation studies

[U-¹⁴C] phenylalanine (phe*) and [O¹⁴CH₃] sinapic acid (sin*) were infused into the cut ends of normal and bm3 maizes (anthesis stage) under or above the last node or at mid-internode, with or without the leaf, in light or in darkness. Radioactivity was measured in the organs, and in phenolic constituents of the cell wall and saponified residues of the bases and tops of the apical inter-node. In both maize genotype labelled under the node the radioactivity was distributed more evenly in the organs with sin* than with phe*. Infusion above the node and at mid-internode greatly increased radioactivity in the bases and tops, respectively. Removal of the leaf only slightly increased the radioactivity, mainly in the bases, and no clear-cut effect of darkness was observed. Phe* labelled the phenolic acids and the three lignin units, but the syringyl units of bm3 maize were only slightly labelled. Sin* specifically labelled the syringyl units, which represented the least condensed fraction of lignins. Both the native and labelled lignins were highly alkali soluble. There were differences in lignin biogenesis between the bases and tops, and between normal and bm3 maizes. The newly formed lignins were slightly different from the native lignins but had similar types of heterogeneity, with variations in the internode and between genotypes similar to those in native lignins. Provided due allowance is made for the distinguishing characteristics of newly formed lignins, the [¹⁴C-lignin] cell walls, which are strongly labelled on complementary structures, seem suitable model substrates for fermentation studies.     

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.     

Cell wall fermentation kinetics are impacted more by lignin content and ferulate cross‐linking than by lignin composition

BACKGROUND: We used a biomimetic model system to ascertain how reductions in ferulate–lignin cross‐linking and shifts in lignin composition influence ruminal cell wall fermentation. Primary walls from maize cell suspensions with normal or reduced feruloylation were artificially lignified with various monolignols previously identified in normal, mutant, and transgenic plants. Cell wall fermentability was determined from gas production during in vitro incubation with rumen microflora and by analysis of non‐fermented polysaccharides. RESULTS: Hemicellulose fermentation lag time increased by 37%, rate decreased by 37%, and the extent declined by 18% as cell wall lignin content increased from 0.5 to 124 mg g^?1. Lignification increased lag time for cellulose fermentation by 12‐fold. Ferulate–lignin cross‐linking accounted for at least one‐half of the inhibitory effect of lignin on cell wall fermentation. Incorporating sinapyl p‐coumarate, a precursor of p‐coumaroylated grass lignin, increased the extent of hemicellulose fermentation by 5%. Polymerizing varying ratios of coniferyl and sinapyl alcohols or incorporating 5‐hydroxyconiferyl alcohol, coniferaldehyde, sinapyl acetate, or dihydroconiferyl alcohol into lignin did not alter the kinetics of cell wall fermentation. CONCLUSION: The results indicate that selection or engineering of plants for reduced lignification or ferulate–lignin cross‐linking will improve fiber fermentability more than current approaches for shifting lignin composition. Copyright © 2008 Society of Chemical Industry     

Cyclodimers of p-coumaric and ferulic acids in the cell walls of tropical grasses

Cell walls from leaves and stems of three tropical grasses (Setaria anceps cv Nandi (Schumach) Moss), pangola grass Digitara decumbens Stent and spear grass Heteropogon contortus (L) Beauv ex Roemer & Schultes were extracted with alkali, and ether-soluble fractions were prepared from the acidified solutions. Cyclobutane dimers derived from p-coumaric acid (CA: 3-(4-hydroxypheny) propenoic acid) and ferulic acid (FA: 3-(4-hydroxy-3-methoxyphenyl) propenoic acid) were found in all plant residues. Structural information on the dimers was obtained by gas chromatography-mass spectrometry. Stem cell walls differed from those of leaf in having major dimer components which contained residues tentatively identified as coniferyl alcohol (3-(4-hydroxy-3-methoxyphenyl)-2-propen-l-ol). Species differences in leaf cell wall cyclodimers were evident. Pangola and setaria grasses had dimers mainly derived from FA, whereas spear grass had over 70% of dimers derived from CA. Di-esterification of some dimers in the cell walls was confirmed by the release of dimers containing the reduced forms of CA and FA (coumaryl and coniferyl alcohols respectively) when leaf cell walls were extracted with borohydride. Head to tail dimerisation giving derivatives of truxillic acid (t-2, c-4-diphenyl-r-l, t-3-cyclobutanedicarboxylic acid) predominated, although evidence was obtained for the presence of head-to-head coumaric acid dimers (derivatives of truxinic acid, t-3, t-4-diphenyl-rl, c-2-cyclobutanedicarboxylic acid) in setaria stem and spear grass leaf. The results suggested that cyclodimers in grass cell walls occurred in cross-linking structures of varying complexity between macromolecules, where they would possibly contribute to fibre strength and thus have an adverse efect on the nutritional value of forage.     

