Reduction in Rumen Tetracycline-Insensitive Bacteria during a Grain Challenge Using the Isoflavone Biochanin A.

The isoflavone biochanin A was previously shown to promote weight gain in growing steers by selectively inhibiting rumen bacteria-like growth-promoting feed antibiotics. The hypothesis that biochanin A inhibited the action of drug efflux pumps was tested by enumerating tetracycline-insensitive bacteria from steers in a subacute rumen acidosis (SARA) challenge. Steers (n = 3/group) treatment groups were forage only, SARA control, SARA with monensin (0.2 g d-1), and SARA with biochanin A (6.0 g d-1). As the steers were stepped up from the forage-only basal diet to 70% cracked corn, the number of rumen bacteria enumerated on two tetracycline-containing media types (nutrient glucose agar and tetracycline, and bile esculin azide and tetracycline) increased (p < 0.05) from as little as 1.7(105) to as great as 6.7(106) cfu mL-1 on the nutrient glucose agar in the SARA and monensin control groups. The biochanin A group maintained the same number of tetracycline-insensitive bacteria as the forage-only controls (p > 0.05). The effects were similar to the more selective media type, but the differences were smaller. These results support the hypothesis that biochanin A inhibits the activity of drug efflux pumps in vivo.

Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method.

Cool-season pasture grasses contain arabinoxylans (AX) as their major cell wall hemicellulosic polysaccharide. AX structural differences may influence enzymatic degradability, but this relationship has not been fully explored in the AX from the vegetative tissues of cool-season forages, primarily because only limited AX structural characterization has been performed in pasture grasses. Structural profiling of forage AX is a necessary foundation for future work assessing enzymatic degradability and may also be useful for assessing forage quality and suitability for ruminant feed. The main objective of this study was to optimize and validate a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method for the simultaneous quantification of 10 endoxylanase-released xylooligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) in cool-season forage cell wall material. The following analytical parameters were determined or optimized: chromatographic separation and retention time (RT), internal standard suitability, working concentration range (CR), limit of detection (LOD), limit of quantification (LOQ), relative response factor (RRF), and quadratic calibration curves. The developed method was used to profile the AX structure of four cool-season grasses commonly grown in pastures (timothy, Phleum pratense L.; perennial ryegrass, Lolium perenne L.; tall fescue, Schedonorus arundinaceus (Schreb.) Dumort.; and Kentucky bluegrass, Poa pratensis L.). In addition, the cell wall monosaccharide and ester-linked hydroxycinnamic acid contents were determined for each grass. The developed method revealed unique structural aspects of the AX structure of these forage grass samples that complemented the results of the cell wall monosaccharide analysis. For example, xylotriose, representing an unsubstituted portion of the AX polysaccharide backbone, was the most abundantly-released oligosaccharide in all the species. Perennial rye samples tended to have greater amounts of released oligosaccharides compared to the other species. This method is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.

Water- and Ethanol-Soluble Carbohydrates of Temperate Grass Pastures: a Review of Factors Affecting Concentration and Composition.

Temperate grasses contain both water- and ethanol-soluble carbohydrates. Water-soluble carbohydrates (WSCs) of temperate grasses include glucose, fructose, sucrose, and fructans (fructose-based polymers) of varying lengths. Ethanol-soluble carbohydrates (ESCs) consist of glucose, fructose, sucrose, and shorter fructans. WSCs and ESCs have been implicated in equine pasture-associated laminitis and other metabolic disorders. In this paper, the peer-reviewed literature of the past decade was summarized for selected factors influencing concentrations and composition of WSCs and ESCs in temperate grasses. WSC concentrations tended to increase under cool temperatures and during cooler seasons. WSC and ESC concentrations generally increased from morning to evening with a range of -20 to 74 g/kg DM for WSCs. Cultivar choice had variable effects on WSC concentrations. Frequent defoliation usually lowered WSC and fructan concentrations. Nitrogen application increased or decreased WSC concentrations, depending on the amount applied and the grass cultivars. Water stress had variable effects on WSC concentration and composition. Multiple factors should be considered before assuming how certain management or environmental conditions will affect WSCs, ESCs, or individual carbohydrates.

