Cytoplasm Of Protoplasts Research Articles

The cytosolic protein GRP1 facilitates abscisic acid- and darkness-induced stomatal closure in Salvia miltiorrhiza

By screening an expressed sequence tag (EST) library of Salvia miltiorrhiza, we detected an acidic protein, SmGRP1, with no significant similarities to the other sequences in public databases. SmGRP1 encodes a peptide of 151 amino acids, 33.77 % of which are glutamic acid residues, and the peptide was positive according to “stains-all” staining. Expression analysis revealed that SmGRP1 was expressed in all examined tissues of S. miltiorrhiza but was most highly expressed in the leaves and stems. Without a signal peptide, SmGRP1 localized to the cytoplasm in protoplasts in subcellular localization experiments. SmGRP1 expression was prominently enhanced by ABA and darkness treatments; the protein could also be induced by high temperature, NaCl, and dehydration treatments, while low temperature suppressed its expression. Furthermore, although there were no obvious phenotypic differences in SmGRP1 overexpression and SmGRP1 knockdown mutants compared with control plants under normal culture conditions, the stomata of the knockdown lines remained open when treated with ABA, darkness, NO, and H2O2. In addition, the water loss rate of the knockdown mutants was faster than that of the control lines and overexpression mutants when exposed to air. These observations indicate that SmGRP1 is a novel acidic protein with potential calcium-binding capability and is involved in stomatal movement and stress resistance.

Genome-Wide Analysis of the Fasciclin-Like Arabinogalactan Protein Gene Family Reveals Differential Expression Patterns, Localization, and Salt Stress Response in Populus.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) involved in plant growth, development and response to abiotic stress. Although many studies have been performed to identify molecular functions of individual family members, little information is available on genome-wide identification and characterization of FLAs in the genus Populus. Based on genome-wide analysis, we have identified 35 Populus FLAs which were distributed on 16 chromosomes and phylogenetically clustered into four major groups. Gene structure and motif composition were relatively conserved in each group. All the members contained N-terminal signal peptide, 23 of which included predicted glycosylphosphatidylinositol (GPI) modification sites and were anchored to plasma membranes. Subcellular localization analysis showed that PtrFLA2/20/26 were localized in cell membrane and cytoplasm of protoplasts from Populus stem-differentiating xylem. The Ka/Ks ratios showed that purifying selection has played a leading role in the long-term evolutionary period which greatly maintained the function of this family. The expression profiles showed that 32 PtrFLAs were differentially expressed in four tissues at four seasons based on publicly available microarray data. 18 FLAs were further verified with qRT-PCR in different tissues, which indicated that PtrFLA1/2/3/7/11/12/20/21/22/24/26/30 were significantly expressed in male and female flowers, suggesting close correlations with the reproductive development. In addition, PtrFLA1/9/10/11/17/21/23/24/26/28 were highly expressed in the stems and differentiating xylem, which may be involved in stem development. To determine salt response of FLAs, qRT-PCR was performed to analyze the expression of 18 genes under salinity stress across two time points. Results demonstrated that all the 18 FLAs were expressed in root tissues; especially, PtrFLA2/12/20/21/24/30 were significantly induced at different time points. In summary, this study may lay the foundation for further investigating the biological functions of FLA genes in Populus trichocarpa.

Radial F-actin configurations are involved in polarization during protoplast germination and thallus branching of Macrocystis pyrifera (Phaeophyceae, Laminariales)

The involvement of actin filaments (AFs) in polarization preceding the development of local outgrowths in protoplast-derived cells (PDCs), as well as in branch initials of Macrocystis pyrifera gametophytes was investigated after AF staining with Rhodamine–Phalloidin (Rh–Ph). A well-developed network of randomly distributed AF arrays was found to traverse the cortical cytoplasm of protoplasts and thallus cells. In some thallus cells, the cortical AFs were largely reorganized and radial AF configurations were formed in the prospective sites of branch emergence. Similar AF configurations were found in PDCs at the stage of formation of rhizoid-like protrusions (RLPs), as well as in young thalli regenerated from protoplasts. The pointed-radial AF formations seemed to change into circular radial structures, from the periphery of which AFs were radiating out. These AF structures were observed at the base of the outgrowths resulting in branch or RLP formation. At later stages the above AFs disappeared and the normal AF network was developed. In parallel, AF caps were found at the tip of the developing RLPs or young branches. The sites of convergence of the radial AFs might be considered as putative AF nucleating or organizing sites, or both. Treatment with Latrunculin B and Jasplakinolide resulted in the inhibition of new RLP formation in regenerating protoplasts, showing that dynamic actin is necessary for polarity establishment and the formation of RLP. The probable mechanism(s) by which the radial AF arrays are involved in polarization of the examined cells, defining the site of cell protrusion(s), are discussed.

Perception of Gibberellin and Abscisic Acid at the External Face of the Plasma Membrane of Barley (Hordeum vulgare L.) Aleurone Protoplasts.

