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Addressing lignin composition and content via Arabidopsis arogenate dehydratase knockout and over-expression genotypes.

Following the down-selection of 14 Arabidopsis thaliana arogenate dehydratase (ADT) knockout and over-expression (OE) genotypes, the most highly contrasting quadruple knockout adt3/4/5/6 and ADT OE genotypes were subjected to proteomics, metabolomics, and scanning electron microscopy (SEM) analyses as needed, with results compared to Columbia wild-type (WT). The basal adt3/4/5/6 stem cross-sections, ∼70% lignin content reduced, exhibited buckled vessel cell walls and partially detached xylary fibers, in contrast to WT and ADT4m/5 m OE genotypes that did not. Anatomical defects primarily resulted from guaiacyl lignin level reductions in vessels with concomitant increased stem syringyl:guaiacyl (S/G) ratios. Phenylpropanoid and various upstream shikimate-chorismate pathway enzyme abundances, as well as specific monolignol oxidases (laccases/peroxidases), generally increased in adt3/4/5/6 at different stem and rosette leaf growth/development stages, relative to WT. Opposite effects were largely observed with the ADT5m OE genotype. By contrast, flavonoid and glucosinolate pathway enzyme amounts varied. Such enzyme abundance increases were overall unproductive as adt3/4/5/6 was unable to restore WT, ADT4 OE, ADT5 OE, ADT5m OE, and ADT4m/5 m OE secondary metabolite (lignin, phenylpropanoid, lignan, flavonoid, phenolic acid, and glucosinolate) levels. Conversely, ADT OE genotypes did not significantly increase programmed lignin levels or alter S/G compositions. In sum, proteomics analyses of adt3/4/5/6 and adt5 'perceived' that lignin and low molecular weight secondary metabolite amounts were not at 'programmed' levels as for WT and ADT OE genotypes but observed increases in relevant pathway protein abundances were futile. Notably though, proteomics analyses did not lead to predicting that lignin and associated biochemical pathways would have reduced metabolite levels, relative to WT and ADT OE genotypes. Genotype adt3/4/5/6, possibly the highest lignin level reduced genotype reported, did not utilize other phenolics to compensate. By contrast, the differential temporal and spatial deposition of cell wall oxidases again indicate the exquisite control over lignin deposition, and our lack of knowledge of precise lignin structure and assembly in subcellular regions of the lignified cell walls.

Lignin

Factors underlying a latitudinal gradient in the S/G lignin monomer ratio in natural poplar variants.

The chemical composition of wood plays a pivotal role in the adaptability and structural integrity of trees. However, few studies have investigated the environmental factors that determine lignin composition and its biological significance in plants. Here, we examined the lignin syringyl-to-guaiacyl (S/G) ratio in members of a Populus trichocarpa population sourced from their native habitat and conducted a genome wide association study to identify genes linked to lignin formation. Our results revealed many significant associations, suggesting that lignin biosynthesis is a complex polygenic trait. Additionally, we found an increase in the S/G ratio from northern to southern geographic origin of the trees sampled, along with a corresponding metabolic and transcriptional reprogramming of xylem cell wall biosynthesis. Further molecular analysis identified a mutation in a cell wall laccase genetically associated with higher S/G ratios that predominate in trees from warmer lower latitudes. Collectively, our findings suggest that lignin heterogeneity arises from an evolutionary process enabling poplar adaptation to different climatic challenges.

Populus

Dietary fiber components: relationship to the rate and extent of ruminal digestion.

A mathematical model can serve as a useful reference for describing the mechanisms involved in digestion and for discussing the factors that influence the rate and extent of ruminal digestion. Ruminal digestion can be divided into four components: digestion rate, digestion lag, potential extent of digestion, and passage rate. Each component affects the apparent extent of digestion in a distinct manner and is influenced by separate factors. Digestion rate is directly related to apparent extent of digestion. It is not influenced by chemical entities presently being measured, but may be related to the morphological, crystalline, or physical nature of fiber. It may also be influenced by factors that inhibit or stimulate ruman microbial growth and their fiber-degrading enzymes. Digestion lag is inversely related to apparent extent of digestion; however, factors influencing it are poorly defined. The may include factors affecting microbial populations and their attachment to fiber prior to digestion; or the digestion lag may be related to the chemical or physical alteration of fiber that must occur before digestion can begin. The potential extent of digestion is directly related to apparent extent of digestion and is influenced by plant fiber composition, primarily. Lignin, and possibly silica, functions to limit the potential extent of digestion. Rate of passage essentially competes with rate of digestion for fiber particles as they pass through the rumen; therefore it is inversely related to the apparent extent of digestion. Passage rate is associated with feed intake level and particle size, although other factors such as type of diet and animal physiology may be important.

