Halina Gabryś

Influence of calcium on blue-light-induced chloroplast movement in Lemna trisulca L.

The presence of calcium is essential for chloroplast movement induced by blue light in Lemna trisulca L. The regulatory role of calcium was confirmed by the inhibition of chloroplast movement by cytochalasin B and trifluoperazine. The calcium concentration in tissues was modified by ethylene glycol-bis(2-aminoethylether)-N,N,N′, N′-tetraacetic acid (EGTA), the calcium ionophore A23187 and La3+. Only a long period of incubation (12h) in EGTA or La3+ caused distrubances in chloroplast movement. This indicates that calcium influx is not essential for chloroplast movement. Those conditions that dramatically changed the internal calcium concentration, either applications of calcium ionophore A23187 and EGTA, or ionophore and La3+, markedly decreased the amplitude of response to blue-light pulses. This demonstrates that disturbances of chloroplast movement are observable only when internal stores of calcium are affected by Ca2+-antagonists. We suggest that the calcium involved in blue-light-induced chloroplast movement is derived from intracellular stores. The addition of Mg2+ to EGTA buffer counteracted its effect, indicating that Mg2+, as well as Ca2+, might possibly be involved in chloroplast movement.

Kinetics of chromium(V) formation and reduction in fronds of the duckweed Spirodela polyrhiza — a low frequency EPR study

The uptake of chromate by the duckweed Spirodela polyrhiza was investigated with atomic absorption spectroscopy and the reduction of Cr(VI) to Cr(V) was measured using low frequency EPR spectroscopy. The biphasic kinetics of the uptake was fitted to parameters of a proposed kinetic model. Another model was developed to simulate chromate reduction. The first step of chromate reduction was found to be much faster than the uptake of Cr(VI) from the free space. Most probably, this step occurs already in the cell wall or on the cell membrane surface. Further reduction of Cr(V) to Cr(III) was estimated to be slower. The disappearance of the Cr(V) signal, following transfer of the plants into a Cr-free solution, lasted several tens of hours; the kinetics was mono- or biexponential depending on the length of Cr loading. The rate constants for Cr reduction in living plants were determined for the first time.

Blue light signalling in chloroplast movements

Chloroplast movements are among the mechanisms allowing plants to cope with changes in their environment. Chloroplasts accumulate at illuminated cell areas under weak light while they avoid areas exposed to strong light. These directional responses may be controlled by blue and/or red light, depending on the plant group. In terrestrial angiosperms only the blue light perceived by phototropins is active. The last decade has seen a rapid development of studies on the mechanism of directional chloroplast movements, which started with an identification of the photoreceptors. A forward genetic approach has been used to identify the components which control chloroplast movements. This review summarizes the current state of research into the signalling pathways which lead to chloroplast responses. First, the molecular properties of phototropins are presented, followed by a characterization both of proteins which are active downstream of phototropins and of secondary messengers. Finally, cross-talk between light signalling involved in chloroplast movements and other signalling pathways is discussed.

Light-induced chloroplast movements in Lemna trisulca. Identification of the motile system

Abstract In mesophyll cells of the water plant Lemna trisulca L. chloroplasts redistribute in response to blue light. In the present study it is shown that an actin depolymerizing agent cytochalasin D, a crosslinker of actin subunits in F-actin m-maleimidobenzoic acid N -hydroxysuccinimide ester (MBS) as well as N -ethylmaleimide (NEM)—a sulfhydryl group reagent, are potent inhibitors of these blue light-induced chloroplast movements in Lemna . Extraction with cold, buffered glycerol solution preserves light-induced chloroplast arrangements within cells producing permeabilized cell models. ‘Reactivation’ of these cell models by Mg-ATP results in remarkable movements which can be inhibited by treatment with NEM and cytochalasin D. Immunofluorescence microscopy demonstrates that a component which is associated with isolated Lemna chloroplasts cross-reacts with antibodies directed against bovine myosin. These results indicate that a contractile actomyosin system is involved in blue light-induced chloroplast movements in Lemna and a putative motor protein, similar to myosin, is associated with the surface of Lemna chloroplasts.

Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study.

