Reiko Nagai

Regulation of Intracellular Movements in Plant Cells by Environmental Stimuli

Publisher Summary This chapter describes regulation of intracellular movements in plant cells (cytoplasm, chloroplasts, and nucleus) by environmental stimuli (light, low temperature, wounding, and chemicals). It also reviews possible receptor systems specific for each type of environmental stimulus, a motile apparatus that functions as a specific effector, and a possible signal-transduction chain that conveys information between a receptor system and an effector system. In plant cells, various forms of intracellular movements such as the orientation movement of chloroplasts, the traumatotactic or premitotic migration of the nucleus, and streaming of the cytoplasm, are observed. These movements are thought to be essential for accomplishment of efficient photosynthesis, cell division at a suitable site, and appropriate delivery of substances that are required for growth and differentiation of plant cells. A spatially well-organized motile apparatus drives these movements in plant cells. When streaming is induced by light irradiation, the streaming is referred to as photodinesis. The distribution of organelles in the cytoplasm does not change after the perception of light and the response is independent of the direction of irradiation. For cytoplasmic streaming to occur, at least two conditions must be fulfilled— a motive force must be generated and the cytoplasmic matrix must have appropriate mechanical properties. Streaming of the cytoplasm can be accelerated through an increase in the motive force or a decrease in the cytoplasmic viscosity.

The plant cytoskeleton.

Particles that can nucleate microtubules in vitro have been isolated from higher plant cells. Observations of living cells injected with fluorescent probes have improved our understanding of plant cytoskeleton dynamics. Despite growing recognition of the need for biochemical studies on cytoskeleton-associated proteins, little progress has been made in this field in the past year, although plant lamins have been isolated and partially characterized.

ORIENTATION MOVEMENTS OF CHLOROPLASTS IN Vallisneria EPIDERMAL CELLS: DIFFERENT EFFECTS OF LIGHT AT LOW‐ and HIGH‐FLUENCE RATE*

Abstract— Epidermal cells of Vallisneria gigantea have a large central vacuole which is surrounded by a thin layer of cytoplasm. The chloroplasts are distributed over all six cytoplasmic layers of an approximate cuboid. In low‐intensity light, the accumulation of chloroplasts in the side facing the outer periclinal wall (the P side) continues for several hours. Red light (650 nm) shows the highest effect and induces such an accumulation even at a fluence rate of only 0.02 W/m2. In response to high‐intensity light, the chloroplasts move to the sides that face the anticlinal walls (the A sides) within a few tens of minutes. Blue light (450 nm) is most effective in inducing this movement. At a fluence rate of 1.51 W/m2, the reaction is induced in only half of the specimens. Neither red nor blue light can induce any orientation movement in the presence of 100 μg/ml of cytochalasin B.

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil, EGTA, W-7, and TFP on the action potential

SummaryThe cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La3+ or verapamil (a calcium channel blocker) or removal of external Ca2+ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca2+ channel blockers could not be removed by addition of CaCl2 to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca2+ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.

Dynamic changes in the organization of microfilaments associated with the photocontrolled motility of chloroplasts in epidermal cells ofVattisneria

SummaryUsing time-lapse video microscopy, we performed a semiquantitative investigation of the movement of chloroplasts on the cytoplasmic layer that faces the outer periclinal wall (P side) of epidermal cells of leaves of the aquatic angiospermVallisneria gigantea Graebner. Under continuous irradiation with red light (650 nm, 0.41 W/m2), the movement of chloroplasts on the P side was transiently accelerated within 5 min. The increased movement began to decrease at around 20 min and fell below the original level after 40 to 60 min of irradiation with red light. The acceleration and deceleration of movement of chloroplasts on the P side seemed to lead directly to the increase and the subsequent decrease in the rate of migration of chloroplasts from the P side to the anticlinal layers of cytoplasm, which are responsible for the accumulation of chloroplasts on the P side, as we demonstrated previously. In the presence of inhibitors of photosynthesis, the accelerated movement of chloroplasts was maintained for as long as the chloroplasts were irradiated with red light. The rapid acceleration and deceleration of the movement of chloroplasts could be observed repeatedly with sequential irradiation with red and then far-red light (746 nm, 0.14 W/m2). Concomitantly with the loss of motility of chloroplasts on the P side, a dynamic change in the configuration of microfilaments, from a network to a honeycomb, occurred on the P side.

Dynamic changes in the actin cytoskeleton during the high-fluence rate response of theMougeotia chloroplast

SummarySince photo-induced orientation movement of a single, ribbon-shaped chloroplast in each cell of the filamentous green algaMougeotia is inhibited in the presence of cytochalasin B, actin is thought to be involved in the process of chloroplast movements. However, this possibility remains to be proved. A specific class of cytoplasmic filaments, which emerge from the advancing front of the moving chloroplast, can be seen by differential interference contrast (DIC) microscopy. However, no one has yet succeeded in defining the nature of these filaments. We have been able to stain the actin filaments (AFs) associated with the moving chloroplast with fluorescein-conjugated phalloidin (FP) after pre-treatment withm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). No filamentous structures were observed in cells that had been pre-irradiated with low-fluence rate red light. However, transversely oriented fluorescent filaments appeared at the front edge of the moving chloroplast when it began to rotate under irradiation with high-fluence rate white light. These filaments disappeared after completion of the orientation movement, suggesting the simultaneous appearance of AFs and the orientation movement of the chloroplast. Thick cytoplasmic strands connecting the edge of the chloroplast with the parietal cytoplasm were often seen by DIC microscopy before and after completion of the high-fluence rate orientation movement. These thick cytoplasmic strands could not be stained by FP, but were often stained by 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), suggesting that they are transvacuolar strands that include endoplasmic reticulum.

