Localization of beta-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells.

The distribution of beta-glucan synthases between plasma membranes and intracellular membranes of suspension-cultured Italian-ryegrass (Lolium multiflorum Lam.) endosperm cells was examined. Highly purified plasma membranes prepared from protoplasts were only slightly enriched in beta-glucan synthases assayed at 10 microM- and 1 mM-UDP-glucose. Most beta-glucan synthase was associated with intracellular membranes. These membranes were fractionated on a linear sucrose density gradient and were resolved into different membrane fractions containing beta-glucan synthases. Beta-Glucan synthases assayed at 10 microM-UDP-glucose were found in a fraction banding at a density of 1.11 g . cm-3, but most of the beta-glucan synthase assayed at 1 mM-DDP-glucose was at a density of 1.04 g . cm-3.     

Biosynthesis of arabinogalactan-protein in Lolium multiflorum (Italian ryegrass) endosperm cells. Subcellular distribution of galactosyltransferases.

Intracellular membranes from protoplasts of Italian-ryegrass (Lolium multiflorum) endosperm cells have been fractionated on sucrose density gradients and identified on the basis of putative-marker-enzyme assays. Galactosyltransferases capable of incorporating galactose from UDP galactose into 66% ethanol-soluble products are associated with all membrane fractions. Affinity chromatography of the ethanol-insoluble products on (murine myeloma protein J539)-Sepharose reveals that the enzymes responsible for the synthesis of polymers containing (1----6)-beta-D-galactose residues are associated exclusively with subcellular fractions enriched in Golgi-derived membranes. This suggests that the Golgi apparatus plays an important part in the synthesis of the carbohydrate component of the ryegrass arabinogalactan-protein.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5 regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.Abbreviations 2,4-D 2,4 Dichlorophenoxyacetic acid - GUS Glucuronidase - Hm Hygromycin - HPH Hygromycin phosphotransferase - MS medium Murashige and Skoog medium - PCR Polymerase chain reaction - X-Gluc 5-Bromo-4-chloro--indolyl--D-glucuronic acid     

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene. Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998     

Factors affecting parasitism of stem-borer larvae in Italian ryegrass (Lolium multiflorum)

Factors affecting parasitism of stem-boring larvae which attack Italian ryegrass were studied. Stem-boring larvae dissected out of tillers from a range of Italian ryegrass cultivars showed parasitism of between 10–65%. However, the level of attack by Chasmodon apterus Nees, a ground-dwelling braconid which dominated the parasitoid complex, did appear to be affected by the plant cultivar, independently of the level of stem-borer infestation.Italian ryegrass cultivars, newly-sown on land previously sown to barley and which had received conventional seed-bed cultivations showed low levels of attack by C. apterus, but increased levels of attack by Halticoptera circulus Walker. The use of the pesticide phorate adversely affected parasitism by c. apterus.

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Résumé L'étude porte sur les facteurs influençant le parasitisme par des hyménoptères des larves mineuses des tiges du ray-gras Italien. Le niveau du parasitisme dans les larves extraites des talles de 13 cultivars différents du ray-gras Italien est identique. Toutefois, le niveau d'attaque par Chasmodon apterus, majoritaire dans le complexe des parasites, semble être affecté par le cultivar, quel que soit le niveau d'infestation par les mineuses des tiges. Les effectifs des larves de mineuses des tiges et de C. apterus semblent augmenter parallèlement, le maximum étant atteint avec le cultivar Tiara. Bien que la densité des mineuses soit plus importante pour d'autres cultivars, le niveau de parasitisme y est moindre. Les cultivars de ray-gras Italiens semées après une culture d'orge, et recevant une fertilisation conventionnelle au semis, sont peu attaqués par C. apterus, probablement parce que ce parasite colonise les champs lentement; par contre le niveau d'attaque dû à Halticoptera circulus est accru. Les traitements pesticides à base de phorate affectent négativement le parasitisme par C. apterus.

     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Improvement of persistence and yield of Italian ryegrass (Lolium multiflorum) through pesticide use

The present two field experiments, involving plots of Italian ryegrass sown in 1976, 1980 and 1984 confirmed results of earlier work in which the yield and persistence of Italian ryegrass was greatly increased by the application of pesticide. A range of pesticide treatments were used. The responses to application of permethrin applied three times per year average 8% for the three years of the first experiment and 5% for the five years of the second experiment. Such responses appeared cost-effective, but the overall environmental impact of such applications is not known.     

