Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Exudation of alcohol and aldehyde sugars from roots of defoliated Lolium perenne L. grown under sterile conditions

Root exudates were collected over a 27 day period from defoliated and non-defoliated Lolium perenne L. plants grown under sterile conditions in microlysimeters. Eleven individual sugars, including both aldehyde and alcohol sugars, were identified and quantified with a gas chromatograph-mass spectrometer (GC-MS). There was no change in the number of sugars present between 7 and 27 days, but the exudation of alcohol sugars decreased rapidly at about day 12. Xylose and glucose were present in the largest amounts. Defoliation initially increased the total amount of sugars in the exudates, but continuous defoliation reduced total sugar exudation by 16% and induced changes in the exudation patterns of individual sugars. Defoliation enhanced exudation of erythritol, threitol, and xylitol, reduced exudation of glucose and arabitol, but had little effect on the amounts of other sugars exuded. The more complex 6 C, 5 OH aldehyde sugars, especially glucose, showed changes earlier and to a greater extent (17 days), than the 5 C, 4 OH (xylose and ribose) and 6 C 4 OH (fucose) aldehyde groups. These findings confirm the general finding that repeated defoliation reduces the quantity of total sugars exuded, but the pattern of release of individual sugars is complex and variable.

Splanchnic metabolism of nitrogenous compounds and urinary nitrogen excretion in steers fed alfalfa under conditions of increased absorption of ammonia and L-arginine supply across the portal-drained viscera

Effects of increased ammonia and/or arginine absorption across the portal-drained viscera (PDV) on net splanchnic (PDV and liver) metabolism of nitrogenous compounds and urinary N excretion were investigated in six cathetenzed Hereford x Angus steers (501 +/- 1 kg BW) fed a 75% alfalfa:25% (as-fed basis) corn-soybean meal diet (0.523 MJ of ME/[kg BW0.15.d]) every 2 h without (27.0 g of N/kg of dietary DM) and with 20 g of urea/kg of dietary DM (35.7 g of N/kg of dietary DM) in a split-plot design. Net splanchnic flux measurements were obtained immediately before beginning and ending a 72-h mesenteric vein infusion of L-arginine (15 mmol/h). For 3 d before and during arginine infusion, daily urine voided was measured and analyzed for N composition. Feeding urea increased PDV absorption (P < 0.01) and hepatic removal (P < 0.01) of ammonia N, accounting for 80% of increased hepatic urea N output (P < 0.01). Numerical increases in net hepatic removal of AA N could account for the remaining portion of increased hepatic urea N output. Arginine infusion increased hepatic arginine removal (P < 0.01) and hepatic urea N output (P < 0.03) and switched hepatic ornithine flux from net uptake to net output (P < 0.01), but numerical changes in net hepatic removal of ammonia and AA N could not account fully for the increase in hepatic urea N output. Increases in urine N excretion equaled quantities of N fed as urea or infused as arginine. Estimated salivary urea N excretion was not changed by either treatment. Urea cycle regulation occurs via a complex interaction of mechanisms and requires N sources other than ammonia, but the effect of increased ammonia absorption on hepatic catabolism of individual AA in the present study was not significant.

Splanchnic metabolism of nutrients and hormones in steers fed alfalfa under conditions of increased absorption of ammonia and L-arginine supply across the portal-drained viscera

Effects of increased ammonia and/or arginine absorption on net splanchnic (portal-drained viscera [PDV] plus liver) metabolism of nonnitrogenous nutrients and hormones in cattle were examined. Six Hereford x Angus steers (501 +/- 1 kg BW) prepared with vascular catheters for measurements of net flux across the splanchnic bed were fed a 75% alfalfa:25% (as-fed basis) corn and soybean meal diet (0.523 MJ of ME/[kg BW(0.75.)d]) every 2 h without (27.0 g of N/kg of DM) and. with 20 g of urea/kg of DM (35.7 g of N/kg of DM) in a split-plot design. Net flux measurements were made immediately before and after a 72-h mesenteric vein infusion Of L-arginine (15 mmol/h). There were no treatment effects on PDV or hepatic 02 consumption. Dietary urea had no effect on splanchnic metabolism of glucose or L-lactate, but arginine infusion decreased net hepatic removal Of L-lactate when urea was fed (P < 0.01). Net PDV appearance of n-butyrate was increased by arginine infusion (P < 0.07), and both dietary urea (P < 0.09) and arginine infusion (P < 0.05) increased net hepatic removal of n-butyrate. Dietary urea also increased total splanchnic acetate output (P < 0.06), tended to increase arterial glucagon concentration (P < 0.11), and decreased arterial ST concentration (P < 0.03). Arginine infusion increased arterial concentration (P < 0.07) and net PDV release (P < 0.10) and tended to increase hepatic removal (P < 0.11) of insulin, as well as arterial concentration (P < 0.01) and total splanchnic output (P < 0.01) of glucagon. Despite changes in splanchnic N metabolism, increased ammonia and arginine absorption had little measurable effect on splanchnic metabolism of glucose and other nonnitrogenous components of splanchnic energy metabolism.

Optimisation of the aqueous extraction conditions of phenols from meadowsweet (Filipendula ulmaria L.) for incorporation into beverages

Meadowsweet was extracted in water at a range of temperatures (60–100 °C), and the total phenols, tannins, quercetin, salicylic acid content and colour were analysed. The extraction of total phenols followed pseudo first-order kinetics, the rate constant (k) increased from 0.09 ± 0.02 min−1 to 0.44 ± 0.09 min−1, as the temperature increased from 60 to 100 °C. An increase in temperature from 60 to 100 °C increased the concentration of total phenols extracted from 39 ± 2 to 63 ± 3 mg g−1 gallic acid equivalents, although it did not significantly affect the proportion of tannin and non-tannin fractions. The extraction of quercetin and salicyclic acid from meadowsweet also followed pseudo first-order kinetics, the rate constant of both compounds increasing with an increase in temperature up until 90 °C. Therefore, the aqueous extraction of meadowsweet at temperatures at or above 90 °C for 15 min yields extracts high in phenols, which may be added to beverages.

Optimisation of the extraction and processing conditions of chamomile (Matricaria chamomilla L.) for incorporation into a beverage

The total phenols, apigenin 7-glucoside, turbidity and colour of extracts from dried chamomile flowers were studied with a view to develop chamomile extracts with potential anti-inflammatory properties for incorporation into beverages. The extraction of all constituents followed pseudo first-order kinetics. In general, the rate constant (k) increased as the temperature increased from 57 to 100 °C. The turbidity only increased significantly between 90 and 100 °C. Therefore, aqueous chamomile extracts had maximum total phenol concentration and minimum turbidity when extracted at 90 °C for 20 min. The effect of drying conditions on chamomile extracted using these conditions was determined. A significant reduction in phenol concentration, from 19.7 ± 0.5 mg/g GAE in fresh chamomile to 13 ± 1 mg/g GAE, was found only in the plant material oven-dried at 80 °C (p ⩽ 0.05). The biggest colour change was between fresh chamomile and that oven-dried at 80 °C, followed by samples air-dried. There was no significant difference in colour of material freeze-dried and oven-dried at 40 °C.