Ethanol-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentration by Near-Infrared Reflectance Spectroscopy (NIRS) and Evaluation of Effects of Cultivar and Management.

Ethanol-soluble carbohydrates (ESCs) of cool-season grasses include mono- and disaccharides and sometimes short-chain fructans, which may exacerbate the risk of pasture-associated laminitis. A calibration for prediction of ESC concentrations by near-infrared reflectance spectroscopy (NIRS) was developed from 323 samples of four cool-season grass species (orchardgrass, Kentucky bluegrass, tall fescue, and perennial ryegrass) across 10 cultivars collected in central Kentucky in the morning and afternoon over two growing seasons. The calibration, which had accuracy above 95%, was used to predict ESC concentrations of 1,532 samples from the second growing season. ESC concentrations increased in the afternoon compared to the morning across all cultivars. In the majority of samples, ESC concentrations were not affected by nitrogen application to plots. Use of NIRS has the potential to evaluate management and cultivar effects on ESC concentrations in cool-season grass pastures.

Water-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentrations by Near-Infrared Reflectance Spectroscopy and Evaluation of Effects of Genetics, Management, and Environment.

An excess of water-soluble carbohydrates (WSCs) can present a risk for horses prone to pasture-associated laminitis or some other metabolic conditions. Determining WSC concentrations in commonly grazed cool-season grasses, at different times of day and under different fertilization treatments, can help optimize grazing times and management strategies. The goals of this study were to develop a near-infrared reflectance spectroscopy (NIRS) calibration curve for WSC and to apply it to four cool-season grass species. Tall fescue, Kentucky bluegrass, orchardgrass, and perennial ryegrass (10 cultivars) were sampled every two to four weeks from plots with or without added nitrogen, in the morning and afternoon. WSCs were quantified colorimetrically for a sample subset, and these values were used to develop an NIRS calibration predicting WSC concentrations with 90% accuracy. An interaction of species, nitrogen treatment, time of day, and harvest date influenced WSC concentrations in 10 harvests (P = .040). A modest positive relationship was observed between photosynthetically active radiation and WSC concentration when morning and afternoon samples were included (r = 0.503; P = .024). On nine harvest dates, perennial ryegrass or tall fescue were highest in WSC. High-WSC cultivars included "Aberzest" and "Calibra" perennial ryegrass, "Ginger" Kentucky bluegrass, and "Bronson" and "Cajun II" tall fescue. Water-soluble carbohydrates did not exceed 150 g/kg freeze-dried weight, possibly due to assay method, sampling times, or defoliation. The results suggest that minimizing WSC intake for horses may be possible by cultivar choice, grazing time, or mowing frequency.

Isoflavone supplementation, via red clover hay, alters the rumen microbial community and promotes weight gain of steers grazing mixed grass pastures.

Biochanin A, an isoflavone present in the pasture legume red clover (Trifloium pratense L.), alters fermentation in the rumen of cattle and other ruminants. Biochanin A inhibits hyper-ammonia-producing bacteria and promotes cellulolytic bacteria and fiber catalysis in vitro and ex vivo. Consequently, biochanin A supplementation improves weight gain in grazing steers. Red clover contains biologically active isoflavones that may act synergistically. Therefore, the objective was to evaluate the effect of two levels of red clover hay on growth performance and the microbial community in growing steers grazing mixed grass pastures. A grazing experiment was conducted over 2 early growing seasons (2016 and 2017) with 36 cross-bred steers and twelve rumen-fistulated, growing Holstein steers for evaluation of average daily gain and rumen microbiota, respectively. Steers were blocked by body weight and assigned to pastures with one of four treatments: 1) pasture only, 2) pasture + dry distillers' grains (DDG), 3) pasture + DDG + low level of red clover hay (~15% red clover diet), or 4) pasture + DDG + high level of red clover hay (~30% red clover diet). DDG were added to treatments to meet protein requirements and to balance total protein supplementation between treatments. All supplementation strategies (DDG ± red clover hay) increased average daily gains in comparison to pasture-only controls (P < 0.05), with a low level of red clover supplementation being the most effective (+0.17 kg d-1 > DDG only controls; P < 0.05). Similarly, hyper-ammonia-producing bacteria inhibition (10-100-fold; P < 0.05), fiber catalysis (+10-25%; P < 0.05) and short chain fatty acid concentrations were greatest with the low red clover supplement (+~25%; P < 0.05). These results provide evidence that lower levels or red clover supplementation may be optimal for maximizing overall microbial community function and animal performance in grazing steers.