Animals

Effect of alkaline hydrogen peroxide treatment on cell wall composition and digestion kinetics of sugarcane residues and wheat straw.

Our objective was to characterize changes in cell wall composition and digestibility of sugarcane bagasse, pith from bagasse, and wheat straw after treatment with alkaline hydrogen peroxide (AHP). The AHP treatment solution contained 1% H2O2 (wt/vol) maintained at pH 11.5 with NaOH. The H2O2 in solution amounted to 25% of the quantity of substrate treated. After treatment, residues were washed and dried. Detergent fiber composition, total fiber components (neutral sugars, uronic acids, Klason lignin, and noncore lignin phenolic acids), IVDMD, in vitro digestion kinetics of NDF, and monosaccharide digestibilities (24 and 120 h) were determined. Total fiber (TF) and NDF concentrations of all treatment residues were increased (P less than .05) over control substrates by AHP because of greater losses of cell solubles than of cell wall constituents. Hemicellulose:cellulose ratio in NDF of treatment residues was decreased (P less than .05) by AHP for all substrates, but the neutral sugar composition of TF did not agree with this preferential loss of hemicellulose components. Klason lignin, ADL, and esterified noncore lignin, especially ferulic acid, were reduced (P less than .05) by AHP, whereas etherified noncore lignin composition was unchanged. Treatment increased (P less than .05) IVDMD, extent of NDF digestion, and monosaccharide digestibilities of all crop residues. The rate of NDF digestion was increased (P less than .05) for the sugarcane residues but not for wheat straw. Alkaline hydrogen peroxide improved crop residue digestibility, probably as a result of the removal of core and noncore lignin fractions.

Animal Feed

Gastrointestinal implications in pigs of wheat and oat fractions. 1. Digestibility and bulking properties of polysaccharides and other major constituents.

The present work was undertaken to study the gastrointestinal effects of wheat and oat dietary fibre (DF) using 40-50 kg pigs cannulated in the terminal ileum. The variables studied were: chemical characteristics of the DF, ileal and faecal digestibility of nutrients and bulking properties of polysaccharides and other major constituents. The wheat products studied included refined wheat flour and wheat fractions rich in the following botanical components: aleurone, pericarp/testa and bran. The oat products used were rolled oats and oat bran. The products varied considerably in DF content (g/kg dry matter) and composition; non-starch polysaccharides (NSP) and Klason lignin content ranged from 34 and 1 g/kg respectively in wheat flour, to 465 and 92 g/kg in pericarp/testa. The main NSPs in the wheat were arabinoxylans (AX) (64-69%) and cellulose (15-31%) and in oats mixed linked beta(1----3; 1----4-D-glucans (beta-glucans; 46-63%) and AX (28-32%). The lowest content of soluble NSP was found in the lignified wheat fractions (bran and pericarp/testa) and the highest in oat bran. Eight diets were produced using the wheat and oat products and studied in two series of experiments using wheat flour as the DF-depleted control. The diets in Expt 1 were based on wheat flour and three iso-DF enriched diets prepared by adding DF from the fractions rich in wheat aleurone, pericarp/testa or bran. In Expt 2, oat bran was added to wheat flour to achieve the same DF intake level as in Expt 1. This series also included diets based on rolled oats and rolled oats plus oat bran. Starch was almost completely digested in the small intestine (0.97-1.00). However, there was a tendency to a slightly lower digestibility of oat starch compared with wheat starch. The recovery of wheat NSP in ileal digesta was 82-104% compared with 64-66% for oats. The low recovery of NSP in oat diets was primarily due to the low recovery of beta-glucans (25-36%). In the large intestine NSP and starch residues were extensively degraded. For the DF-depleted control diets or diets based on oats, 8-17% NSP survived breakdown while in the diets enriched with aleurone, pericarp/testa or bran fractions, NSP recovery was 33, 50 and 38% respectively. Fermentative breakdown of carbohydrates in the large intestine was estimated to contribute between 10 and 24% of the energy for maintenance. Energy derived from the inflow of organic acids from the ileum contributed an additional 1-4% of maintenance energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animal Feed

Selection of GhTT2-A07 promoter enhances fiber quality in improved cotton varieties.