Roles of structural phenylpropanoids in forage cell wall digestion

Phenolic constituents (lignins and phenolic acids) and carbohydrates are assembled in a tight architecture which differs according to the plant species. During cell wall digestion, the hydrolysis kinetics differ between carbohydrates and seem to depend chiefly on the content and organisation of tissue phenolics. Among the phenylpropanoids, ferulic acid is released more quickly than p-coumaric acid. Lignins remain largely in the cell walls. They also undergo transformations, chiefly solubilisation as lignin-carbohydrate complexes. The limiting effect of lignins on cell wall degradation increases with increasing content. However, their effect on degradation might also depend on qualitative factors such as lignin structure and polymer organisation in walls and tissues. When various grasses (normal and selected genotypes), or grasses and legumes are compared, correlations between certain factors such as lignin uncondensed fraction, syringyl units or phenolic acids contents and cell wall degradation emerge but not clear causal relationship has been shown. Nonetheless, other structural characteristics, related to the alkali reactivity of lignins, seem to have a stronger influence on cell wall degradation. Phenylpropanoids seem to act mainly as a physical and (bio)chemical barrier to the action of the microbial enzymes. In addition, their reactivity as phenolic compounds and their hydrophobicity seem to play a role. Digestion is not limited only by phenolics. The factors that limit glycanolysis—the accessibility, crystallinity and capillary structure of cellulose and the branching of hemicelluloses—seem to have little or no effect on cell wall degradation in vivo. In contrast, other antiquality substances (tannins, cutin and silica), plant antomy, environmental factors, factors modulating microbial growth and animal physiology influence cell wall utilisation. Future research in this field should focus on the effects of phenolic structure and of cell wall and tissue organisation on carbohydrate degradation.     

Impact of accessibility and chemical composition on cell wall polysaccharide degradability of maize and lucerne stems

Although lignification of forages is generally accepted as limiting cell wall degradability, prediction of degradation from cell wall composition is often difficult when forages are of similar maturity. It has been proposed that rumen microbe accessibility to potentially degradable cell walls is limited by the presence of non‐disrupted cells in forage particles with lignified middle lamella/primary walls acting as barriers to microbial access. We tested this accessibility hypothesis by evaluating the impact of reducing particle size of maize and lucerne stems to the level of individual cells by ball‐milling, in order to eliminate accessibility as a limiting factor. While cell wall concentration and composition were not influenced by ball‐milling compared with grinding to pass a 1 mm screen in a cyclone‐type mill, degradability of total cell wall polysaccharides was dramatically increased. However, only those polysaccharides (cellulose and xylan) which are most abundant in cell types with lignified middle lamella/primary and secondary walls increased in degradability owing to particle size reduction. Degradability of pectins, which are abundant in non‐lignified tissues in lucerne, did not respond to ball‐milling. Contrary to our expectations, ball‐milled forages showed fewer correlations for cell wall composition with degradability than observed for the larger‐particle‐size grinding treatment. Many components of the cell wall were correlated with polysaccharide degradation for the cyclone‐ground samples; however, the results were inconsistent as to which cell wall components were correlated with degradation among and within forages. This observation does not clarify the role of cell wall chemical structure as a limiting agent to wall degradation in the absence of accessibility barriers, but this study does provide support for the hypothesis that lignified middle lamella/primary walls act as barriers to microbial access for degradation. © 2000 Society of Chemical Industry     

The newly extended maize internode: A model for the study of secondary cell wall formation and consequences for digestibility