Seasonal and Diurnal Variation in Water-Soluble Carbohydrate Concentrations of Repeatedly Defoliated Red and White Clovers in Central Kentucky.

Nonstructural carbohydrates of pasture plants, comprising water-soluble carbohydrates (WSCs) and starch, may contribute to excessive consumption of rapidly fermentable carbohydrates by grazing horses. Seasonal and diurnal variation in WSCs were studied in red (Trifolium pratense L.) and white clovers (Trifolium repens L.) subjected to a typical management regime of rotationally grazed horse pastures. Two red and two white clover cultivars from monoculture plots were harvested after 4 weeks of growth from April to October of 2015, in the morning and afternoon of each harvest date. Water-soluble carbohydrates were quantified for each harvest, and starch was quantified for two harvests. Mean monthly WSC concentrations ranged from 80 to 99 mg/g (freeze-dried weight basis), whereas mean starch concentrations were 31 and 40 mg/g. In September, white clover had 14% more WSCs than red clover (P < .0001). Water-soluble carbohydrate concentrations were 10% higher in the afternoon than in the morning (P < .0001). Starch concentrations were 290% higher in the afternoon than in the morning (P < .0001), and nonstructural carbohydrate concentrations in the afternoon averaged 150 mg/g. Further studies are needed to determine whether the mixed grass-legume pastures of central Kentucky accumulate enough nonstructural carbohydrates to present risk factors for equine metabolic or digestive dysfunction.

Effects of Harvest Date, Sampling Time, and Cultivar on Total Phenolic Concentrations, Water-Soluble Carbohydrate Concentrations, and Phenolic Profiles of Selected Cool-Season Grasses in Central Kentucky.

Grasses are a source of nutrients for grazing horses. However, water-soluble carbohydrates (WSCs) of grasses have been implicated in some equine health issues. Grasses also contain phenolic compounds, whose sensory and antimicrobial properties may affect forage intake by horses and horse health. The goals of this study were to assess factors affecting phenolic and WSC concentrations in selected cultivars of cool-season grasses and profile the phenolic compounds. Total phenolics and WSC were quantified in "Linn" and "Calibra" perennial ryegrass (PRG), "Cajun II" tall fescue (Cajun TF), "Persist" orchardgrass (OG), and "Ginger" Kentucky bluegrass (BG), collected in the morning and afternoon of late April, early May, and late May. WSCs were higher in the afternoon (P < .0001), and afternoon concentrations differed among cultivars (P = .011) and decreased by late May (P < .0001). Total phenolics, initially highest in Calibra PRG and Persist OG, decreased or remained constant from late April to late May, except in Persist OG (P < .0001). Total phenolics decreased in the afternoon in Persist OG and Calibra PRG (P = .015). High-performance liquid chromatography revealed similar phenolic profiles in Calibra PRG, Linn PRG, and Cajun TF. These differed from the profiles of Ginger BG and Persist OG. Major peaks were characterized by liquid chromatography coupled to mass spectrometry. The cultivars had a diversity of phenolic compounds possibly worth exploring for properties that may interact with those of WSC to impact equine health.

Liberation of recalcitrant cell wall sugars from oak barrels into bourbon whiskey during aging.

Oak barrels have been used by humans for thousands of years to store and transport valuable materials. Early settlers of the United States in Kentucky began charring the interior of new white oak barrels prior to aging distillate to create the distinctively flavored spirit we know as bourbon whiskey. Despite the unique flavor and cultural significance of "America's Spirit", little is known about the wood-distillate interaction that shapes bourbon whiskey. Here, we employed an inverse method to measure the loss of specific wood polysaccharides in the oak cask during aging for up to ten years. We found that the structural cell wall wood biopolymer, cellulose, was partially decrystallized by the charring process. This pyrolytic fracturing and subsequent exposure to the distillate was accompanied by a steady loss of sugars from the cellulose and hemicellulose fractions of the oak cask. Distinct layers of structural degradation and product release from within the barrel stave are formed over time as the distillate expands into and contracts from the barrel staves. This complex, wood-sugar release process is likely associated with the time-dependent generation of the unique palate of bourbon whiskey.