Modern cultivated cotton fibers are predominantly white with enhanced quality compared to their wild ancestors. However, the molecular mechanisms and evolutionary drivers linking fiber color to quality remain least focused. In this study, we identified FQC1 (Fiber Quality and Color 1), a major quantitative trait locus (QTL) on chromosome A07 that concurrently regulates both fiber quality and pigmentation. Through map-based cloning, we revealed that Gossypium hirsutum TRANSPARENT TESTA2-A07 (GhTT2-A07), an R2R3-MYB transcription factor, resides within this locus. GhTT2-A07 modulates fiber development by directly activating genes in the general phenylpropanoid pathway, thereby promoting the metabolic flux toward downstream secondary metabolites. Variations in the GhTT2-A07 promoter led to its reduced expression in modern white cotton cultivars. This down-regulation suppresses the accumulation of S/G/H-type lignin monomers and proanthocyanidins, resulting in altered secondary cell wall composition and ultimately enhancing the quality of mature white fibers. Population genetic analyses further indicate that the white-fiber allele GhTT2-A07W has been fixed in modern breeding genotypes, underscoring the impact of artificial selection during cotton domestication. Overall, our study elucidates the biochemical and molecular mechanisms underlying fiber quality and pigmentation in cotton, clarifies the selection criteria for high-quality white fibers in modern cultivars, and provides a theoretical basis for future targeted genetic improvement of cotton fibers.

Alleles

Evaluation of Myrothecium verrucaria as a nutrient source for ruminants.

The soil saprophytic fungus Myrothecium verrucaria was cultivated from glucose, starch, or xylan as the carbon source, and the biomass was compared with three selected feedstuffs. Fungal biomass was analyzed for nitrogen, protein, lipid, water soluble fraction, hemicellulose, cellulose, lignin, and residual ash, and the in vitro dry matter disappearance was determined. The chemical composition of M. verrucaria varied with substrate. In nitrogen content, the fungal biomass was more similar to alfalfa hay than to milo stalks or wheat straw. Content of water soluble fraction of fungal biomass was between those of alfalfa hay and milo stalks; content of hemicellulose was between those of milo stalks and wheat straw. The fungal fractions identified as cellulose and lignin varied widely with substrate. The fungal biomass was less digestible than alfalfa hay but more digestible than milo stalks or wheat straw. There may be potential uses of this organism in upgrading nutritive value of low-quality forages.

Animal Feed

High-Density Genome-Wide Association Mapping Identifies Candidate Loci Associated with Maize Stalk Cell Wall Composition.

Maize (Zea mays L.) stalk cell wall composition is a key determinant of forage digestibility, lodging resistance, and biomass utilization efficiency. Although previous genome-wide association studies (GWAS) have identified loci associated with lignin (LIG), cellulose (CEL), and hemicellulose (HC), advances in genomic resources provide an opportunity to revisit existing phenotypic datasets at substantially higher resolution. Here, we re-analyzed a maize association panel consisting of 341 diverse inbred lines using an expanded genotype dataset containing 10.77 million SNPs, two derived compositional indices (CEL/HC and [LIG/(CEL + HC)], and six complementary GWAS models. Across all traits and models, we identified 855 unique significant SNPs associated with 579 candidate genes. Among the traits examined, LIG/(CEL + HC) yielded the greatest number of associations, suggesting that indices representing the relative balance among cell wall components may better capture the genetic architecture of cell wall composition than individual component measurements alone. Integration of multiple GWAS models with functional enrichment, haplotype, and selective sweep analyses prioritized three biologically relevant candidate genes encoding a MYB58 transcription factor, the glycosyltransferase Xt9, and a putative xyloglucan 6-xylosyltransferase. Haplotype analysis revealed significant effects of Xt9 and the xyloglucan 6-xylosyltransferase on cell wall composition, while selective sweep analysis identified Xt9 as a target of repeated selection during maize domestication, ecological adaptation, and modern breeding. Although these candidate genes provide promising targets for future investigation, the associations identified here are based on a single association panel and require functional and independent population validation. Collectively, our results demonstrate how high-density genotyping combined with complementary GWAS models can refine candidate associations and generate testable hypotheses from existing phenotypic datasets.

cell wall composition

Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay.

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.

Alcohol Oxidoreductases

Comparison of the binding of various bile acids and bile salts in vitro by several types of fiber.

The binding in vitro of the sodium salts of cholic acid, chenodeoxycholic acid, deoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid by alfalfa, bran, cellulose, lignin, and cholestyramine was measured. Cholestyramine bound an average of 81.3% of all the bile acids and salts tested whereas cellulose bound only negligible amounts (1.4%). Of the other substances tested, lignin bound 29.2%, alfalfa, 15.9% and bran, 9.0%. No distinct pattern of binding was discerned. It is therefore apparent that the validity of statements concerning the effect of fiber on bile salf metabolism rests upon the specificity of the composition of the fiber involved and the bile acids or salts tested.

