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Isolation of plasma membrane from protoplasts of Lolium multiflorum (ryegrass) endosperm cells.

ABSTRACT: Plasma membranes have been isolated from protoplasts of suspension-cultured ryegrass (Lolium multiflorum) endosperm cells. The protoplast membrane is coated before cell disruption with murine myeloma protein J539, a galactose-binding immunoglobulin A. The plasma membrane is labelled with 125I by using chemically or enzymically catalysed iodination techniques, or, more conveniently, by using 125I-labelled myeloma protein J539, which enables the membrane to be simultaneously coated and labelled. Protoplast lysis is effected by gentle mechanical means after swelling in hypo-osmotic medium. The plasma-membrane fraction is recovered at low centrifugal forces by fractionation of cell lysates on a discontinuous sucrose/sorbitol gradient. The plasma-membrane fraction is enriched 96-fold on a protein basis with respect to the specific radioactivity of 125I-labeled myeloma protein J539 in the homogenate. Electron microscopy showed long membrane profiles often associated with one another.

SUBMITTER: Schibeci A

PROVIDER: S-EPMC1158515 | biostudies-other | 1982 Sep

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1153383 | biostudies-other

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

Project description: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.

| S-EPMC1154139 | biostudies-other

Positive regulations of adipogenesis by Italian ryegrass [Lolium multiflorum] in 3T3-L1 cells.

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| S-EPMC4057822 | biostudies-literature

Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells.

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| S-EPMC1133664 | biostudies-other

Comparative study of Cd uptake and tolerance of two Italian ryegrass (<i>Lolium multiflorum</i>) cultivars.

Project description:Cadmium (Cd) is one of the most toxic heavy metals and is difficult to be removed from contaminated soil and water. Italian ryegrass (Lolium multiflorum), as an energy crop, exhibits a valuable potential to develop Cd polluted sites due to its use as a biofuel rather than as food and forage. Previously, via a screening for Cd-tolerant ryegrass, the two most extreme cultivars (IdyII and Harukaze) with high and low Cd tolerance during seed germination, respectively, were selected. However, the underlying mechanism for Cd tolerance was not well investigated. In this study, we comparatively investigated the growth, physiological responses, and Cd uptake and translocation of IdyII and Harukaze when the seedlings were exposed to a Cd (0-100 ?M) solution for 12 days. As expected, excess Cd inhibited seedling growth and was accompanied by an accumulation of malondialdehyde (MDA) and reduced photosynthetic pigments in both cultivars. The effects of Cd on the uptake and translocation of other nutrient elements (Zn, Fe, Mn and Mg) were dependent on Cd concentrations, cultivars, plant tissues and elements. Compared with Harukaze, IdyII exhibited better performance with less MDA and higher pigment content. Furthermore, IdyII was less efficient in Cd uptake and translocation compared to Harukaze, which might be explained by the higher non-protein thiols content in its roots. Taken together, our data indicate that IdyII is more tolerant than Harukaze, which partially resulted from the differences in Cd uptake and translocation.

| S-EPMC5628607 | biostudies-literature

Comparative proteomic analyses reveal the proteome response to short-term drought in Italian ryegrass (Lolium multiflorum).

Project description:Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport.

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Interactions between Cellulose and (1,3;1,4)-β-glucans and Arabinoxylans in the Regenerating Wall of Suspension Culture Cells of the Ryegrass Lolium multiflorum.

Project description:Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are composed of several polysaccharides, of which cellulose forms the main fibrillar network. Enmeshed between these fibrils of cellulose are non-cellulosic polysaccharides (NCPs), pectins, and proteins. This study investigates the sequence, timing, patterning, and architecture of cell wall polysaccharide regeneration in suspension culture cells (SCC) of the grass species Lolium multiflorum (Lolium). Confocal, superresolution, and electron microscopies were used in combination with cytochemical labeling to investigate polysaccharide deposition in SCC after protoplasting. Cellulose was the first polysaccharide observed, followed shortly thereafter by (1,3;1,4)-β-glucan, which is also known as mixed-linkage glucan (MLG), arabinoxylan (AX), and callose. Cellulose formed fibrils with AX and produced a filamentous-like network, whereas MLG formed punctate patches. Using colocalization analysis, cellulose and AX were shown to interact during early stages of wall generation, but this interaction reduced over time as the wall matured. AX and MLG interactions increased slightly over time, but cellulose and MLG were not seen to interact. Callose initially formed patches that were randomly positioned on the protoplast surface. There was no consistency in size or location over time. The architecture observed via superresolution microscopy showed similarities to the biophysical maps produced using atomic force microscopy and can give insight into the role of polysaccharides in PCWs.

| S-EPMC7828102 | biostudies-literature

Identification of the valid reference genes for quantitative RT-PCR in annual ryegrass (Lolium multiflorum) under salt stress.