Hops (Humulus lupulus L.) Bitter Acids: Modulation of Rumen Fermentation and Potential As an Alternative Growth Promoter.

Antibiotics can improve ruminant growth and efficiency by altering rumen fermentation via selective inhibition of microorganisms. However, antibiotic use is increasingly restricted due to concerns about the spread of antibiotic-resistance. Plant-based antimicrobials are alternatives to antibiotics in animal production. The hops plant (Humulus lupulus L.) produces a range of bioactive secondary metabolites, including antimicrobial prenylated phloroglucinols, which are commonly called alpha- and beta-acids. These latter compounds can be considered phyto-ionophores, phytochemicals with a similar antimicrobial mechanism of action to ionophore antibiotics (e.g., monensin, lasalocid). Like ionophores, the hop beta-acids inhibit rumen bacteria possessing a classical Gram-positive cell envelope. This selective inhibition causes several effects on rumen fermentation that are beneficial to finishing cattle, such as decreased proteolysis, ammonia production, acetate: propionate ratio, and methane production. This article reviews the effects of hops and hop secondary metabolites on rumen fermentation, including the physiological mechanisms on specific rumen microorganisms, and consequences for the ruminant host and ruminant production. Further, we propose that hop beta-acids are useful model natural products for ruminants because of (1) the ionophore-like mechanism of action and spectrum of activity and (2) the literature available on the plant due to its use in brewing.

Mitigation of Ergot Vasoconstriction by Clover Isoflavones in Goats (Capra hircus).

Ergot alkaloids produced by a fungal endophyte (Epichloë coenophiala; formerly Neotyphodium coenophialum) that infects tall fescue (Lolium arundinaceum) can induce persistent constriction of the vasculature in ruminants, hindering their capability to thermo-regulate core body temperature. There is evidence that isoflavones produced by legumes can relax the vasculature, which suggests that they could relieve ergot alkaloid-induced vasoconstriction and mitigate the vulnerability to severe heat stress in ruminants that graze tall fescue. To test if isoflavones can relieve alkaloid-induced vasoconstriction, two pen experiments were conducted with rumen-fistulated goats (Capra hircus) to determine with ultrasonograpy if isoflavones can (1) promote vascular compliance by countering alkaloid-induced vasoconstriction and (2) relieve already imposed alkaloid-induced vasoconstriction. Goats were fed ad libitum chopped orchardgrass (Dactylis glomerata)-timothy (Phleum pratense) hay prior to conducting the experiments. Measures of carotid and interosseous luminal areas were obtained pre- (baseline) and post-ruminal infusions in both experiments with goats being fed the hay, and for blood flow rate in the carotid artery in Experiment 2. Responses to infusion treatments were evaluated as proportionate differences from baseline measures. Peak systolic velocity, pulsatility index, and heart rate were measured on the last day on treatment (DOT) in Experiment 1, and on all imaging sessions during Experiment 2. For Experiment 1, rumens were infused with ground toxic fescue seed and isoflavones in Phase A and with only the toxic seed in Phase B. The infusion treatments were switched between phases in Experiment 2, which employed a fescue seed extract having an ergot alkaloid composition equivalent to that of the ground seed used in Experiment 1. During Experiment 1, luminal areas of carotid and interosseous arteries in Phase A did not deviate (P > 0.1) from baselines over 1, 2, 3, and 4 DOT, but the areas of both declined linearly from baselines over 1, 2, 3, and 4 DOT in Phase B. By 6, 7, and 8 DOT in Experiment 2, luminal areas of the arteries and flow rate declined from baselines with infusions with the only seed extract in Phase A, but luminal areas and flow rate increased over 4, 5, and 6 DOT with the additional infusion of isoflavones. Peak systolic velocity and heart rate were not affected by treatment in either experiment, but were highest when infused with only ergot alkaloids in both experiments. Treatment with isoflavones was demonstrated to relax the carotid and interosseous arteries and reduce resistance to blood flow. Results indicate that isoflavones can relax persistent vasoconstriction in goats caused by consumption of ergot alkaloids, and mitigate the adverse effect that ergot alkaloids have on dry matter intake.

