ISSN 10214437, Russian Journal of Plant Physiology, 2011, Vol. 58, No. 2, pp. 233–237. © Pleiades Publishing, Ltd., 2011. Original Russian Text © A.A. Bulychev, S.O. Dodonova, 2011, published in Fiziologiya Rastenii, 2011, Vol. 58, No. 2, pp. 202–207.
RESEARCH PAPERS
Role of Cyclosis in Asymmetric Formation of Alkaline Zones at the Boundaries of Illuminated Region in Chara Cells A. A. Bulychev and S. O. Dodonova Department of Biophysics, Faculty of Biology, Moscow State University, Moscow, 119991 Russia; fax: 7 (495) 9391115; email:
[email protected] Received July 16, 2010
Abstract—The role of cytoplasmic streaming in pattern formation at the plasma membrane and chloroplast layer was examined with Chara corallina Klein ex Willd. cells exposed to nonuniform illumination. Our hypothesis was that the exchange of ions and metabolites between the chloroplasts and the cytoplasm in the illuminated cell area alters the composition of the cytosol while the flow of modified cytoplasm induces asym metrical changes in the plasmalemmal transport and fluorescence of chloroplasts in the adjacent shaded areas. The hypothesis was tested by measuring the H+transporting activity of plasmalemma and nonphoto chemical quenching (NPQ) in shaded areas of Chara cells at distances of 1–5 mm on either side of the illu minated region (white light, 1000 μmol/(m2 s), beam width 2 mm). When measured at equal distances on opposite sides from the illuminated region, both pH and NPQ changes differed considerably depending on the direction of cytoplasmic movement at the light–shade boundary. In the region where the cytoplasm flowed out of irradiated area, the formation of alkaline zone (the plasma membrane domain with a high H+ conductance) and NPQ in chloroplasts was observed. In the vicinity of light–shade boundary where the flow was directed from the shade to the illuminated area, neither alkaline zone nor NPQ were formed. The results demonstrate the significance of cyclosis in the transfer of physiologically active intermediate that affects the membrane transport, the functional activity of chloroplasts, and the pattern formation in the plant cell. Keywords: Chara corallina, cytoplasmic streaming, nonuniform illumination, plasmalemma, chloroplasts, alkaline zones, chlorophyll fluorescence, nonphotochemical quenching. DOI: 10.1134/S1021443711020038
INTRODUCTION Cytoplasmic streaming plays an essential role in the plant cell metabolism since it enables lateral distribu tion of substances over long distances at which diffu sion is ineffective [1]. This function is particularly important for giant cells, such as internodes of charac ean algae. The velocity of cytoplasmic streaming in these cells is as high as 100 μm/s, which is the upper limit observed in plant cells. The movement of cyto plasm in Chara cells is determined by interaction of actin filaments attached to immobile chloroplasts and of myosin molecules bound to freely moving endo plasmic organelles [2]. Although the role of rotational cytoplasmic movement in the lateral intracellular transport of solutes is generally recognized, there is only scarce evidence that intact nongrowing cells can maintain cytoplasmic concentration gradients capable of affecting the plasmalemmal transport systems and the activity of cell organelles. Under natural conditions the lateral gradients in cells and tissues can be promoted by inhomogeneous Abbreviations: AZ—alkaline zone, NPQ—nonphotochemical quenching of chlorophyll fluorescence, pH0—local pH in the outer medium near the cell surface.