The response of protoplasts isolated from aleurone layers of barley (Hordeum vulgare L. cv Himalaya) to internally and externally applied hormone was analyzed to localize the site of perception of the hormonal signal. Protoplasts responded to externally applied gibberellic acid (GA3) with increased synthesis and secretion of [alpha]-amylase, transient expression of the glucuronidase reporter gene fused to the hormone-responsive elements of the [alpha]-amylase promoter, and the vacuolation typical of GA3-treated aleurone cells. When up to 250 [mu]M GA3 was microinjected into the protoplast cytoplasm, none of these responses were observed. This did not reflect damage to the protoplasts during the microinjection procedure, since microinjected protoplasts remained responsive to externally applied hormone. Nor did it reflect loss of microinjected GA3 from the protoplast, since 50% of microinjected [3H]GA20 was retained by protoplasts for at least 24 h. Externally applied abscisic acid (ABA) could reverse the stimulation of [alpha]-amylase synthesis and secretion, whereas microinjecting up to 250 [mu]M ABA was ineffective at antagonizing the stimulatory effect of GA3. These results suggest that the site of perception of GA3 and ABA in the barley aleurone protoplast is on the external face of the plasma membrane.

Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts

ABSTRACT The mode of action of cell wall elicitors in the induction of various plant cell responses, such as the activation of host defence mechanisms, is unknown, but signal transduction through cytosolic free calcium ([Ca2+]i) has been suggested. This paper shows that polygalacturonic acid or oligogalacturonides cause a prolonged increase in [Ca2+]i of carrot protoplasts within 20 min of induction. Our data support the view that a special conformation of the oligogalacturonides possessing >9 residues is necessary to induce an elevation in [Ca2+]i. The localization of [Ca2+]i elevation around the periphery of protoplast cytoplasm and the inhibition of the response with verapamil suggest that exogenous Ca2+ is the major source for the rise in [Ca2+]i.

Osmotically induced fluid-phase uptake of fluorescent markers by protoplasts ofChenopodium album

Protoplasts fromChenopodium album suspension culture show large, up to 5-fold, changes in surface area upon hypertonic or hypotonie treatment. These surface area variations cannot be explained by elastic stretching of the plasmalemma. An exchange of membrane material between the plasmalemma and an internal membrane source takes place. Fluid-phase uptake experiments with the fluorescence dyes 5, 6-carboxyfluorescein and Lucifer yellow CH demonstrated that osmotic shrinkage of protoplasts is accompanied by vesicular uptake of the external medium into protoplast cytoplasm. Confocal laser scanning microscopy, as well as conventional fluorescence microscopy, revealed the number, diameter and distribution of the osmocytotic vesicles at different osmotic levels. The rate of osmocytotic vesicle uptake was higher in the presence of calcium chloride than in the presence of EDTA in the external medium. At 6.9 mM calcium chloride we observed a loss of vesicular fluorescence upon returning protoplasts to 0.4 M from 0.8 M sorbitol.

Induction of protoplast formation in the ectomycorrhizal fungus Paxillus involutus by the root rot pathogen Fusarium oxysporum

SUMMARYCo‐culture of the pathogenic fungus Fusarium oxysporum f. sp. pini Schlecht. emend Snyd. & Hans, with the ectomycorrhizal fungus Paxillus involutus Fr. on potato dextrose agar caused no visible morphological changes in the fungi. Co‐culture on modified Melin Norkrans medium in petri plates yielded, however, protoplast formation at or near tips of P. involutus hyphae. No changes were visible in F. oxysporum as a result of co‐culture and the two fungi grew in close association with each other without inhibition. Staining with Calcofluor White M2R New and fluorescein diacetate displayed that, at this stage in development, a wall surrounding the protoplast was absent but a plasma membrane sheathed by an extracellular mucilage was present. Protoplasts lacking definite form adhered closely to adjoining hyphae while those with the characteristic spherical form were easily dislodged from hyphae. Average size of spherical protoplasts was 17–20 μm. Breaks were present in the hyphal wall in regions adjacent to protoplast formation and these may have been pathways for cytoplasmic leakage. Transmission electron microscopy revealed these breaks more clearly, and provided additional evidence of a plasma membrane around protoplast cytoplasm. Protoplasts contained few organelles except vesicles, larger electron dense bodies and occasional nuclei. Hyphae forming protoplasts often lacked cytoplasmic integrity although those not forming protoplasts appeared healthy.

Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62×10-4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes

We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 x 10(5) protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.

Rapid separation of the plastid, mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena

Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-μm aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 μl microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.

Polyethylene glycol-induced transplantation of chloroplasts into protoplasts: An ultrastructural assessment

Mixtures of protoplasts from suspension-cultured cells of Parthenocissus tricuspidata and chloroplasts from Petunia hybrida were treated with polyethylene glycol (PEG). The envelope of intact chloroplasts readily fused with the protoplasts plasmalemma to establish continuity between the chloroplast stroma and the protoplast cytoplasm. Broken chloroplasts became localised in membrane-bounded vesicles in the cytoplasm of the higher plant protoplasts.

Demonstration of ribosomes in mesosomes associated with Bacillus subtilis protoplasts.

The physiological differences between Bacillus subtilis (ATCC 6633) cells derived from a glucose-salts-yeast extract (GSY) medium and those of cells from tryptose broth permitted the identification of variables in protoplasting environments which noticeably affected the clarity of mesosomal ribosomes. They were the sucrose and magnesium ion concentrations and the type of buffer used. The environment suitable for conversion of GSY cells to the protoplast state was a 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, containing 0.6 M sucrose and 0.03 M MgCl(2). Branched mesosomal tubules and a unique organization of vesicles were detected in thin sections and in negative stains of the specimens. Ribosomes were demonstrable in the extruded structures associated with protoplasts that had been prepared according to four fixation schedules and embedded in either of two epoxy plastics. Adjustments in the fixation schedules improved the clarity of the large bodies of protoplast cytoplasm to a degree equivalent to that of their dangling appendages.