Blackpatch of Clover, Cause of Slobbers Syndrome: A Review of the Disease and the Pathogen, Rhizoctonia leguminicola.

Rhizoctonia leguminicola Gough and Elliott is a widely used name for the causal agent of blackpatch disease of red clover (Trifolium pratense L.). This fungal pathogen produces alkaloids (slaframine and swainsonine) that affect grazing mammals. Slaframine causes livestock to salivate profusely, and swainsonine causes neurological problems. Although the blackpatch fungus was classified as a Rhizoctonia species (phylum Basidiomycota), morphological studies have indicated that it is in the phylum Ascomycota, and sequencing data have indicated that it may be a new genus of ascomycete. The effects of the alkaloids on grazing mammals and their biosynthetic pathways have been extensively studied. In contrast, few studies have been done on management of the disease, which requires a greater understanding of the pathogen. Methods of disease management have included seed treatments and fungicides, but these have not been investigated since the 1950s. Searches for resistant cultivars have been limited. This review summarizes the biological effects and biosynthetic precursors of slaframine and swainsonine. Emphasis is placed on current knowledge about the epidemiology of blackpatch disease and the ecology and taxonomy of the pathogen. Possibilities for future research and disease management efforts are suggested.

Soluble Phenolic Compounds in Different Cultivars of Red Clover and Alfalfa, and their Implication for Protection against Proteolysis and Ammonia Production in Ruminants.

Red clover (Trifolium pratense) contains soluble phenolic compounds with roles in inhibiting proteolysis and ammonia production. Alfalfa (Medicago sativa) has been found to have a low phenolic content, but few alfalfa and red clover cultivars have been compared for phenolic content. Total soluble phenolics were quantified by a Folin-Ciocalteu colorimetric assay in nine red clover and 27 alfalfa cultivars. Mean total phenolic contents of red clover and alfalfa were 36.5 ± 4.3 mg/gdw and 15.8 ± 1.4 mg/gdw, respectively, with the greater standard deviation of red clover possibly indicating more diversity in phenolic content. Because different phenolic standards had different response factors in the colorimetric assay, the red clover and 11 alfalfa cultivars were analyzed by HPLC to determine if the differences in total soluble phenolics between genera reflected differences in the amounts of phenolics or in the classes of phenolics responding to the colorimetric assay. Two red clover cultivars differed in total phenolics and phaselic acid. Alfalfa produced different phenolic compounds from red clover, at lower concentrations. Extracts of two red clover cultivars were separated by thin-layer chromatography (TLC), and the bands were assayed for activity against Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium (HAB). Only biochanin A had anti-HAB activity. Inhibitory amounts indicated that five red clover cultivars could be suitable sources of anti-HAB activity.

Thin-layer chromatographic (TLC) separations and bioassays of plant extracts to identify antimicrobial compounds.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium.

Red clover (Trifolium pratense) is rich in phenolic compounds. Both the crude phenolic extract and biochanin A, an isoflavonoid component of the extract, suppress growth of Clostridium sticklandii, a bovine, Gram-positive, ruminal hyper-ammonia producing bacterium (HAB). The purpose of this study was to determine if other components of red clover extract contributed to its anti-HAB activity. Extracts of the Kenland cultivar of red clover, collected 0 h and 24 h after cutting, were separated by normal-phase thin-layer chromatography (TLC) in either ethyl acetate-hexanes (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by either overlaying the plates with agar seeded with C. sticklandii, or setting the excised bands face-down onto plates of bacteria-seeded agar. Biochanin A inhibited C. sticklandii growth on TLC plates when as little as 8 nmol was present in the extract. An antimicrobial band, seen in a previous bioassay, was not found, suggesting that this second compound may be more labile than biochanin A.

Effects of pH, sample size, and solvent partitioning on recovery of soluble phenolic acids and isoflavonoids in leaves and stems of red clover (Trifolium pratense cv. Kenland).