illumination of plant objects [3]. Variations in photo synthetic irradiance are related to partial shading of algal thalli by neighboring plants, by changes in orien tation of incident light beams, and by rapid changes of light and shade in the permanently changing natural environment. According to the literature, the lateral concentra tion gradients of ions and substances can arise in con junction with spatial patterns at the plasma membrane of illuminated cells. It was supposed [4] that the pho tosynthetic fixation of СО2 leads to accumulation of excess ОН– in the cytoplasm; these ions are carried away with a stream of fluid to particular regions of the plasma membrane, where hydroxide ions are released to the outer medium and alkalize the apoplast to pH ~10 [4]. Cyclosis was also included in the theoretical model describing the generation of alternate acid and alkaline zones around illuminated cells of characean algae [5]. The model assumed nonuniform spatial dis tribution of cytoplasmic inorganic carbon concentra tion along with the uniform intracellular distribution of Н+ (ОН–). However, experimental evidence in favor of the aforementioned proposals was limited to disappearance of alkaline zones in longitudinal pH
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profiles upon the action of cytochalasin B, which is known to inhibit the cytoplasmic streaming [4, 5]. Although alkaline zone formation is a lightdepen dent process, these zones often originate at the inter faces between shaded and brightly lit cell areas [6, 7]. Illumination of the cell with a narrow light beam can be employed as a means to elucidate the role of cyclo sis in the formation of spatially heterogeneous proper ties in individual cells. If chloroplasts in the illumi nated region release or deplete some functionally active metabolite, the influence of altered cytoplasmic concentration of this mediator might be observed at a distance from the illuminated zone owing to the trans fer of the mediator by the streaming cytoplasm. It is reasonable to assume that the composition of the cyto plasm flowing out of the illuminated area would be modified by intracellular shuttling of ions and metab olites between the cytosol and photosynthesizing chloroplasts, whereas the cytoplasm approaching the illuminated area from nonilluminated regions would have a composition characteristic of darkened cells. The objective of this study was to verify the sup posed role of cyclosis in maintenance of heteroge neous composition of the cytoplasm in cell regions located at equal distances upstream and downstream in the cytoplasmic flow from the illumination area. We took into account that illumination of Chara cells with a narrow beam induces the appearance of alkaline zones on one or both margins of the illuminated area [5]. It is known that generation of alkaline zone is associated with the increase in the plasma membrane conductance by more than an order of magnitude as compared to the conductance of darkened cell. The main contribution to this high conductance is presum ably due to Н+ or ОН– [8, 9]. The results show that the alkaline zone arises at the light–shade border only in the case when the cytoplasmic movement is directed from the illuminated to the shaded area but it does not appear at the border with the opposite orientation of the fluid stream. MATERIALS AND METHODS Characean algae Chara corallina Klein ex Willd. were grown in an aquarium at room temperature at daylight illumination. Individual fully grown intern odal cells of about 6 cm in length and 0.9–1 mm in diameter were excised and kept at least one day in arti ficial pond water containing 0.1 mM KCl, 1.0 mM NaCl, and 0.1 mM CaCl2. The pH was adjusted to 7.0 by adding about 0.2 mM NaHCO3. Young cells with out calcium depositions were used for experiments. The isolated cell was placed horizontally into a trans parent plexiglas chamber mounted on the stage of an inverted Axiovert 25CFL microscope (Carl Zeiss, Germany). The methods for measuring local pH in the outer medium near the cell surface (pH0) and chloro phyll fluorescence parameters of chloroplasts on cell regions with a diameter ~100 μm were described ear
lier [10]. The coefficient of nonphotochemical quenching NPQ was calculated from the formula (Fm – F m' ) / F m' , where Fm and F m' are maximal yields of chlorophyll fluorescence induced by saturating light pulses in darkness and under conditions of local illu mination, respectively. Measurements were conducted on cell regions that were capable of forming the alkaline zones (AZ) under whole cell illumination. In addition, we selected cell regions where the cytoplasmic streaming was unidi rectional in the observation plane, i.e., where the counterdirected flows occurred in the lower and upper cell halves. Oppositely directed flows are sepa rated by the transparent chloroplastfree line that fol lows a loose helical path. However, for distances of few millimeters, the cytoplasmic flow can be regarded as a linear motion. In the beginning of each experiment, the cell was illuminated with blue light (100 μmol/(m2 s)) directed from the upper light source of the microscope through a SZS22 blue filter. Overall illumination in combina tion with microelectrode pH measurements allowed us to detect AZ and to select the region for subsequent measurements. Local illumination of cell regions was obtained from a white lightemittingdiode (LED) (Luxeon LXK2PWN2S00, Lumileds, United States) con nected to a programmable power source. The lighting protocol was controlled with WinWCP program (Strathclyde Electrophysiology Software). The maxi mal photosynthetic photon flux density provided by this source was 1000 μmol/(m2 s). The selected cell region was illuminated by means of a flexible light guide fixed in the holder of KM1 micromanipulator (Russia). The outer diameter of the light guide with the ferrule was 6 mm; diameter of the fiber optic core was 2 mm. The light