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1.
A novel method for the delivery of the fluorescent dye Lucifer Yellow CH to the cytosol of a source leaf mesophyll cell was devised which utilized a preencapsulation of the dye in phospholipid vesicles (liposomes). The liposomes were easily injected into the vacuoles of leaf cells of Beta vulgaris or Ipomea tricolor, where fusion with the tonoplast resulted in the release of the dye into the cytosol. Subsequent cell-to-cell movement of the dye was readily followed by fluorescence microscopy. Using this liposome technique symplastic continuity from the the mesophyll to the minor veins of the source leaf of Ipomea tricolor was demonstrated. This agreed with ultrastructural studies which demonstrated the presence of plasmodesmata between all cells from the mesophyll to the minor veins. The symplastic movement of dye from the injected mesophyll cell to the minor veins was unaffected by pretreatment of the leaf tissues with 2 millimolar p-chloromercuribenzenesulfonic acid. Pretreatment of the leaf tissues at alkaline pH (3-[N-morpholino] propanesulfonic acid-KOH, pH 8.0) had no apparent effect on dye movement between adjacent mesophyll cells but inhibited the movement of dye into and along the minor veins. Thus, although there were no apparent barriers to symplastic solute movement in this leaf, symplastic barriers could be imposed by the experimental conditions used.  相似文献   

2.
We investigated the phloem loading pathway in barley, by determining plasmodesmatal frequencies at the electron microscope level for both intermediate and small blade bundles of mature barley leaves. Lucifer yellow was injected intercellularly into bundle sheath, vascular parenchyma, and thin-walled sieve tubes. Passage of this symplastically transported dye was monitored with an epifluorescence microscope under blue light. Low plasmodesmatal frequencies endarch to the bundle sheath cells are relatively low for most interfaces terminating at the thin- and thick-walled sieve tubes within this C3 species. Lack of connections between vascular parenchyma and sieve tubes, and low frequencies (0.5% plasmodesmata per μm cell wall interface) of connections between vascular parenchyma and companion cells, as well as the very low frequency of pore-plasmodesmatal connections between companion cells and sieve tubes in small bundles (0.2% plasmodesmata per μm cell wall interface), suggest that the companion cell-sieve tube complex is symplastically isolated from other vascular parenchyma cells in small bundles. The degree of cellular connectivity and the potential isolation of the companion cell-sieve tube complex was determined electrophysiologically, using an electrometer coupled to microcapillary electrodes. The less negative cell potential (average –52 mV) from mesophyll to the vascular parenchyma cells contrasted sharply with the more negative potential (–122.5 mV) recorded for the companion cell-thin-walled sieve tube complex. Although intercellular injection of lucifer yellow clearly demonstrated rapid (0.75 μm s-1) longitudinal and radial transport in the bundle sheath-vascular parenchyma complex, as well as from the bundle sheath through transverse veins to adjacent longitudinal veins, we were neither able to detect nor present unequivocal evidence in support of the symplastic connectivity of the sieve tubes to the vascular parenchyma. Injection of the companion cell-sieve tube complex, did not demonstrate backward connectivity to the bundle sheath. We conclude that the low plasmodesmatal frequencies, coupled with a two-domain electropotential zonation configuration, and the negative transport experiments using lucifer yellow, precludes symplastic phloem loading in barley leaves.  相似文献   

3.
Cell-cell communication in the leaves of Commelina cyanea and other plants   总被引:8,自引:4,他引:4  
Abstract. The fluorescent probes 6–carboxyfluorescein and lucifer yellow CH which do not pass the plasmalemma have been used to examine cell-to-cell communication in the leaf of Commelina cyanea. Dye movement from cell-to-cell occurs in epidermal, spongy and palisade mesophyll, and vascular cells. Dye movement between these tissues was also found. Hence, the epidermis, spongy and palisade mesophyll cells, and vascular tissue are all linked in a continuous symplast. However, dye injected into the epidermal cells rarely moves into guard cells, indicating that these cells are relatively isolated from the surrounding cells. In the same way, guard cells in Vicia faba and the C4 grass Anthephora pubescens also appeared to be isolated from epidermal cells. Thus guard cell isolation from cell-to-cell communication appears to be a common phenomenon. Hence, the ion fluxes required for guard cell function must occur via the apoplast.  相似文献   

4.
We have stripped small (3 × 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.  相似文献   

5.
A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.  相似文献   

6.
An unusual procedure with the dye lucifer yellow has provided stable neuronal marking that survives paraffin embedding and sectioning. Lucifer yellow CH was dissolved in an electrolyte containing formaldehyde and injected into the large intemeurons of a cricket. Intense fluorescence in the axoplasm was retained even after conventional histological procedures.  相似文献   

7.
An unusual procedure with the dye lucifer yellow has provided stable neuronal marking that survives paraffin embedding and sectioning. Lucifer yellow CH was dissolved in an electrolyte containing formaldehyde and injected into the large interneurons of a cricket. Intense fluorescence in the axoplasm was retained even after conventional histological procedures.  相似文献   

8.
Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.  相似文献   

9.
Dye and electrical coupling of endothelial cells in situ   总被引:3,自引:0,他引:3  
J L Bény  F Gribi 《Tissue & cell》1989,21(6):797-802
Electron microscopic studies show that endothelial cells of pig coronary arteries are linked by gap junctions. We investigated the dye and electrical coupling of these junctions in a strip of pig coronary artery in vitro. The membrane potential of two neighbouring (about 0.2 mm) endothelial cells were simultaneously recorded with two microelectrodes. The fluorescent dye lucifer yellow was microiontophoretically injected through one of the microelectrodes. The endothelial cells in situ were dye and electrically coupled. The dye coupling extended parallel to the longitudinal axis of the arteries. We conclude that an electrical message like the bradykinin and substance P hyperpolarizations of the endothelial cells can be conveyed electrotonically by the endothelium along the longitudinal axis of arteries.  相似文献   

10.
Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.  相似文献   

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