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PubMed2003

Identification of distinct steps during tubule formation by the movement protein of Cowpea mosaic virus.

The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement.

Biological Transport↗

PubMed2004

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus.

Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein.

Cell Membrane↗

PubMed2005

Evidence for interaction between the 2a polymerase protein and the 3a movement protein of Cucumber mosaic virus.

The genome of Cucumber mosaic virus consists of three single-stranded RNA molecules, RNAs 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome and have been implicated in movement of the viral RNAs in plants. The 3a movement protein (MP), encoded by RNA 3, is essential for transferring the RNA genomes from infected cells to adjacent cells across the plasmodesmata. Far-Western analysis demonstrated that bacterially expressed 2a polymerase protein directly interacted with the MP. Interaction was confirmed in a yeast two-hybrid assay, and co-immunoprecipitation analysis showed that the MP interacted only with the 2a polymerase protein. A yeast three-hybrid assay showed that the 1a-2a protein interaction relevant for replicase complex formation was not affected by the MP. Although the MP has no affinity for the 1a protein, it interacted indirectly with the 1a protein via the 2a polymerase protein. These results suggest that the replicase complex may be involved in movement through its interaction with the MP.

Cucumovirus↗

PubMed2002

Double labeling of KNOTTED1 mRNA and protein reveals multiple potential sites of protein trafficking in the shoot apex.

Recent reports indicate that several plant mRNAs and proteins are able to traffic intercellularly through plasmodesmata. Localization studies can reveal differences between mRNA and protein localization that would be indicative of such a process. However, subtle differences could be missed when comparing localization in adjacent sections, especially in developmental studies where adjacent sections through immature apical regions may be one or more cells removed from each other. Therefore, we have developed a novel method for double localization of KNOTTED1 mRNA and protein in sections through the maize (Zea mays) shoot apex. The advantage of double labeling is revealed in our demonstration of novel potential sites of cell-to-cell trafficking of KNOTTED1 protein in the shoot apical region. The technique should be applicable to any gene products where the appropriate probes are available and will, therefore, help to determine the extent of protein and/or mRNA trafficking in plants.

Homeodomain Proteins↗

PubMed2003

Diurnal and light-regulated expression of AtSTP1 in guard cells of Arabidopsis.

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.

Arabidopsis↗

PubMed1993

Transpiration Induces Radial Turgor Pressure Gradients in Wheat and Maize Roots.

Previous studies have shown both the presence and the absence of radial turgor and osmotic pressure gradients across the cortex of roots. In this work, gradients were sought in the roots of wheat (Triticum aestivum) and maize (Zea mays) under conditions in which transpiration flux across the root was varied This was done by altering the relative humidity above the plant, by excising the root, or by using plants in which the leaves were too young to transpire. Roots of different ages (4-65 d) were studied and radial profiles at different distances from the tip (5-30 mm) were measured. In both species, gradients of turgor and osmotic pressure (increasing inward) were found under transpiring conditions but not when transpiration was inhibited. The presence of radial turgor and osmotic pressure gradients, and the behavior of the gradient when transpiration is interrupted, indicate that active membrane transport or radial solvent drag may play an important role in the distribution of solutes across the root cortex in transpiring plants. Contrary to the conventional view, the flow of water and solutes across the symplastic pathway through the plasmodesmata cannot be inwardly directed under transpiring conditions.

Journal Article↗

PubMed2005

Nematode infection triggers the de novo formation of unloading phloem that allows macromolecular trafficking of green fluorescent protein into syncytia.

Syncytial feeding complexes induced by the cyst nematode Heterodera schachtii represent strong metabolic sinks for photoassimilates. These newly formed structures were described to be symplastically isolated from the surrounding root tissue and their mechanism of carbohydrate import has repeatedly been under investigation. Here, we present analyses of the symplastic connectivity between the root phloem and these syncytia in nematode-infected Arabidopsis (Arabidopsis thaliana) plants expressing the gene of the green fluorescent protein (GFP) or of different GFP fusions under the control of the companion cell (CC)-specific AtSUC2 promoter. In the same plants, phloem differentiation during syncytium formation was monitored using cell-specific antibodies for CCs or sieve elements (SEs). Our results demonstrate that free, CC-derived GFP moved freely from the phloem into the syncytial domain. No or only marginal cell-to-cell passage of GFP was observed into other root cells adjacent to these syncytia. In contrast, membrane-anchored GFP variants as well as soluble GFP fusions with increased molecular masses were restricted to the SE-CC complex. The presented data also show that nematode infection triggers the de novo formation of phloem containing an approximately 3-fold excess of SEs over CCs. This newly formed phloem exhibits typical properties of unloading phloem previously described in other sink tissues. Our results reveal the existence of a symplastic pathway between phloem CCs and nematode-induced syncytia. The plasmodesmata responsible for this symplastic connectivity allow the cell-to-cell movement of macromolecules up to 30 kD and are likely to represent the major or exclusive path for the supply of assimilates from the phloem into the syncytial complex.