The upper five internodes were collected from maize (Zea mays L) inbred cell lines Co 125 and W401 harvested at the same developmental stage, 5 days after silking. Each internode was dissected into ten equal lengths labelled A (top) to J (base). The youngest cells were found in section J, which contained the intercalary meristem, and the oldest in section A. Internodes 1, 3 and 5 provided material for chemical analysis and internodes 2 and 4 for degradability measurements. Cell wall material accounted for one-third of dry matter in section J, doubling to two-thirds in the upper half of each internode. Only section J exhibited a polysaccharide profile typical of primary cell walls. In all other sections, 1,4-linked glucose (± 46% of cell wall) and xylan largely free from side chains (± 25% of cell wall) predominated. Net accretion of cell wall polysaccharide reached a maximum by segment G and thereafter little additional carbohydrate was deposited. Lignification appeared to be separated from the biogenesis of structural carbohydrate and continued over much of each internode reaching a maximum in section C. Degradability measurements, made using a modified neutral-detergent cellulase digestibility method, showed substantial differences between sections. In line Co 125, cell wall degradability fell from over 95% in the youngest section (J) to approximately 24% in section B. Internode 4 of line W401 failed to show the same pattern of degradabilities, probably because of a sequential rather than simultaneous pattern of internode elongation. Saponifiable p-coumaric acid appeared to provide a more sensitive marker than lignin of the extent of secondary wall development. The inverse relationship between extent of lignification in each section and its degradability confirmed the value of the internode model for the study of secondary wall formation and its biological consequences.     

Effects of an exogenous xylanase gene expression on the growth of transgenic rice and the expression level of endogenous xylanase inhibitor gene RIXI

BACKGROUND: Xylanases have attracted considerable interest in recent years owing to their various applications in industry and agriculture. The use of transgenic plants to produce xylanases is a less expensive alternative to biotechnological programmes. The aim of this study was to elucidate whether introducing a foreign xylanase gene ATX into rice had any adverse effect on plant growth and development. RESULTS: A recombinant xylanase gene ATX was introduced into rice variety Zhonghua 11 through Agrobacterium‐mediated transformation. The T₂ generation of transgenic rice was compared with the control (non‐transgenic plants). Exogenous xylanase gene ATX was expressed in rice, and all examined transgenic lines exhibited xylanase activity. The transgenic lines (T₂, ‘X1‐3’ and ‘X2‐5’) appeared to grow and develop normally. There were no differences in net photosynthetic rate between transgenic rice lines (‘X1‐3’ and ‘X2‐5’) and wild type (WT) rice plants at the heading/flowering stage. Xylanases are key enzymes in the degradation of plant cell walls. Cell wall composition analysis showed that that there were no changes in cell wall polysaccharides in the root apex but some alterations in leaves in transgenic rice plants. The results also showed that the expression of exogenous xylanase gene ATX in rice would increase the expression of endogenous xylanase inhibitor gene RIXI, which could play a role in plant defence. Thus the stress resistance of transgenic rice plants might be improved. CONCLUSION: Exogenous xylanase gene ATX could be successfully expressed in rice, and the exogenous protein had no apparent harmful effects on growth and development in transgenic rice plants. Copyright © 2012 Society of Chemical Industry     

Rates of degradation of plant cell walls measured with a commercial cellulase preparation

The relationship between the degradability, determined with a commercial cellulase preparation, of the cell walls of various plant parts of Italian ryegrass, maize and red clover can be expressed as Y = A - Be^?k₁^t-Cek₂^t, where Y = percentage of cell walls degraded, t = reaction time, k₁ and k₂ are rates of degradation, and A, B, and C are constants where A = B + C. Degradability of the cell walls of Italian ryegrass or maize could be predicted accurately from the absorbance of the filtrate at λ_max 282-288 or 310-324 nm. Treatment of cell walls of barley straw with 0.1 or 1M sodium hydroxide for 7 or 20 h degraded between 12 and 41% of the walls and led to the release of p-coumaric and ferulic acids, the amount increasing with concentration of alkali and treatment time; the less concentrated alkali released more ferulic than p-coumaric acid. Treatment with the cellulase preparation of the residues from alkali treatment showed that they were almost twice as degradable as the untreated walls.     