Project description:Annual ryegrass (Lolium multiflorum) is a cool-season annual grass cultivated worldwide for its high yield and quality. With the areas of saline soil increasing, investigation of the molecular mechanisms of annual ryegrass tolerance under salt stress has become a significant topic. qRT-PCR has been a predominant assay for determination of the gene expression, in which selecting a valid internal reference gene is a crucial step. The objective of present study was to evaluate and identify suitable reference genes for qRT-PCR in annual ryegrass under salt stress. The results calculated by RefFinder indicated that eEF1A(s) was the most stable reference gene in leaves, whereas EF1-a was the least stable; meanwhile, TBP-1 was the most optimal in roots and in all samples, and the eIF-5A shouldn't be utilized for normalization of the gene expression. eEF1A(s) is more suitable than TBP-1 as reference gene in leaves when verified with P5CS1 and Cyt-Cu/Zn SOD genes. We should choose optimal reference genes in specific tissues instead of the most stable one selected from different conditions and tissues.

| S-EPMC6272566 | biostudies-literature

Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress.

Project description:BackgroundAnnual ryegrass (Lolium multiflorum L.) is a commercially important, widely distributed forage crop that is used in the production of hay and silage worldwide. Drought has been a severe environmental constraint in its production. Nevertheless, only a handful of studies have examined the impact of short-term drought stress on annual ryegrass. The aim of this study was to explore how stress-induced core metabolic processes enhance drought tolerance, or adaptation to drought, in annual ryegrass.ResultsWe profiled the transcriptomes, proteomes, and metabolomes of two annual ryegrass genotypes: the drought-resistant genotype "Abundant 10" and drought-susceptible genotype "Adrenalin 11." We identified differentially expressed metabolites and their corresponding proteins and transcripts that are involved in 23 core metabolic processes, in response to short-term drought stress. Protein-gene-metabolite correlation networks were built to reveal the relationships between the expression of transcripts, proteins, and metabolites in drought-resistant annual ryegrass. Furthermore, integrated metabolic pathways were used to observe changes in enzymes corresponding with levels of amino acids, lipids, carbohydrate conjugates, nucleosides, alkaloids and their derivatives, and pyridines and their derivatives. The resulting omics data underscored the significance of 23 core metabolic processes on the enhancement of drought tolerance or adaptation to drought in annual ryegrass.ConclusionsThe regulatory networks were inferred using MCoA and correlation analysis to reveal the relationships among the expression of transcripts, proteins, and metabolites that highlight the corresponding elements of these core metabolic pathways. Our results provide valuable insight into the molecular mechanisms of drought resistance, and represent a promising strategy toward the improvement of drought tolerance in annual ryegrass.

| S-EPMC5789592 | biostudies-literature

Fuzzy Union to Assess Climate Suitability of Annual Ryegrass (Lolium multiflorum), Alfalfa (Medicago sativa) and Sorghum (Sorghum bicolor).

Project description:The Law of the Minimum is often implemented using t-norm or fuzzy intersection. We propose the use of t-conorm or fuzzy union for climate suitability assessment of a grass species using annual ryegrass (Lolium multiflorum Lam.) as an example and evaluate the performance for alfalfa (Medicago sativa L.) and sorghum (Sorghum bicolor L.). The ORF and ANDF models, which are fuzzy logic systems based on t-conorm and t-norm between temperature and moisture conditions, respectively, were developed to assess the quality of climate conditions for crops. The parameter values for both models were obtained from existing knowledge, e.g., the EcoCrop database. These models were then compared with the EcoCrop model, which is based on the t-norm. The ORF model explained greater variation (54%) in the yield of annual ryegrass at 84 site-years than the ANDF model (43%) and the EcoCrop model (5%). The climate suitability index of the ORF model had the greatest likelihood of occurrence of annual ryegrass compared to the other models. The ORF model also had similar results for alfalfa and sorghum. We emphasize that the fuzzy logic system for climate suitability assessment can be developed using knowledge rather than presence-only data, which can facilitate more complex approaches such as the incorporation of biotic interaction into species distribution modeling.

| S-EPMC6033868 | biostudies-literature

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