Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses

Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids.We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSⁿ and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.     

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.     

Effects of biotic and abiotic stress on the growth of three genotypes of Lolium perenne with and without infection by the fungal endophyte Neotyphodium lolii

The combinations of three genotypes of Lolium perenne with and without (i) infection by the fungal endophyte Neotyphodium lolii, (ii) infection by ryegrass mosaic virus and (iii) one of five different forms of abiotic stress were studied in pot experiments in a glasshouse. The five abiotic stress treatments were (i) low pH (compared with ‘optimal’ pH), (ii) cutting plants to a height of 1 cm (compared with 5 cm), (iii) shading (compared with no shading), (iv) cutting plants at 2‐weekly intervals (compared with 6 wk) and (v) low nitrogen applied (compared with ‘high’ nitrogen applied). On average, over the five experiments, the accumulated herbage dry weight was 10% more for N. lolii‐infected plants than uninfected, 22% more for virus‐free plants than infected, and 265% more for ‘unstressed’ plants than for plants with abiotic stress. The effects of N. lolii infection on plant growth when the plants were under abiotic or biotic stress were not consistent.     

Effects of the fungal endophyte, Neotyphodium lolii, on net photosynthesis and growth rates of perennial ryegrass (Lolium perenne) are independent of In Planta endophyte concentration

• Background and Aims Neotyphodium lolii is a fungal endophyteof perennial ryegrass (Lolium perenne), improving grass fitnessthrough production of bioactive alkaloids. Neotyphodium speciescan also affect growth and physiology of their host grasses(family Poaceae, sub-family Pooideae), but little is known aboutthe mechanisms. This study examined the effect of N. lolii onnet photosynthesis (P_n) and growth rates in ryegrass genotypesdiffering in endophyte concentration in all leaf tissues. • Methods Plants from two ryegrass genotypes, Nui D andNui UIV, infected with N. lolii (E+) differing approx. 2-foldin endophyte concentration or uninfected clones thereof (E–)were grown in a controlled environment. For each genotype xendophyte treatment, plant growth rates were assessed as tilleringand leaf extension rates, and the light response of P_n, darkrespiration and transpiration measured in leaves of young (30–45d old) and old (>90 d old) plants with a single-chamber openinfrared gas-exchange system. • Key Results Neotyphodium lolii affected CO₂-limited ratesof P_n, which were approx. 17 % lower in E+ than E– plants(P < 0·05) in the young plants. Apparent photon yieldand dark respiration were unaffected by the endophyte (P >0·05). Neotyphodium lolii also decreased transpiration(P < 0·05), but only in complete darkness. There wereno endophyte effects on P_n in the old plants (P > 0·05).E+ plants grew faster immediately after replanting (P < 0·05),but had approx. 10 % lower growth rates during mid-log growth(P < 0·05) than E– plants, but there was noeffect on final plant biomass (P > 0·05). The endophyteeffects on P_n and growth tended to be more pronounced in NuiUIV, despite having a lower endophyte concentration than NuiD. • Conclusions Neotyphodium lolii affects CO₂ fixation,but not light interception and photochemistry of P_n. The impactof N. lolii on plant growth and photosynthesis is independentof endophyte concentration in the plant, suggesting that theendophyte mycelium is not simply an energy drain to the plant.However, the endophyte effects on P_n and plant growth are stronglydependent on the plant growth phase.     

Hot alkali-labile linkages in the walls of the forage grass Phalaris aquatica and Lolium perenne and their relation to in vitro wall digestibility

The factors affecting in vitro dry matter digestibility (IVDMD) of fully mature internodes of 150 lines of the forage grass, Phalaris aquatica, and internodes of 100 lines of perennial ryegrass (Lolium perenne), harvested just after anthesis, were investigated. The relationships between IVDMD and the contents of acetyl bromide lignin, and ester-ether linkages between lignin and wall polysaccharides, measured by hydroxycinnamic acids (HCAs) released by 4 M NaOH at 170 degrees C respectively, were determined. The regression analysis gave r(2)=0.05 and 0.03 for the relation between IVDMD and lignin content and r(2)=0.51 and 0.53 for the relation between IVDMD and the content of hot alkali-labile HCA (predominantly ferulic acid) for phalaris and ryegrass, respectively. These observations are interpreted in terms of the restricted accessibility of polysaccharide hydrolysing enzymes to their substrates in the forage cell walls by the covalent cross-linking of wall polymers through HCAs.     

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.     

The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne)

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.     

Fatality of house crickets on perennial ryegrasses infected with a fungal endophyte

Plants of perennial ryegrass, Lolium perenne L., infected with an endophytic fungus, Acremonium loliae Latch et al. were toxic to house crickets, Acheta domesticus L. Death followed progressive inactivity, culminating in 100% cricket mortality by 84 h. The epithelial lining of the crop and proventriculus of affected crickets was progressively loosened and detached, resulting in complete failure of the alimentary process.Besides the house cricket which is an acridid, antibiosis of a number of coleopterous and lepidopterous pests on endophyte-infected ryegrasses has been previously reported. Thus, the endophyte is an important source of insect resistance which plant breeders can incorporate into new crop varieties.