guide was positioned vertically and illuminated the cell from above; its location was finely adjusted with micrometric screws of the manip ulator. The light guide edge was moved close to the upper cell surface; the distance between the center of analyzed region (diameter ~100 μm) and the margin of illuminated zone was varied from 1 to 5 mm (Fig. 1). Figure 1 shows schematically the positions of cell regions illuminated alternatively by bright white light (positions 1 and 2) with respect to the central area of measurements (zone A). After formation of AZ under whole cell illumina tion, the cell was placed in darkness, which caused the pH0 to decrease for 5–10 min toward the pH value in the bulk solution. When the pH0 lowered to 8.0–8.3, local illumination was applied to one or the other side from the area where pH and chlorophyll fluorescence were measured. During each experiment we tracked changes in pH0 and fluorescence parameters in zone А, which were induced by local illumination of regions 1 or 2 for 200–240 s. The major part of experiments was performed on the lower cell side that is readily
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Fig. 1. Schematic view of a part of Chara corallina cell used for experiments. Local changes of apoplastic pH (pH0) and chlorophyll fluorescence parameters were measured on the central microscopic region A with a diameter ~100 μm. The regions of local illumination corresponding to alternative light guide positions 1 and 2 are shown as circular areas located at equal distances from the measurement zone A; these regions were located upstream in the cytoplasmic flow (Pos. 1) and downstream in the fluid flow (Pos. 2) with respect to the zone A. Arrows show the direction of cytoplasmic streaming in the analyzed lower side of the internode. Symbol R designates the distance between the center of measurement zone and the border of the fiberoptic core. The block pH0 designates the system for pH measurements with microelectrode sensor positioned in the zone A.
accessible for microscopic observations with an inverted microscope. In addition, we measured pH0 on the upper cell side where the cytoplasmic streaming had opposite direction with respect to the vector of movement on the lower cell side. The velocity of cyto plasmic streaming was 90–10 μm/s. Figures display the results of representative experi ments performed in 4–5 replicates on different cells. RESULTS Illumination of Chara corallina cell with a light beam (white light, 1000 μmol/(m2 s), beam width 2 mm at the light guide output) provides the means to generate AZ in the shaded region at a distance of few millimeters from the light–shade border. Our interest was focused on the possible role of vectorial cytoplas mic streaming in the formation of AZ at the interface between illuminated and shaded areas. We found that the formation of AZ depends on mutual orientation of analyzed and illuminated areas with respect to the vec tor of cytoplasmic flow. When the light guide was posi tioned at equal distances on one or the other side from analyzed region, the AZ formation occurred on the condition that illuminated area was located upstream in the cytoplasmic flow regarding the analyzed area (light guide position 1), i.e., when the examined shaded region was perfused with the cytoplasm flowing from the illuminated cell part (Fig. 2, curve 1). In the case of light guide position 2, when the analyzed shaded region А was flushed by the cytoplasm flowing from the darkened cell part, illumination did not pro duce AZ formation (Fig. 2, curve 2). In this case pH0 RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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went on decreasing throughout the period of local illu mination toward the pH level in the bulk solution. The amplitude of lightinduced pH rise was almost independent on the distance between the illuminated and analyzed areas in the range from 1 to 4 mm; how ever, the duration of the lag period preceding the AZ emergence was substantially prolonged with this dis tance. When the gap between illuminated and ana lyzed regions was 5 mm, no AZ was formed on the time scale examined. In some experiments we measured pH0 changes in the upper cell half, where the cytoplasmic flow was directed oppositely with respect to orientation of flow in the lower cell half. It should be noted that the upper and lower cell halves are exposed to different irradi ances because the upper chloroplast layer attenuates the intensity of transmitted light approximately three fold. During measurements of pH0 on the upper cell half, we observed a fast (3 × 10–2 pH unit/s) and large (1.5 pH units) shift of pH0 upon illumination when the light guide was located in position 2. When the light guide was in position 1, the pH increase upon local illumination was slow (the maximal rate 2 × 10–3 pH unit/s) and its extent was relatively small (0.55 pH units). This result shows that the requirements for AZ formation were identical both for lower and the upper cell halves. It is known that high local pH values under whole cell illumination correlate with the increased non photochemical quenching (NPQ) in chloroplasts at a certain range of light intensities [11]. We measured NPQ in the analyzed shaded region on the lower cell side upon alternate illumination of regions situated No. 2
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Time, s Fig. 2. Dependence of alkaline zone formation in the ana lyzed region A on mutual orientation of illuminated and analyzed areas with respect to the vector of cytoplasmic streaming. (1) Local changes of pH0 at the site A upon illumination of the region located 4 mm upstream in the cytoplasmic flow (light guide position 1); (2) upon illumination of the region located 4 mm downstream in the cytoplasmic flow (light guide position 2). Arrows ↑ and ↓ mark the moments of switching light on and off, respectively (white light, 1000 μmol/(m2 s)).