Protease-sensitive anchoring of microfilament bundles provides tracks for cytoplasmic streaming in Vallisneria

SummaryIn mesophyll cells of the aquatic angiospermVallisneria gigantea Graebner, the endoplasm streams rotationally along the cell walls normal to the leaf surface in situ. Bundles of microfilaments anchored in the ectoplasm serve as tracks for the cytoplasmic streaming. In single mesophyll cells isolated by enzymatic digestion, hypertonic treatment induces abnormal streaming concomitant with plasmolysis, specifically at one or both of the shorter sides of an approximate rectangle. The disorderly arrangement of microfilaments in such cells has been confirmed by fluorescence microscopy of cells stained with FITC-phalloidin. While inhibitors of proteases added to the enzyme solution used for isolation of cells suppress the disturbance of rotational streaming, exogenously applied protease promotes it. The results suggest that bundles of microfilaments in the ectoplasm are stabilized by protease-sensitive factor(s) in the presence of the cell wall.

Green algal microtubule-associated protein with a molecular weight of 90 kDa which bundles microtubules

SummaryCytoplasmic streaming in the freshwater, coencytic green alga,Dichotomosiphon tuberosus, is regulated by light. Conspicuous changes are observed in the number of microtubules cross-linked together in bundles when the cytoplasmic streaming is modulated by light. In an attempt to identify the cross-linker, we stainedD. tuberosus cells with antibodies specific for several different microtubules-associated proteins (MAPs) from vertebrates. Antibodies raised against bovine adrenal 190 kDa MAP stained the algal cells, and the pattern of staining was quite similar to that obtained with tubulin-specific antibodies. Examination by immunoelectron microscopy revealed that the antibodies specific for the 190 kDa microtubule-associated protein (MAP) were located along the microtubules. Western blotting demonstrated that the antibodies crossreacted with a peptide fromD. tuberosus with a molecular weight of about 90 kDa. This peptide was heat-stable, a property shared by the bovine 190 kDa MAP. Moreover, this 90 kDa peptide, crossreacted with antibodies raised against a synthetic peptide, identical to the tubulin-binding domain found in the 190 kDa MAP and in a tau protein. Partially purified 90 kDa protein fromD. tuberosus has the ability to bundle microtubules when mixed with a tubulin fraction fromD. tuberosus, in the presence of taxol. These results suggest that the 90 kDa protein fromD. tuberosus is a MAP that bundles microtubules.

Cytoplasmic filaments and their assembly into bundles inPhysarum plasmodium

SummaryFilaments were visualized in the endoplasmic drop ofPhysarum polycephalum. The drop was obtained through puncture of the wall of the plasmodial capillary. A glycerinated specimen of the drop of endoplasm could spread over the surface of aqueous solution in a thin film, which has the outstanding merit of observing cytoplasmic filaments by means of negative staining technique. When the cytoplasmic filaments found on the film was treated with HMM, characteristic arrowhead structures were formed demonstrating that they represented actin filaments. On addition of Mg-ATP, three groups of aggregates were observed: 1. bundles of actin filaments, 2. bundles characteristic of myosin aggregates and 3. loose masses in which actin filaments and myosin aggregates came together.In sectioned specimens, filaments dispersed in the endoplasm without forming large bundles were recognized not infrequently together with other cell organelles. Bundles of filaments were developed in the peripheral region of the drop as gelation of cytoplasm was produced there. They were well preserved in glycerinated specimens and reacted with rabbit HMM to make arrowhead complexes. Among these decorated filaments, non-decorated thicker filaments were also observed.The above observation has made it clear that the fibrillar structures in the peripheral ectoplasmic region of the drop were formed by the assembly of the free filaments in the endoplasm.

A polarized light and electron microscopic study of the birefringent fibrils inPhysarum plasmodia

SummaryThe birefringent fibrils in thin-spread plasmodium ofPhysarum polycephalum have been investigated with both polarizing and electron microscopes. The birefringent fibrils were classified into three groups by polarized light microscopy. The first type of fibril is observed in the advancing frontal region as a mutual orthogonal array. The birefringence changes rhythmically in accordance with the shuttle streaming. The second type of birefringent fibril is located in the strand region and runs parallel or somewhat oblique to the strand axis. The third type is observed in the strand region always perpendicular to the streaming axis. Electron microscopy confirmed that all these fibrils are composed of microfilaments, which range in densities in the cross view of the fibril from 1.2 to 1.7 × 103/μm2 (1.5 × 103/(xm2 on the average).