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Résumé Des expériences ont été faits sur des grillons, Acheta domesticus L., avec des plantes de raygrass vivaces, Lolium perenne L., qui étaient, ou non-infectées par un champignon endophyte, Acremonium loliae Latch, Christensen et Samuels. Les grillons se nourrisant de raygrass infectés ont montré un degré de mortalité beaucoup plus elevé, en 48 heures, que les grillons se nourrissant de raygrass non-infectés. En l'espace de 84 heures, les grillons se nourrissant des trois sortes de raygrass infectés ont montré un pourcentage de mortalité de 100%. La mort a suivi l'inactivité progressive qui est symptomatique des inhibiteurs métaboliques.Les dissections de grillons paralysés ont révélé l'existence d'un estomac antérieur distendu et plus transparent. Le proventricule aussi était transparent, mais moins que l'estomac antérieur. La transparence de l'estomac antérieur, spécialement le jabot, a résulté d'une extension progressive et du ramollissement de l'épithélium intestinal et des groupes de muscles longitudinaux insérés dedans, et éventuellement ces cellules sont éliminées. Ces profonds changements histopathologiques inhibent apparemment le fonctionnement de l'estomac antérieur du grillon avec comme résultat l'inhibition de tout le processus alimentaire.En plus, des excroissances ressemblant à des tumeurs ont été observées dans les jabot des grillons, qui avaient été nourris d'un des trois raygrass infectés par le champignon endophyte. Apparemment, ces excroissances anormales ont été les résultats de l'activation prématurée des groupes de nidi. Quand elles étaient pleinement formées, ces masses se sont séparées de l'épithélium du jabot et ont obstrué la lumière. L'étude de l'étologie et de la pathologie de ces excroissances anormales requiert un travail supplémentaire.Les raygrass infectés par un champignon endophyte sont toxiques pour un certain nombre d'éspèces différentes d'insectes, tels que le Argentine stem weevil Listronotus bonariensis (Kuschel), le bluegrass billbug Sphenophorus parvulus (Gyllenhal) et les larves de sod webworm lépidoptères (plusieurs orthographes). Ainsi, l'endophyte est une source importante de résistance des insectes que les éleveurs de plantes peuvent incorporer donc les nouvelles variétés de cultures.

     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

Association of a fungal endophyte in perennial ryegrass with antibiosis to larvae of the southern armyworm, Spodoptera eridania

Larvae of the Southern armyworm Spodoptera eridania Cramer (Lepidoptera:Noctuidae), feeding on perennial ryegrasses (Lolium perenne L.) infected with an endophytic fungus (Acremonium loliae Latch, Christensen and Samuels), had a much lower survival rate (7–13%) than larvae feeding on endophyte-free ryegrasses (82–90%). Death of the larvae on endophyte-infected entries occurred rapidly between 144 h and 168 h of feeding. This corresponded with armyworms feeding on the base of the plant, where endophyte concentration is highest. Twenty-four hours after the start of the bioassay the larval mass and rate of larval development were significantly higher on endophytic entries. From 48–144 h no differences were seen, but after 144 h the mass of larvae on endophyte-infected grasses sharply declined. Observations from this bioassay further substantiate the association between A. loliae-infected ryegrass and antibiosis to several lepidopterous and coleopterous insect pests.

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Résumé Le ray-grass vivace (Lolium perenne), contaminé par le champignon endophyte Acremonium loliae Latch, Christensen & Samuels, a présenté une augmentation de la resistance à de nombreux coléoptères et lépidoptères nuisibles. Cette note examine les réactions de Spodoptera eridania Cramer (Lépido., Noctuidae) alimenté sur trois lignées de ray-grass contaminées par le champignon et trois lignées saines. Après 168 h d'alimentation sur ray-grass contaminé, les chenilles presentent une très forte mortalité; la survie n'est que de 7 à 13% contre 82 à 90% pour le ray-grass sain. Le décès brutal des chenilles correspond à leur alimentation sur la base de la plante ou la concentration du champignon est la plus forte. Les chenilles consomment constamment, broutant plus des 2/3 du feuillage du ray-grass; les broutements des six séries ne différaient pas significativement.Au bout de 24 h, la nombre de chenilles passées du 3ème au 4ème stade, et l'augmentation de poids sont plus élevés pour les séries sur plante contaminée, ce qui suggère un avantage initial pour les chenilles en présence de champignon endophyte, l'analyse en poids sec a montré que l'augmentation de poids initial est réel. Entre 48 et 144 h, cependant, le nombre de 4ème stade et le poids des chenilles sont les mêmes dans les deux séries. Après 144 h, le poids des chenilles sur ray-grass contaminé diminue significativement; aucune n'était parvenue au 5ème stade, contre 11% sur ray-grass sain. Nous n'avons pas observé de signes apparents de neurotoxicité. Au lieu de cela, il ya a eu interaction avec un processus physiologique fondamental, ce qui a provoqué une forte perte de poids larvaire et la mort, indiquant l'intervention d'inhibiteurs métaboliques.

     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

Mathematical model of fructan biosynthesis and polymer length distribution in plants

Background and Aims

There are many unresolved issues concerning the biochemistry of fructan biosynthesis. The aim of this paper is to address some of these by means of modelling mathematically the biochemical processes.

Methods

A model has been constructed for the step-by-step synthesis of fructan polymers. This is run until a steady state is achieved for which a polymer distribution is predicted. It is shown how qualitatively different distributions can be obtained.

Key Results

It is demonstrated how a set of experimental results on polymer distribution can by simulated by a simple parameter adjustments.

Conclusions

Mathematical modelling of fructan biosynthesis can provide a useful tool for helping elucidate the details of the biosynthetic processes.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC-MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772-2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a beta-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O(9)-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.     

Distribution of littorine and other alkaloids in the roots of datura species

Littorine, various tropoyl and tigloyl esters and cuscohygrine are components of the roots of all nine Datura species studied. The genus exhibits a uniform alkaloid spectrum and its inclusion, with Solandra, in the tribe Datureae is supported on phytochemical grounds.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization

Background and Aims

To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately.

Methods

Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines.

Results

A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species.

Discussion

The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.