Transformations of (14C-lignin) cell walls of wheat by rumen microorganisms

The lower halves of apical internodes of wheat harvested at the flowering stage were labelled with [U-¹⁴C] phenylalanine (phe) or with [O¹⁴CH₃] sinapic acid (sin). Cell wall residues (CWR) and saponified residues (SR) were incubated in a fermenter simulating the rumen for 7 days with rumen fluid or without microorganisms (controls). PheCWR was labelled in all lignin units (measured as aldehydes from nitrobenzene oxidation), in phenolic acids and slightly in proteins. Labelling of pheSR was more lignin-specific. SinCWR and sinSR were specifically labelled in syringyl units of lignin. The fermentation of CWR resulted in phenylpropane-derived unit losses in the following decreasing order: ferulic acid>p-coumaric acid>syringaldehyde>vanillin>p-hydroxybenzaldehyde. If allowance is made for slight losses in controls, 61, 52, 61 and 63% of the phenylpropanes of pheCWR, sinCWR, pheSR and sinSR, respectively, were transformed into an acid-precipitable fraction, an acid-soluble fraction and ¹⁴CO_2. The comparison of pheCWR and sinCWR degradation showed that syringyl units were solubilised into acid-precipitable molecules to a greater extent than the other lignin units; demethylation of the syringyl units of lignins was also evident from the different productions of ¹⁴CO₂. Alkali-resistant lignins of SR were mainly transformed into acid-precipitable molecules and were weakly degraded. Lignin solubilisation and degradation seem to be governed by different mechanisms which depend on both cell wall structure and rumen microflora.     

Phenolic components and degradability of the cell walls of the brown midrib mutant,bm3, of Zea mays

Cell walls separated from the leaf blade, leaf sheath and stem of the brown midrib mutant, bm₃, of Zea mays were more degradable by a commercial cellulase than the corresponding part of the isogenic normal inbred cultivar (Tr). The walls of each part of the mutant when compared with the corresponding part of the normal cultivar contained less lignin and bound phenolic components released by treatment with NaOH. The major phenolic components detected were trans-p-coumaric and trans-ferulic acids together with small amounts of their cis isomers and diferulic acid. Cell walls of stem of the mutant contained a total of 17.3 mg g^?1 of these bound acids compared with 9.8 mg g^?1 for leaf sheath and 3.5 mg g^?1 for leaf blade: there was more than twice as much p-coumaric acid in cell walls of stem as in those of leaf sheath and more than seven times as much as in those of leaf blade. When cell walls of the stem from the mutant or the normal cultivar were treated with NaOH their degradability by cellulase was highly correlated with the amounts of phenolic components released by the alkali.     

Volatile production in tomato fruit with modified alcohol dehydrogenase activity

Using a low shear maceration procedure, headspace volatiles were collected and analysed from tomato fruit which had been genetically modified to enhance or reduce the activity of alcohol dehydrogenase 2 (ADH2). Fruit with enhanced activity showed up to three times the activity of control plants while down‐regulated fruit showed about 3% of the control ADH activity. The aldehyde to alcohol ratios for (Z )‐3‐hexenal : (Z)‐3‐hexenol and 3‐methylbutanal : 3‐methylbutanol were calculated and shown to differ for down‐regulated fruit compared to control fruit. Up‐regulation showed no significant increase in either ratio. The amounts of (Z)‐3‐hexenol or 3‐methylbutanol were not significantly increased by up‐regulation. Down‐regulation caused a significant decrease in the amount of (Z )‐3‐hexenol but no increase in (Z )‐3‐hexenal. In contrast, the amount of 3‐methylbutanal increased while the amount of 3‐methylbutanol did not change significantly. Since 3‐methylbutanal and its alcohol are formed over the whole ripening period while (Z)‐3‐hexenal is formed only in the short period when tomato fruit are macerated, a potential explanation for these differences can be proposed. © 1999 Society of Chemical Industry     

The Production of Low-Alcohol Wines by Aerobic Yeasts

Aerobic yeasts of the genera Pichia and Williopis are commonly regarded as spoilage yeasts of beer and wine by causing turbidity, a surface film of yeast growth and often an excessive estery flavour. However, their ability to utilise sugars oxidatively for cell growth with the production of estery and other flavours of wine with only minimal production of ethanol suggests a method for the production of low-alcohol wines of pleasant “fermented” flavour without the need for additional equipment to remove alcohol by dialysis, reverse osmosis or distillation, or without the excessive sweetness remaining from arrested fermentation. Three strains of Pichia and one of Williopsis were examined for their ability to produce approximately 3%(v/v) ethanol and a good estery and fruity flavour. With normal anaerobic fermentation conditions, or with gentle stirring to prevent formation of a surface film, excessive amounts of alcohol were produced from grape juice of 15% or 20% (w/v) initial sugar concentration. However, an acceptably flavoured wine of alcohol content < 3% was produced by agitation and aeration during fermentation. The ethanol formed in the early stages of culture was oxidised to a final level < 3%, with the production of cell mass and an acceptable flavour.     

