Linear Ion Trap MSⁿ of Enzymatically Synthesized 13C-Labeled Fructans Revealing Differentiating Fragmentation Patterns of β (1-2) and β (1-6) Fructans and Providing a Tool for Oligosaccharide Identification in Complex Mixtures

Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as <i>Lolium perenne</i>. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MSⁿ method for the separation and detection of fructan isomers and present here evidence for specific MSⁿ fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MSⁿ analysis of ¹³C labeled fructan oligomers produced by <i>L. perenne</i> fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards

Plant species, nitrogen status and endophytes are drivers of soil microbial communities in grasslands

Context: There is concern that the introduction of ‘novel’ plant germplasm/traits could outpace our capacity to measure and so assess their impacts on soil microbial communities and function.Aim: This study aimed to investigate the effects of plant species/functional traits, nitrogen (N) fertilisation and endophyte infection on grassland soil microbial communities within a short time span of 2 years.Methods: Two field experiments with monoculture plots were conducted in a common soil. Experiment 1 compared grasses and legumes, using two cultivars of perennial ryegrass (Lolium perenne) that varied in fructan content, along with the legumes white clover (Trifolium repens) and bird’s-foot trefoil (Lotus pedunculatus) that varied in tannin content. Grass treatments received high and low N application levels. Experiment 2 compared the presence/absence of Epichloë strains in ryegrass, tall fescue (Schedonorus phoenix) and meadow fescue (Schedonorus pratensis). Soil microbial communities were analysed by using high-throughput sequencing of DNA isolated from bulk soil cores.Key results: Higher abundance of ligninolytic fungi was found in grass soils and pectinolytic fungi in legume soils. Levels of N fertilisation and fructan in ryegrass had only minor effects on soil fungal communities. By contrast, N fertilisation or fixation had a strong effect on bacterial communities, with higher abundance of nitrifiers and denitrifiers in high-N grass soils and in legume soils than in low-N grass soils. Epichloë affected soil microbiota by reducing the abundance of certain fungal phytopathogens, increasing mycorrhizal fungi and reducing N-fixing bacteria.Conclusions: Chemical composition of plant cell walls, which differs between grasses and legumes, and presence of Epichloë in grasses were the main drivers of shifts in soil microbial communities.Implications: Impacts of farming practices such as mono- or poly-culture, N fertilisation and presence of Epichloë in grasses on soil microbial communities should be considered in pasture management.</p

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

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

RT-qPCR of <i>E. festucae</i> iron-regulated genes and <i>Nox</i> genes in <i>ΔsidN</i> infected perennial ryegrass.

<p>Relative mean abundance relative to wild-type (fold difference displayed) of iron regulated gene expression (<i>ftrA</i>, <i>fetC</i>, <i>hapX</i>) and Nox gene expression (<i>noxA</i>, <i>noxB</i>, <i>noxR</i>, <i>racA</i>) in perennial ryegrass infected with Δ<i>sidN</i> mutants 54 and 85 are shown. For <i>ftrA</i>, <i>fetC</i>, <i>noxA</i> and <i>racA</i> results have a p-value of <0.001 and for <i>hapX</i>, <i>noxB</i> and <i>noxR</i>, the p-values are 0.002, 0.005 and 0.017 respectively. Error bars indicate SED.</p

<i>ΔsidN</i> mutants display abnormal hyphal morphologies on water-agar.

<p>A. Mycelia of wild-type <i>E. festucae</i> Fl1 (WT) on water agar. B. Mycelia of <i>ΔsidN</i> 85 mutant proliferating on water agar by lateral branches. C. Close up of <i>ΔsidN</i> 85 mutant mycelia showing hyphal convolutions and swellings. Bar is 50 µM.</p

Elevated ergovaline levels detected in Δ<i>sidN</i> infected plants.

<p>HPLC analysis of ergovaline production was carried out on perennial ryegrass pseudostems infected with wild-type <i>E. festucae</i> Fl1 (WT), <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>), and complemented <i>ΔsidN</i> strains (C-<i>sidN</i> 1 and C<i>-sidN</i> 2). The numbers of plant reps used for analysis were 3–5 for each sample. Error bars indicate standard deviation.</p

Colonies of <i>ΔsidN</i> mutants are sensitive to hydrogen peroxide on DM.

<p>Values given are ratios of radial growth measurements of colonies grown for 7 days at 22°C on DM (defined medium) or PD (potato dextrose) medium supplemented with 0.7 mM H₂O₂ versus DM or PD. Statistics were generated from an analysis of variance. LSD is the Least Significant Difference between any two means and the SED represents the Standard Error of the Difference.</p