Animals↗

PubMed1994

The Use of Fluorescent Tracers to Characterize the Post-Phloem Transport Pathway in Maternal Tissues of Developing Wheat Grains.

Various polar fluorescent tracers were used to characterize the pathways for apoplastic and symplastic transport in the "crease tissues" (i.e. the vascular strand, chalaza, nucellus, and adjacent pericarp) of developing wheat (Triticum aestivum L.) grains. With mostly minor exceptions, the results strongly support existing views of phloem unloading and post-phloem transport pathways in the crease. Apoplastic movement of Lucifer yellow CH (LYCH) from the endosperm cavity into the crease was virtually blocked in the chalazal cell walls before reaching the vascular tissue. However, LYCH could move slowly along the cell wall pathway from the chalaza into the vascular parenchyma. Slow uptake of LYCH into nucellar cell cytoplasm was observed, but no subsequent symplastic movement occurred. Carboxyfluorescein (CF) imported into attached grains moved symplastically from the phloem across the chalaza and into the nucellus, but was not released from the nucellus. In addition, CF moved in the opposite direction (nucellus to vascular parenchyma) in attached grains. Thus, the post-phloem symplastic pathway can accommodate bidirectional transport even when there is an intense net assimilate flux in one direction. When fresh sections of the crease were placed in fluorochrome solutions (e.g. LYCH or pyrene trisulfonate), dye was rapidly absorbed into intact cells, apparently via unsealed plasmodesmata. Uptake was not visibly reduced by cold or by respiratory inhibitors, but was greatly reduced by plasmolysis. Once absorbed, the dye moved intercellularly via the symplast. Based on this finding, a size-graded series of fluorescein-labeled dextrans was used to estimate the size-exclusion limits (SEL) for the post-phloem symplastic pathway. In most, and perhaps all, cells of the crease tissues except for the pericarp, the molecular diameter for the SEL was about 6.2 nm. The SEL in much of the vascular parenchyma may be smaller, but it is still at least 3.6 nm. Channel diameters would likely be about 1 nm larger, or about 4.5 to 7.0 nm in the vascular parenchyma and 7.0 nm elsewhere. These dimensions are substantially larger than those for "conventional" symplastic connections (about 3 nm), and would have a greater than proportionate effect on the per channel diffusive and hydraulic conductivities of the pathway. Thus, relatively small and probably ultrastructurally undetectable adjustments in plasmodesmatal structure may be sufficient to account for assimilate flux through the crease symplast.

Journal Article↗

PubMed2005

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection.

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.

Cells, Cultured↗

PubMed2006

Cross-species translocation of mRNA from host plants into the parasitic plant dodder.

An intriguing new paradigm in plant biology is that systemically mobile mRNAs play a role in coordinating development. In this process, specific mRNAs are loaded into the phloem transport stream for translocation to distant tissues, where they may impact on developmental processes. However, despite its potential significance for plant growth regulation, mRNA trafficking remains poorly understood and challenging to study. Here, we show that phloem-mobile mRNAs can also traffic between widely divergent species from a host to the plant parasite lespedeza dodder (Cuscuta pentagona Engelm.). Reverse transcription-polymerase chain reaction and microarray analysis were used to detect specific tomato (Lycopersicon esculentum Mill.) transcripts in dodder grown on tomato that were not present in control dodder grown on other host species. Foreign transcripts included LeGAI, which has previously been shown to be translocated in the phloem, as well as nine other transcripts not reported to be mobile. Dodders are parasitic plants that obtain resources by drawing from the phloem of a host plant and have joint plasmodesmata with host cortical cells. Although viruses are known to move between dodder and its hosts, translocation of endogenous plant mRNA has not been reported. These results point to a potentially new level of interspecies communication, and raise questions about the ability of parasites to recognize, use, and respond to transcripts acquired from their hosts.

Arabidopsis↗

PubMed2006

Common plantain. A collection of expressed sequence tags from vascular tissue and a simple and efficient transformation method.

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-beta-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Arabidopsis↗

PubMed1968

Translocation of assimilates and phosphate in detached bean leaves.

(14)C-assimilates were accumulated by the veins in the blades and transported basipetally into the petioles of detached leaves of Red Kidney bean (Phaseolus vulgaris L.). Neither process was greatly affected by mild moisture stress, age of fully enlarged leaves, or period in the dark prior to exposure to (14)CO(2). However, both vein loading and transport into petioles were greatly reduced by oxygen deficiency. The basipetal transport of (32)PO(4) also did not appear to be greatly reduced by 6 or 8 days of darkness prior to the application of phosphate-(32)P, followed by a transport period of 1 day in the dark. Endothall at 5 x 10(-3)m was effective in stopping basipetal flow of (32)P. It is considered that transport in leaves may be powered by forces in the plasmodesmata of the cell walls between the border parenchyma and phloem.