Several extraction parameters were tested to determine optimal conditions for extracting phenolics from leaves and stems of red clover (Trifolium pratense L. cv. Kenland), with the goal of using extracts in bioassays and in assessment of phenolic profiles. HPLC-UV profiles were compared before and after partitioning a methanolic extract of soluble phenolics with ethyl acetate-ethyl ether (1:1, v/v). The effect of extract pH on the partitioning of phenolics into the ethyl acetate-ethyl ether (EtOAc-Et2O) phase was evaluated, and several tissue weights were extracted to determine a minimum amount that could be extracted without loss of information. HPLC profiles of soluble phenolics were similar in the methanolic extracts and the partitioned EtOAc-Et2O extracts. However, recoveries in unpartitioned extracts were 2- to 4-fold greater than in the acidified, partitioned extracts. Also, recovery was considerably affected by the pH to which extracts were adjusted prior to partitioning. In extracts acidified to pH 2, recoveries were 2- to 7-fold higher than in extracts partitioned at pH 6. In extracts prepared from 250, 120, or 60 mg of tissue, peak areas of methanolic extracts were directly proportional to the amount of tissue extracted.

A validated method for gas chromatographic analysis of gamma-aminobutyric acid in tall fescue herbage.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in animals that is also found in plants and has been associated with plant responses to stress. A simple and relatively rapid method of GABA separation and quantification was developed from a commercially available kit for serum amino acids (Phenomenex EZ:faast) and validated for tall fescue (Festuca arundinacea). Extraction in ethanol/water (80:20, v/v) at ambient temperature yielded detectable amounts of GABA. Clean separation from other amino acids in 28 min was achieved by gas chromatography (GC) with flame ionization detection (FID), using a 30 m, 5% phenyl/95% dimethylpolysiloxane column. The identity of the putative GABA peak was confirmed by GC with mass spectrometric (MS) detection. The relatively small effects of the sample matrix on GABA measurement were verified by demonstrating slope parallelism of GABA curves prepared in the presence and absence of fescue extracts. Limits of quantification and detection were 2.00 and 1.00 nmol/100 microL, respectively. Method recoveries at two different spike levels were 96.4 and 94.2%, with coefficients of variation of 7.3 and 7.2%, respectively.

A functional genomics investigation of allelochemical biosynthesis in Sorghum bicolor root hairs.

Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.

Increased alpha-tocopherol content in soybean seed overexpressing the Perilla frutescens gamma-tocopherol methyltransferase gene.

Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, gamma-tocopherol, the biosynthetic precursor to alpha-tocopherol, is the predominant form found in the seed, whereas alpha-tocopherol is the most bioactive component. This suggests that the final step of the alpha-tocopherol biosynthetic pathway catalyzed by gamma-tocopherol methyltransferase (gamma-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active alpha-tocopherol form could have significant health benefits. In order to increase the soybean seed alpha-tocopherol content, the gamma-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens gamma-TMT gene resulted in a 10.4-fold increase in the alpha-tocopherol content and a 14.9-fold increase in the beta-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that alpha-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the alpha-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.

Detoxification and transcriptome response in Arabidopsis seedlings exposed to the allelochemical benzoxazolin-2(3H)-one.

Benzoxazolin-2(3H)-one (BOA) is an allelochemical most commonly associated with monocot species, formed from the O-glucoside of 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one by a two-step degradation process. The capacity of Arabidopsis to detoxify exogenously supplied BOA was analyzed by quantification of the major known metabolites BOA-6-OH, BOA-6-O-glucoside, and glucoside carbamate, revealing that detoxification occurs predominantly through O-glucosylation of the intermediate BOA-6-OH, most likely requiring the sequential action of as-yet-unidentified cytochrome P450 and UDP-glucosyltransferase activities. Transcriptional profiling experiments were also performed with Arabidopsis seedlings exposed to BOA concentrations, representing I(50) and I(80) levels based on root elongation inhibition assays. One of the largest functional categories observed for BOA-responsive genes corresponded to protein families known to participate in cell rescue and defense, with the majority of these genes potentially associated with chemical detoxification pathways. Further experiments using a subset of these genes revealed that many are also transcriptionally induced by a variety of structurally diverse xenobiotic compounds, suggesting they comprise components of a coordinately regulated, broad specificity xenobiotic defense response. The data significantly expand upon previous studies examining plant transcriptional responses to allelochemicals and other environmental toxins and provide novel insights into xenobiotic detoxification mechanisms in plants.