Fig. 3. Development of nonphotochemical quenching in chloroplasts of the examined zone А as a function of mutual position of the illuminated and analyzed areas with respect to the vector of cytoplasmic streaming. (1) Changes of NPQ in zone А upon illumination of a dis tant cell region located 4 mm upstream in the cytoplasmic flow (light guide position 1); (2) the lack of NPQ upon illu mination of a distant cell region located 4 mm downstream in cytoplasmic flow (light guide position 2). Arrows ↑ and ↓ mark the moments of switching the local light on and off, respectively (white light, 1000 μmol/(m2 s)).
upstream or downstream in the cytoplasmic flow at equal distances from the measurement area, in the same way as it was made during pH measurements (Fig. 3). It is seen that the local illumination in posi tion 1, from which the “irradiated” cytoplasm flowed to the measurement area, induced the increase in NPQ to nearly 0.6 (Fig. 3, curve 1). The similar local illumination of the cell in position 2, when the ana lyzed area was flushed with nonirradiated cytoplasm, induced no significant increase in NPQ (curve 2).
of the light beam directed to one or another side from the measurement area. Similarly, the explanations based on circulation of local electric currents between illuminated and shaded areas can be disregarded because the pathways of these currents should be sym metrical on the both sides of the illumination zone. Measurements of pH0 beneath and above the cell show that local illumination can produce two AZ situ ated on the opposite sides of the light spot. One of them resides on the lower cell half and the other occu pies the upper half. Moreover, at each level (on the upper and lower cell sides) only one AZ is initially formed, namely, in the location where the “irradiated” cytoplasm enters the shaded zone. The velocity of AZ expansion around the site of its initial appearance was found at least 2–3 times lower than the velocity of cytoplasmic streaming. Appar ently, the broadening of AZ after its primary formation is limited by some factors other than the rate of medi ator transport from the illuminated region. Under conditions of nonuniform illumination, AZ originates in the area with very weak light intensity. The lack of full darkness at the light–shade boundary is caused by light scattering from cell structures (cell wall, chloroplasts, other organelles and inclusions), as well as by partial light reflection from the bottom of cell chamber and from the microscope objective lens. According to our estimates, the intensity of light trans mitted through the cell at a distance of 2 mm from the
DISCUSSION The results of this work show that the formation of cell regions with high pH0 and high NPQ in the chlo roplast layer depends on the transfer of cytoplasm from the illuminated zone to the shaded region. Apparently, the cytoplasm flowing out of brightly illu minated zone contains a physiologically active inter mediate, i.e., the product of ion or metabolite exchange between illuminated chloroplasts and the cytoplasm. It should be pointed out that the excretion from illuminated chloroplasts into the cytoplasmic flow of a substance promoting formation of AZ and NPQ is phenomenologically indistinguishable from the uptake by illuminated plastids of the inhibitor for these processes. The possibility of diffusion transport of the intermediate was ruled out because the proper ties of diffusion cannot account for anisotropic effects
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fiberoptic bundle was ≤5 μmol/(m2 s) and declined steeply with the increasing distance from the light– shade boundary. This weak scattered light seems to be essential for the AZ development, because the alkaline zones cannot be formed in complete darkness. It is known that the plasma membrane conductance in the area of AZ is 10–15 times higher than in the darkened cell [8, 9]. Thus, dim light is presumably an important factor of AZ formation at the light–shade border. This assumption is supported by observation of NPQ devel opment at a distance of few millimeters from the illu minated area (Fig. 3). It is commonly accepted that NPQ is absent in darkadapted photosynthesizing objects. A considerable NPQ level observed at low light intensity suggests that the intermediate released into the cytoplasm and transported with the streaming fluid substantially enhances NPQ in dim light. The possibility that chemical intermediates can propagate along the conducting bundles of vascular plants was repeatedly discussed in relation to the ori gin of variation potentials and the transmission path ways for signals promoting system resistance in plants [12, 13]. The present study demonstrates the propaga tion of a functionally active intermediate at the single cell level. The results provide evidence that chloro plasts exposed to intense light modify the composition of the cytoplasm, which is carried away by the stream and exerts its action on functioning of chloroplasts and the transport systems of the plasma membrane at a sig nificant distance from the illumination region. The nature of the laterally transported intermediate responsible for the formation of AZ in the apoplast and NPQ in chloroplasts is presently unknown. This intermediate can be represented by ions, as Lucas and Dainty supposed [4] or by photosynthates that impede the reactions of carbon dioxide assimilation in weakly illuminated regions at the light–shade boundaries. ACKNOWLEDGMENTS We are grateful to N.A. Krupenina for the support and helpful discussion. This work for supported by the Russian Foundation for Basic Research, project no. 100400968a. REFERENCES 1. VerchotLubicz, J. and Goldstein, R.E., Cytoplasmic Streaming Enables the Distribution of Molecules and Vesicles in Large Plant Cells, Protoplasma, 2010, vol. 240, pp. 99–107. 2. Shimmen, T. and Yokota, E., Cytoplasmic Streaming in Plants, Curr. Opin. Cell Biol., 2004, vol. 16, pp. 68–72.