Bile Acids and Salts

[Nutrient composition of some newly bred high protein and/or high lysine grains and their digestibility determined on growing pigs].

With the view to complementing the feedstuff data store and, consequently, to further improving the GDR Feed Evaluation System, some newly bred high-protein and/or high-lysine cereal varieties and strains (spring barley, winter wheat, maize) were studied for their nutrient composition and digestibility. Apart from from WEENDER's feed analysis technique, more recent methods were applied to determine total fat (after HCl treatment), carbohydrates (enzymatic method), lignin and amino acids. The digestibility of the nutrients was determined using growing pigs of different live weight, the test rations being made up of the cereals under and supplementations of limiting amino acids as well as vitamins and minerals. In comparison with the values from currently applied tables, the newly bred strains and varieties proved to have markedly higher contents of crude protein, digestible crude protein, lysine and energetic feed equivalents. Compared to crude fat, the total fat values proved markedly higher in the barley and wheat samples. The readily soluble and easily hydrolizable carbohydrates found with the new analysis procedure suggested, were 100% digestible in all cereal samples used. Lignin proved the constituent most difficult to digest and must be regarded as virtually undigestible in the case of pigs.

Animal Nutritional Physiological Phenomena

Dietary fibre and colonic neoplasia.

Dietary plant fibre, or plantix, is thought to play a significant role in the pathogenesis of colon cancer in humans. It is a complex polymeric substance that has several distinct components resistant to hydrolysis by the digestive enzymes of humans. These components include cellulose, hemicelluloses, pectins, lignin, gums, mucilages and, in certain instances, algal polysaccharides. These polymers have different physicochemical properties, and recent evidence from experimental studies in animals treated with carcinogens suggests that some may exert protective effects in the intestine and others may enhance colon carcinogenesis. This review synthesizes information on the chemical composition, methods of analysis and physicochemical properties of dietary plant fibre and reviews available studies examining the role of fibre in colonic neoplasia in animals and humans.

Animals

Fertilizer calcium as a factor affecting the voluntary intake, digestibility and retention time of pangola grass (Digitaria decumbens) by sheep.

1. Pangola grass (Digitaria decumbens) grown with and without calcium fertilizer was cut at three stages of regrowth to measure voluntary intake of dry matter (DM) and digestibility of various components of the dried-grass diet by sheep kept in metabolism crates. To determine the extent of a simple Ca deficiency half the sheep on each diet was supplemented with 1-4 g Ca/d. Retention times of the various dietary components in the reticulo-rumen were also determined. 2. Feeding a Ca supplement had no effect on voluntary intake or digestibility. 3. Ca fertilizer increased the Ca content of the grass from 2-2 to 3-8 g/kg DM and DM digestibility from 0-455 to 0-476 (P less than 0-01) due to an increase in the digestibility of the hemicellulose. 4. Voluntary intake was increased from 38-8 to 43-2 g/kg body-weight0.75 per d by Ca fertilizer due to an 18% reduction in the period of time the DM was retained in the reticulorumen. 5. Regressions relating voluntary intake to DM digestibility for the Ca-fertilized and control grass were significantly different (P less than 0-01). When compared at the same DM digestibility the voluntary intake of the Ca-fertilized grass was 2-6 g/kg body-weight0.75 per d higher than that of the control. 6. It was concluded that Ca fertilizer increased both DM digestibility and voluntary intake as a result of changes in the structural composition of the grass and not by a simple increase in the Ca content of the diet.

Animal Feed

Effect of white rot basidiomycetes on chemical composition and in vitro digestibility of oat straw and alfalfa stems.