Fungal pretreatment of wheat straw: Effects on the biodegradability of cell walls,structural polysaccharides,lignin and phenolic acids by rumen microorganisms

Disappearance of cell wall components of untreated straw and straw treated with the ligninolytic white-rot fungi Phanerochaete chrysosporium, Dichomitus squalens and Cyathus stercoreus were determined during the course of rumen digestion of samples in nylon bags. The first fungus degraded hemicelluloses and cellulose non-selectively, adversely affecting the digestion rate of crude cell walls. Dichomitus squalens and C. stercoreus preferentially degraded hemicelluloses and lignin, affording cell wall degradation rates 1.5 times higher than in native straw. Furthermore, the extent of cell wall digestion was also significantly enhanced. Both strains improved the extent of cellulose digestion, whereas the potentially degradable xylan fraction remained unchanged. Polysaccharide digestion rates were influenced in different ways depending on the strain tested: straw degraded by C. stercoreus showed an increase in cellulose digestion rate by 50%, whereas residual arabinose units were slowly degraded. Xylan was degraded 1.8 times faster in straw decayed' by D. squalens, while cellulose digestion remained unchanged. Phanerochaete chrysosporium depressed both xylan and cellulose digestion rates. Fungal-treated lignins were solubilised in the rumen faster than in untreated straw, whereas only treatment by C. stercoreus resulted in higher lignin losses. Esterified phenolic acids were extensively degraded by all three fungi. Residual ferulic and p-coumaric acids accumulated during rumen digestion, although only the former decreased in the original straw.     

Analysis of β-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography

We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)-β-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a ‘knr4 disrupted’ tetrad demonstrated that mutant cell walls contained much less (1→3)-β-glucan and (1→6)-β-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.     

Cell wall compositions of raw and cooked corms of taro (Colocasia esculenta)

The monosaccharide compositions of parenchyma cell walls of raw and cooked corms of taro, Colocasia esculenta cv Tausala Pink, were determined. The cell wall constituents were sequentially extracted using CDTA, Na₂CO₃, 1 M KOH, 4 M KOH and water to leave a final residue (α‐cellulose). The monosaccharide compositions of the cell walls and cell wall fractions from the raw and cooked corms were consistent with the presence in these cell walls of large amounts of cellulose and pectic polysaccharides. The monosaccharide composition of the cell walls of the raw corms resembled the monosaccharide compositions of primary cell walls of other non‐commelinoid monocotyledons and dicotyledons. Cooking of the corms resulted in alteration of the cell walls, with solubilisation of pectic polysaccharides occurring earlier in the sequential fractionation and possibly changes in the extractability of xyloglucans and/or xylans. © 2000 Society of Chemical Industry     

Lignin and Hydroxycinnamic Acids in Walls of Brown Midrib Mutants of Sorghum,Pearl Millet and Maize Stems

The characteristics of walls from stems of brown-midrib ( bmr ) mutants from Sorghum bicolor (L) Moench bmr6 and bmr18 (watery- to milky-grain stage), Pennisetum americanum (L) Leeke KS81-1089 (soft-dough stage) and Zea mays L bm3 (early-dent stage) with respect to the types of linkages of hydroxycinnamic acids to wall polymers and to structural features of their lignins were investigated. The lignin content of all mutants, determined using the acid detergent lignin procedure, was significantly lower than that of their normal counterparts. There was, however, no significant differences in total lignin contents between bmr and normal lines as determined by the acetyl bromide procedure or the sum of the acid-insoluble (Klason) lignin and acid-soluble lignin. It is suggested that this behaviour could be explained if bmr mutants are characterised by higher amounts of lignin with a lower degree of polymerisation than normal lines. The lowered S/V ratio and lowered total yield of alkaline nitrobenzene oxidation products in lignin from bmr mutants was confirmed. No etherified p -coumaric acid was found in any sample tested, except the normal line of pearl millet. The concentration of etherified ferulic acid, which is probably involved in ester-ether bridges between lignin and polysaccharides, was lower in bmr mutants than in the normal plants. The low content of ferulic acid bridges in bmr mutants may contribute to the elevated digestibilities of their stems.