Light-induced chloroplast avoidance movement has long been considered to be a photoprotective mechanism. Here, we present an experimental model in which this function can be shown for wild type Arabidopsis thaliana. We used blue light of different fluence rates for chloroplast positioning, and strong red light inactive in chloroplast positioning as a stressing light. The performance of photosystem II was measured by means of chlorophyll fluorescence. After stressing light treatment, a smaller decrease in photosystem II quantum yield was observed for leaves with chloroplasts in profile position as compared with leaves with chloroplasts in face position. Three Arabidopsis mutants, phot2 (no avoidance response), npq1 (impaired zeaxanhtin accumulation) and stn7 (no state transition), were examined for their chloroplast positioning and chlorophyll fluorescence parameters under identical experimental conditions. The results obtained for these mutants revealed additional stressing effects of blue light as compared with red light.

Chloroplast translocations in Lemna trisulca L.induced by continuous irradiation and by light pulses

The analytical model describing the steady state position of chloroplasts in dependence of fluence rate as well as the chloroplast response to single strong light pulses has been proposed. The model is based on the following assumptions: 1. Irradiation of the cell generates the state X in the cell membrane region, proportional to the local fluence rate. After switching on the light, the value of X increases exponentially with the time constant of about 3 min. The dark decay of X is also exponential with the same time constant. The level of X controls all kinds of chloroplast arrangements. 2. The state X generates two further states: Y1 and Y2, the first of them representing attraction forces for chloroplasts and the second representing repulsion forces. Empirical equations have been found for both Y states. The fluence rate response curve can be described with the use of functions Y1 and Y2. 3. The kinetic analysis requires the introduction of two additional functions Z in order to account for delays and time dispersion of the chloroplast movement in response to driving and resistance factors. The computer program for the proposed model was developed and the results of calculations were compared with experimental data (fluence rate response curve and pulse effects) with satisfactory agreement. Initially no attempt was made to ascribe any physical meaning to the postulated states. Some suggestions in this respect are mentioned in the discussion.

Blue-light-induced chloroplast orientation in Mougeotia. Evidence for a separate sensor pigment besides phytochrome.

Chloroplast orientation in the green alga Mougeotia has been induced by unidirectional red or blue light, given continuously during one hour. In addition, part of the preparations obtained scattered strong far-red light simultaneously with the orienting light. This far-red light completely abolished the response to red light, consistent with phytochrome as the sensor pigment for orientation in Mougeotia. In blue light, however, the response was completely insensitive to far-red light, thus pointing to a different sensor pigment in the shortwavelength region.

AtomicJ: An open source software for analysis of force curves

We present an open source Java application for analysis of force curves and images recorded with the Atomic Force Microscope. AtomicJ supports a wide range of contact mechanics models and implements procedures that reduce the influence of deviations from the contact model. It generates maps of mechanical properties, including maps of Youngs modulus, adhesion force, and sample height. It can also calculate stacks, which reveal how samples response to deformation changes with indentation depth. AtomicJ analyzes force curves concurrently on multiple threads, which allows for high speed of analysis. It runs on all popular operating systems, including Windows, Linux, and Macintosh.

Actin cytoskeleton in Arabidopsis thaliana under blue and red light.

Background information. Actin cytoskeleton is the basis of chloroplast‐orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F‐actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F‐actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor® 488‐conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4π·area/perimeter2), was determined for all actin formations which were measured in fluorescence confocal images.

Recruitment of myosin VIII towards plastid surfaces is root-cap specific and provides the evidence for actomyosin involvement in root osmosensing

The existence of a cell wall-plasma membrane-cytoskeleton (WMC) continuum in plants has long been postulated. However, the individual molecules building such a continuum are still largely unknown. We test here the hypothesis that the integrin-based multiprotein complexes of animal cells have been replaced in plants with more dynamic entities. Using an experimental approach based on protoplast digestion mixtures, and utilising specific antibodies against Arabidopsis ATM1 myosin, we reveal possible roles played by plant-specific unconventional myosin VIII in the functioning of WMC continuum. We demonstrate rapid relocation (less than 5 min) of myosin VIII to statolith surfaces in maize root-cap cells, which is accompanied by the reorganisation of actin cytoskeleton. Upon prolonged stimulation, myosin VIII is also recruited to plasmodesmata and pit-fields of plasmolysing root cap statocytes. The osmotic stimulus is the major factor inducing relocation, but the cell wall-cytoskeleton interactions also play an important role. In addition, we demonstrate the tight association of myosin VIII with the surfaces of chloroplasts, and provide an indication for the differences in the mechanisms of plastid movement in roots and leaves of plants. Overall, our data provide evidence for the active involvement of actomyosin complexes, rooted in the WMC continuum, in the cellular volume control and maintenance of spatial relationships between cellular compartments.