Journal Article↗

PubMed1971

Electrical potential differences in cells of barley roots and their relation to ion uptake.

Single cell electropotentials of barley (Hordeum vulgare L., cv. ;Compana') root cortex were measured at different external concentrations of KCl in the presence of Ca(2+). The roots were low in salt from seedlings grown on 0.5 mm aerated CaSO(4) solution. Thus, the conditions were equivalent to those used to define the dual mechanisms found with radioactive tracer-labeled ion uptake. In 0.5 mm CaSO(4) alone, there is an increase with time of cell negativity from about -65 millivolts 15 minutes after cutting segments to about -185 millivolts in 6 to 8 hours. Two possible hypotheses, not mutually exclusive, are offered to explain this aging effect: that cutting exposes plasmodesmata which are leaky initially but which seal in time, and that some internal factors, e.g., hormones diffusing from the apex, have a regulatory effect on the cell potential, an influence which becomes dissipated in isolated segments and permits the development of a higher potential difference. In any case changes in selective ion transport must be involved. The cell potentials at KCl concentrations above 2.0 mm are more negative than would be expected for a passive diffusion potential. It is suggested that this discrepancy may be due to an electrogenic pump or to a higher K(+) concentration in the cytoplasm than in the remainder of the cell, or perhaps to both. Whether there is a clear relationship between cell potential and mechanisms 1 and 2 of cation transport depends upon whether the cell potentials of freshly cut or of aged tissue represent the values relevant to intact roots.

Journal Article↗

PubMed1976

Potassium-linked Chloride Fluxes during Rhythmic Leaf Movement of Albizzia julibrissin.

Transverse sections of Albizzia pulvinules were examined with an electron microprobe to determine ion fluxes associated with turgorcontrolled leaflet movements. K(+) and Cl(-) concentrations are high in the flexor and low in the extensor region of closed pulvini. Both ions migrate out of the flexor and into the extensor during opening as previously described for K(+). The distribution of these elements is significantly correlated in each phase of the rhythmic cycle examined, but only 50 to 60% of the ionic charge of potassium is balanced by chloride. This value increases to 65 to 85% if one considers only the mobile fraction of the potassium.The increase in concentration of both ions in the extensor region precedes the decrease in the flexor, thus indicating that there must be a storage reservoir for K(+) and Cl(-). The inner cortex is suggested as such a reservoir, and plasmodesmata are discussed as a probable pathway for ion movement.

Journal Article↗

PubMed1980

Movement of C-labeled Assimilates into Kernels of Zea mays L: III. AN ANATOMICAL EXAMINATION AND MICROAUTORADIOGRAPHIC STUDY OF ASSIMILATE TRANSFER.

Basal tissue of developing maize kernels was examined by light micros-copy and by scanning and transmission electron microscopy. Plasmodesmata occur in pedicel and endosperm parenchyma but were not seen between the placento-chalazal cells and basal endosperm transfer cells. A layer of noncellular material separates the transfer cells from the placentochalazal cells. Microautoradiography of (14)C-labeled assimilates entering the kernels revealed that incoming sugars are not confined to the apoplast, but rather are present in the cytoplasm and vacuoles of pedicel and endosperm cells. No specific accumulation of radioactivity was seen in any particular tissue, although at later sampling times, a higher grain density in the pedicel than the endosperm indicated a general buildup of sugars in the pedicel. A possible model for sugar movement into developing kernels is discussed.

Journal Article↗

PubMed1981

Reduction of Hydraulic Conductivity during Inhibition of Exudation from Excised Maize and Barley Roots.

The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is shown to reduce the hydraulic conductivity of barley, maize, mung bean, and onion roots. In barley and maize, the reduction in exudation from excised roots is partly due to the reduction in the permeability of the root to water (I(p)), but it can be inferred that the rate of salt release to the xylem, is also inhibited. The action of CCCP on L(p) is suggested to be mainly in blocking the symplasmic pathway at the plasmodesmata.

Journal Article↗

PubMed1982

Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK.

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H(+) excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H(+) uptake, but osmoregulation can still occur, and enhanced H(+) uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H(+) rather than affecting only H(+) or counter ion uptake.Plasmolysis alone does not inhibit fusicoccin-enhanced H(+) excretion, and the rehydration step must be rapid and in the cold for maximum inhibition. This suggests that the plasma membrane is perturbed, possibly due to release or rearrangement of membrane protein. Compared to corresponding osmolarities of sorbitol or NaCl, polyethylene glycol 4000 is much less effective during the plasmolysis step; the possibility is discussed that the ineffectiveness of polyethylene glycol 4000 is due to its preservation of plasmodesmata.

Journal Article↗

PubMed1986

Symplastic Transport in Ipomea tricolor Source Leaves : Demonstration of Functional Symplastic Connections from Mesophyll to Minor Veins by a Novel Dye-Tracer Method.

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.

Journal Article↗