Barbara G. Pickard

Changing Patterns of Localization of the Tobacco Mosaic Virus Movement Protein and Replicase to the Endoplasmic Reticulum and Microtubules during Infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent “viral factories.” The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Action potentials in higher plants

Introduction ............................................................................................................. 172 Some Reasonably Well Characterized Action Potentials in Plants with Rapid Motor Activity ......................................................................................................... 173 Propagating Action Potentials Induced in Ordinary Plants by Altering the Temperature or Osmotic Environment, and by Cutting, Mashing, or Heating Tissue ..................................................................................................................... 178 Certain Other Evoked Action Potentials ........................................................ 188 Pollination ...................................................................................................... 188 Frictional Stimulation of Seedlings ......................................................... 189 Elicitation by Pulses of Current ............................................................ 190 Spontaneous, Localized Activity ..................................................................... 190 Sequelae of Action Potentials ............................................................ 192 Acknowledgments and Notes ............................................................................. 196 Note Added in Proof ....................................................................................... 196 Literature Cited .............................................................................................. 198

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

Gravitropism in a starchless mutant of Arabidopsis : Implications for the starch-statolith theory of gravity sensing.

The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10o curvature developed in mutants rotated on a clinostat following a 5 min induction at 1 · g, compared with 14o in the WT. Curvatures induced during intervals from 2.5 to 30 min were 70% as great in the mutant as the WT. Thus under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Arabidopsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70–80% as much curvature as the WT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the WT. Thus, starch is also unnecessary for gravity reception in these tissues.

A stretch-activated anion channel in tobacco protoplasts.

Stretch‐activated ion channels have been described in animal cells, where they might serve as mechanoreceptors, baroreceptors or osmoreceptors, as well as in yeast and bacteria, where osmoregulatory functions have been suggested. Here we report a large conductance, stretch‐activated, anion‐selective channel in protoplasts of a higher plant, tobacco, and discuss its possible role in osmoregulation.

Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

SummaryArabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.

Receptor potentials and action potentials in Drosera tentacles.

SummaryVoltage fluctuations identified as receptor potentials can be detected with electrodes applied to the mucilage surrounding the head of a tentacle of Drosera intermedia if the head is stimulated by contact with a live insect, by the touch of a clean, inert object, or by application of salt solutions. Associated with a low receptor potential are action potentials, which occur at a frequency dependent on the magnitude of the receptor potential. These action potentials can be detected with electrodes applied to any region of the stalk of the tentacle. Inflection of the lower stalk follows the occurrence of action potentials. Inflection is minute for isolated action potentials but large and rapid when several occur within a brief interval.The apparent amplitude of action potentials recorded from the stalk is independent of receptor potential amplitude, but that of action potentials recorded from the mucilage commonly decreases as the receptor potential deviates from the baseline and increases as it returns. It is suggested that variation of apparent amplitude of the action potentials may result from postulated variation in the resistance of receptor membranes.

Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts.

SummaryUsing higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal β1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not β1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specificity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105–110 and 115–125 kDa. These bands are again recognized by the visualizationi antibody, which was raised against the extracellular domain of chicken β1 integrin, and are also reconized by an antibody against the intracellular domain of chicken β1 integrin. Because β1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronectin are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesioni sites in animals.

Properties of action potentials in Drosera tentacles.

SummaryAction potentials of Drosera tentacles resemble those of vertebrate peripheral nerves in that they appear to be comprised of relatively uniform spikes, variable shoulders or negative after-potentials, and variable positive after-potentials. The peaking of the spike corresponds to a period of great refractoriness, while action potentials of low amplitude may be fired readily during the negative after-potential. The action potentials fired during the negative after-potential appear to be unlike those of peripheral nerves in that they are of abnormally brief duration. Also apparently different from the case in peripheral nerves is the dependence of the duration of an action potential on the interval separating it from the preceding action potential.Action potentials propagate from the neck of the stalk to its base at about 5 mm s-1 at room temperature. Propagation may be reversed artificially, consistent with the possibility that the neuroid cells are electrically coupled.

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath.

SummaryCovisualizations with wide-field computational opticalsectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is antigenicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-β1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of β1 integrin and an antibody against the matrix region of β1 integrin. The latter antibody also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.