Five white rot basidiomycetes were evaluated for their potential to improve ruminal degradation of oat straw and alfalfa stems. Phanerochaete chrysosporium (PC), Scytinostroma galactinum (SG), Phlebia tremellosa (PT), Phellinus pini (PP), and Pholiota mutabilis (PM) were incubated on oat straw and alfalfa stems for 30 d at 28 degrees C and 90% relative humidity. Detergent fiber and total fiber components (neutral sugars, uronic acids, Klason lignin [KL], and ester- and ether-linked non-core lignin phenolics), core lignin nitrobenzene oxidation products, and IVDMD were determined. Electron microscopy of KMnO4-stained and cellulase/colloidal gold-labeled sections was used to monitor fungal activity. Large losses of DM were noted for all fungal species on both substrates. Lignin (KL and ADL) was removed (P less than .05) from oat straw by PC and PT treatment, but no net loss of lignin was observed for fungal treatment of alfalfa stems. Cell-wall polysaccharides were removed from both substrates by fungal activity. Only PC increased (P less than .05) IVDMD of oat straw, and SG, PT, PP, and PC treatment decreased (P less than .05) IVDMD of alfalfa stems, presumably because the fungi removed the most readily fermentable polysaccharides. Transmission electron microscopy using KMnO4 staining showed a nonselective white rot attack. Cytochemical studies using colloidal gold-labeled exo- and endocellulases were used to map the location of cellulose in the cell wall before and after decay by the white rot fungi. All the white rot fungi tested had eroded and thinned cell walls. Residual cell walls were well-labeled; both endo- and exocellulose-colloidal gold identified the cellulosic wall material that remained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed

[Mutagenic activity of the azo dye Disperse Orange 2K and its basic component Reax dispenser].

Mutagenic activity of inhaled dispersed orange 2K nitrodye (DON 2K) and Reax' disperser was evaluated. Chronically inhaled DON 2K (with disperser) in concentration 28.6 mg/m3 is a genetic danger for an individual and population, being also a selective by injurious++ to the red blood cells. Chronically inhaled Reax' disperser (25.7 and 5.8 mg/m3) shows low mutagenic activity in somatic tissue. Thus, the mutagenic effect of DON 2K with disperser (composition is constant) is explained by nitrodye and not a disperser.

Administration, Inhalation

The effect of pelleting on the voluntary intake and digestibility of leaf and stem fractions of three grasses.

1. Leaf is eaten in greater quantities than stem of similar digestibility. To determine whether this difference is caused by physical or chemical factors, leaf and stem fractions from Digitaria decumbens, Chloris gayana and Setaria splendida were fed ad lib, to sheep in the chopped and pelleted forms. Pellets were made from leaf and stem which had been ground through a screen with 3 mm holes. All sheep received a protein and mineral supplement. 2. Voluntary intake of chopped leaf was 34 percent higher than that of the chopped stem fraction (40-3 and 30-0 g/kg body-weight 0-75 respectively, P smaller than 0.01) although dry matter digestibility ratios were similar (0-478 and 0-450 respectively, P greater than 0-05). The higher intake of leaf was associated with a larger surface area (13 400 and 5200 mm2/g for chopped leaf and stem respectively), lower bulk density (60 and 180 kg/m3 respectively) and lower neutral-detergent fibre (706 and 724 g/kg respectively), acid-detergent fibre (383 and 413 g/kg respectively) and lignin (42 and 59 g/kg respectively) contents. Chopped leaf was retained in the reticulo-rumen for a shorter time than the stem fraction (19.9 and 26.4 h respectively). 3. Grinding and pelleting increased the voluntary intake of the leaf fraction by 88 percent and the stem fraction by 60 percent. This increased voluntary intake caused by grinding and pelleting was not accompanied by any significant changes in the chemical composition of the diet. Grinding and pelleting reduced the time that the food was retained in the reticulo-rumen and this change appeared sufficient to account for the observed increases in voluntary intake. 4. It was concluded that the higher intake of the leaf fraction of grasses is caused by differences in retention time of food in the reticulo-rumen. These differences in retention time are caused by differences in physical properties and not chemical composition.

Animal Feed

Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.

There is a need to standardize the NDF procedure. Procedures have varied because of the use of different amylases in attempts to remove starch interference. The original Bacillus subtilis enzyme Type IIIA (XIA) no longer is available and has been replaced by a less effective enzyme. For fiber work, a new enzyme has received AOAC approval and is rapidly displacing other amylases in analytical work. This enzyme is available from Sigma (Number A3306; Sigma Chemical Co., St. Louis, MO). The original publications for NDF and ADF (43, 53) and the Agricultural Handbook 379 (14) are obsolete and of historical interest only. Up to date procedures should be followed. Triethylene glycol has replaced 2-ethoxyethanol because of reported toxicity. Considerable development in regard to fiber methods has occurred over the past 5 yr because of a redefinition of dietary fiber for man and monogastric animals that includes lignin and all polysaccharides resistant to mammalian digestive enzymes. In addition to NDF, new improved methods for total dietary fiber and nonstarch polysaccharides including pectin and beta-glucans now are available. The latter are also of interest in rumen fermentation. Unlike starch, their fermentations are like that of cellulose but faster and yield no lactic acid. Physical and biological properties of carbohydrate fractions are more important than their intrinsic composition.

Animal Feed