The elimination of ryegrass mosaic virus from Lolium multiflorum by meristem tip culture

Meristem tips were cultured from Lolium multiflorum plants infected with ryegrass mosaic virus (RMV). Meristem tips within the size range O'2-fi mm long were cultured on media with or without 2 ,4-D at 1 mg/1. The plants that regenerated in culture showed no symptoms over a 10 month period and no RMV particles were observed by electron microscopy. It was concluded that RMV had been eliminated. The method should prove useful in eliminating the virus from desirable genotypes used as parent material for seed production.     

Isolation of intact plastids from protoplasts from castor bean endosperm

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose “Onozuka” R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.     

Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.)

Summary Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.) has been accomplished. After an enzyme incubation, ovules disintegrated into loose cells upon mechanical manipulation. The egg cells could be identified between the bulk of sporophytic cells derived from the macerated ovules. The morphology of the isolated egg cell corresponds to the morphology of the egg cell in situ and is comparable to the morphology of egg cells of other monocotyledons and angiosperms. Two hours after isolation the egg cells were still viable. The protocol proved reproducible and the yield was determined at 10%.     

The effects of mixing Italian ryegrass (Lolium multiflorum) with perennial ryegrass (L. perenne) or red clover (Trifolium pratense) on the incidence of viruses

Mixing the ryegrass mosaic virus (RMV) resistant perennial ryegrass (Lolium perenne) cv. Endura with the susceptible Italian ryegrass (L. multiflorum) cv. RvP decreased infection of RvP wth RMV from 37% when grown alone to 22% when mixed. However, Endura yielded less than RvP and there was no yield benefit from mixing the two cultivars. Mixing red clover (Trifolium pratense) cv. Hungaropoly with RvP had no detectable effect on RMV incidence in RvP but did decrease the incidence of red clover necrotic mosaic virus in Hungaropoly from 9% to 1% and of white clover mosaic virus from 53-5% to 41%. The yield of the mixture was equal to that of RvP grown alone but given nitrogen fertiliser. The numbers of eriophyid mites, including Abacarus hystrix the vector of RMV, on ryegrass leaves were similar in pure and mixed swards. It is concluded that with herbage crops, the common practice of sowing mixtures of species may help control virus diseases.     

Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3

For effective remediation of contaminants, plant-endophyte partnership is a promising field to be explored. Generally endophytic bacteria assist their host plant by withstanding the stress induced by the contaminants. The objective of this study was to explore the suitability of plant-bacterial partnership for chlorpyrifos (CP) remediation using ryegrass and a CP degrading endophyte, Mesorhizobium sp. HN3 which belongs to plant growth promoting rhizobia. The inoculated yfp-tagged Mesorhizobium sp. HN3 efficiently colonized in the rhizosphere, enhanced plant growth and degradation of CP and its metabolite 3,5,6 trichloro-2-pyridinol (TCP). Significantly lower CP residues were observed in the roots and shoots of plants vegetated in inoculated soil which might be attributed to the efficient root colonization of HN3yfp. These results suggest the involvement of Mesorhizobium sp. HN3yfp in CP degradation inside the roots and rhizosphere of plants and further emphasize on the effectiveness of endophytic bacteria in stimulating the remediation of pesticide contaminants. This is the first report which demonstrates the efficacy of bacterial endophyte for degradation of CP residues taken up by the plant and enhanced remediation of chlorpyrifos contaminated soil.     

The role of silica in protecting Italian ryegrass (Lolium multiflorum) from attack by dipterous stem-boring larvae (Oscinellafiit and other related species)

The importance of the silica content of the plant as a protection against dipterous stem-borers was studied. Levels of silica in the stubble were inversely related to the degree of infestation by stem-borers in 13 varieties of established swards of Italian ryegrass and also in newly-sown swards of four varieties. There were significant reductions in larval colonisation of Italian ryegrass (cv. RvP) grown on soil watered with sodium silicate solution. A small range of silica content in the stubble was related to large differences in the level of attack by stem-borers.     

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum)

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.     

Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.)

Background

Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs) in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency.

Results

Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene.

Conclusions

Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.     

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.