Characterization of Proanthocyanidins from Seeds of Perennial Ryegrass (Lolium perenne L.) and Tall Fescue (Festuca arundinacea) by Liquid Chromatography–Mass Spectrometry

Perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) are forage species of the grass family (Poaceae) that are key components of temperate pasture-based agricultural systems. Proanthocyanidins (PAs) are oligomeric flavonoids that, when provided as part of a farm animal’s diet, have been reported to improve animal production and health. Up to now, forage grasses have been deemed not to produce PAs. This paper reports for the first time the detection of polymerized PAs in aqueous methanolic extracts of seed tissue of both perennial ryegrass and tall fescue, using LC-MS/MS. We have determined the structure of the PAs to be <i>trans</i>-flavan-3-ol-based, consisting predominately of afzelechin and catechin and linked primarily by B-type bonds. Investigations into the leaf tissue of both species failed to detect any PAs. This discovery opens the possibility of using genetic engineering tools to achieve tannin accumulation in leaf tissue of perennial ryegrass and tall fescue

Form and Function in Spoken Discourse : An analysis of inconsistencies in language form and function

A large number of language functions cannot readily be predicted simply by a consideration of their forms. There are a relatively limited number of language forms that are utilized by an infinite number of complex communicative functions involved in human linguistic interaction. The gap that exists between language forms and functions is bridged by contextual factors including speaker/listener cooperation, shared knowledge, intonation, conventional social norms and others. These factors help speakers communicatively bridge the correspondence gap between form and function and help give otherwise ambiguous forms their functionally intended meaning. In the first section of this article the author has made a summary of the arguments of the various approaches to the analysis of discourse on the issue of linguistic form and function. In the final sections the author has chosen a sample of spoken text and commented on examples, which demonstrate that the functions are not predictable from linguistic forms used by the interlocutors

Additional file 4: of Low pyrrolizidine alkaloid levels in perennial ryegrass is associated with the absence of a homospermidine synthase gene

Genetic markers used for linkage mapping of the LpDHS, LpHSS1 and LpHSS2 genes in a F1 population of perennial ryegrass. (PDF 428 kb

Additional file 5: of Low pyrrolizidine alkaloid levels in perennial ryegrass is associated with the absence of a homospermidine synthase gene

Tall Fescue FaDHS and FaHSS1 translated protein sequences derived from ESTs retrieved from GenBank. (FASTA 1 kb

Additional file 8: of Low pyrrolizidine alkaloid levels in perennial ryegrass is associated with the absence of a homospermidine synthase gene

Statistical analysis of thesinine-rhamnoside traits in perennial ryegrass. A) Frequency distribution of untransformed thesinine-rhamnoside BLUP’s. B) Pearson correlation coefficients among levels of thesinine-rhamnoside PAs. (PDF 537 kb

An Extracellular Siderophore Is Required to Maintain the Mutualistic Interaction of <i>Epichloë festucae</i> with <i>Lolium perenne</i>

<div><p>We have identified from the mutualistic grass endophyte <i>Epichloë festucae</i> a non-ribosomal peptide synthetase gene (<i>sidN</i>) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of <i>sidN</i> eliminated biosynthesis of epichloënin A <i>in vitro</i> and <i>in planta</i>. During iron-depleted axenic growth, Δ<i>sidN</i> mutants accumulated the pathway intermediate N⁵-<i>trans</i>-anhydromevalonyl-N⁵-hydroxyornithine (<i>trans</i>-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of <i>Lolium perenne</i> (perennial ryegrass) with Δ<i>sidN</i> mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of Δ<i>sidN</i> mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.</p></div

Synthesis of epichloënin A is dependent on <i>sidN</i>.

<p>LC-MS analysis showing MS¹extracted ion chromatograms for both epichloënin A (a, <i>m/z</i> 542) and ferriepichloënin A (b, <i>m/z</i> 569) in supernatant and mycelium from two week old iron-depleted cultures of wild-type <i>E. festucae</i> Fl1 (WT), <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>), and a complemented <i>ΔsidN</i> strain (C-<i>sidN</i>). Note scale for supernatant is 10× of that for mycelium.</p

Iron depletion renders Δ<i>sidN</i> mutants incapable of axenic vegetative growth.

<p>A. 16-day-old cultures of wild-type <i>E. festucae</i> Fl1 (WT) and Δ<i>sidN</i> mutant strains (<i>ΔsidN</i> 54 and <i>ΔsidN</i> 85) were grown on iron depleted defined media (DM), and DM media supplemented with 100 µM BPS, or 100 µM BPS and culture filtrate from WT (BPS CF), or 100 µM BPS and 20 µM FeS0₄ (BPS Fe²⁺) or 100 µM BPS and 20 µM FeCl₃ (BPS Fe³⁺) respectively. B. Radial growth measurements of WT, complement (C-sidN) and Δ<i>sidN</i> 85 were determined by inoculating mycelial plugs in triplicate onto DM media, or DM supplemented with 100 µM BPS, DM with 100 µM BPS and 20 µM FeS0₄ (Fe2+) and DM with 100 µM BPS and 20 µM FeCl₃ (Fe3+) respectively. Colonies were measured at 10 days. The results represent the mean of three independent experiments. The radial growth is normalized to that of WT grown on DM media.</p

The Endophyte-Grass symbiotic interaction phenotype is disrupted in <i>ΔsidN</i> infected plants.

<p>A. Phenotypes of perennial ryegrass plants infected with wild-type <i>E. festucae</i> Fl1 (WT), complemented <i>ΔsidN</i> strain (C-<i>sidN</i>) and <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>). B. Root systems of <i>sidN</i> infected plants are reduced compared to WT infections. A perennial ryegrass plant infected with <i>E. festucae</i> (WT) is compared against three Δ<i>sidN</i> 85 infected plants displaying increased levels of plant stunting from left to right. Plants are 14 weeks old. C, D and E. Light micrograph DIC images of aniline-blue stained hyphae of wild-type <i>E. festucae</i> Fl1 (WT). C. <i>ΔsidN</i> mutant 85. D. <i>ΔsidN</i> mutant 54. E. in mature leaf sheaths.</p

Abnormalities in the hyphal distribution and ultrastructure of <i>ΔsidN</i> mutants in perennial ryegrass plants.

<p>A. Light micrographs of 1 µM cross sections of the inner leaf sheath of perennial ryegrass infected with wild-type <i>E. festucae</i> Fl1 (WT) and <i>ΔsidN</i> mutant 85 (85) are shown. The top panel is a cross section of mesophyll cells, whereas the lower panel is a close up of vascular tissue. Representative hyphae are indicated by arrows and the circle on the 85 panel indicates epiphyllous hyphae. Inserts show higher magnification of the endophyte hyphae indicated by the arrowheads in the main panels. Bars = 20 µM. B. Transmission electron micrographs of cross sections of endophyte hyphae in the intercellular spaces of perennial ryegrass. Wild-type <i>E. festucae</i> Fl1 (WT), complemented <i>ΔsidN</i> strain (C-<i>sidN</i>) <i>ΔsidN</i> mutant 54 (54), <i>ΔsidN</i> mutant 85 (85) are shown. Samples shown were photographed from leaf sheath sections, with WT, C-<i>sidN</i>, and <i>ΔsidN</i> 85 hyphae located in mesophyll tissue, whereas <i>ΔsidN</i> 54 is in sclerenchma tissue. c, cytoplasm, v, vacuole. Bars = 500 nm.</p