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3. Pieruschka, R., Schurr, U., Jensen, M., Wolff, W.F., and Jahnke, S., Lateral Diffusion of CO2 from Shaded to Illuminated Leaf Parts Affects Photosynthesis Inside Homobaric Leaves, New Phytol., 2006, vol. 169, pp. 779–788. 4. Lucas, W.J. and Dainty, J., Spatial Distribution of Functional OH– Carriers along a Characean Intern odal Cell: Determined by the Effect of Cytochalasin B – on H14C O 3 Assimilation, J. Membr. Biol., 1977, vol. 32, pp. 75–92. 5. Bulychev, A.A., Polezhaev, A.A., Zykov, S.V., Plyusnina, T.Yu., Riznichenko, G.Yu., Rubin, A.B., Jantoβ, W., Zykov, V.S., and Müller, S.C., LightTriggered pH Band ing Profile in Chara Cells Revealed with a Scanning pH Microprobe and Its Relation to SelfOrganization Phe nomena, J. Theor. Biol., 2001, vol. 212, pp. 275–294. 6. Bulychev, A.A., Cherkashin, A.A., Rubin, A.B., and Müller, S.C., Distribution of Acid and Alkaline Zones on the Cell Surface of Chara corallina under Stationary and Local Illumination, Russ. J. Plant Physiol., 2002, vol. 49, pp. 715–722. 7. Bulychev, A.A. and Vredenberg, W.J., SpatioTemporal Patterns of Photosystem II Activity and PlasmaMem brane Proton Flows in Chara corallina Cells Exposed to Overall and Local Illumination, Planta, 2003, vol. 218, pp. 143–151. 8. Smith, J.R. and Walker, N.A., Effects of pH and Light on the Membrane Conductance Measured in the Acid and Basic Zones of Chara, J. Membr. Biol., 1985, vol. 83, pp. 193–205. 9. Bulychev, A.A. and Krupenina, N.A., Transient Removal of Alkaline Zones after Excitation of Chara Cells Is Associated with Inactivation of High Conduc tance in the Plasmalemma, Plant Signal. Behav., 2009, vol. 4, pp. 24–31. 10. Bulychev, A.A. and Kamzolkina, N.A., Effect of Action Potential on Photosynthesis and Spatially Distributed H+ Fluxes in Cells and Chloroplasts of Chara corallina, Russ. J. Plant Physiol., 2006, vol. 53, pp. 1–9. 11. Krupenina, N.A. and Bulychev, A.A., Action Potential in a Plant Cell Lowers the Light Requirement for Non Photochemical EnergyDependent Quenching of Chlorophyll Fluorescence, Biochim. Biophys. Acta, 2007, vol. 1767, pp. 781–788. 12. Stahlberg, R., Cleland, R.E., and Volkenburgh, E., Slow Wave Potentials – a Propagating Electrical Signal Unique to Higher Plants, Communication in Plants, Baluška, F., Mancuso, S., and Volkmann, D., Eds., Berlin: SpringerVerlag, 2006, pp. 291–308. 13. Rhodes, J.D., Thain, J.F., and Wildon, D.C., Signals and Signalling Pathways in Plant Wound Responses, Communication in Plants, Baluška, F., Mancuso, S., and Volkmann, D., Eds., Berlin: SpringerVerlag, 2006, pp. 391–401.
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