A. A. Cherkashin

Comparative study on photosynthetic activity of chloroplasts in acid and alkaline zones of Chara corallina

A novel experimental approach has been applied to investigate the relationship between pH banding in Chara cells and photosynthetic activity of chloroplasts located in cell regions adjacent to acid and alkaline bands. The combination of pH microelectrode technique with pulse amplitude modulation (PAM) microfluorimetry enabled parallel measurements of longitudinal pH profiles and chlorophyll fluorescence yield in acid and alkaline zones of individual Chara cells. The scanning with a pH-microelectrode along the cell length revealed the light-dependent pH pattern, i.e., alternating acid and alkaline bands with pH differences as large as 2 - 3 pH units. In parallel, measurements of chlorophyll fluorescence yield under actinic light were performed using PAM microfluorometry. It was found that the effective photochemical yield of photosystem II is substantially higher in acid than in alkaline zones. The results clearly show that the banding pattern is not confined solely to the plasmalemma but is also exhibited in alternating photosynthetic performance of the underlying chloroplast layer. Apparently, the acid regions enriched with CO2 ensure sufficient flow of this substrate to the Calvin cycle reactions, thus promoting the photosynthetic rate, whereas the alkaline zones devoid of CO2 favor radiative losses of absorbed solar energy in chloroplasts.

Discontinuously propagating waves in the bathoferroin-catalyzed Belousov–Zhabotinsky reaction incorporated into a microemulsion

Three new types of discontinuously propagating waves are reported in the bathoferroin-catalyzed Belousov-Zhabotinsky (BZ) reaction dispersed in water-in-oil Aerosol OT microemulsion. Jumping waves (JWs) are typically observed at or above room temperature and develop from the familiar trigger waves. Bubble waves (BWs) typically emerge from trigger or JWs at similar temperatures, while rotating waves (RWs) evolve from JW at higher temperatures (>40 degrees C). All these waves propagate discontinuously in a saltatory fashion. Other characteristic features include a discontinuous front for BW consisting of small concentric waves (bubbles) and lateral rotation of annular RWs. All three types of waves, as well as segmented but continuously propagating waves, can coexist. A simple model that is able to describe both jumping and segmented waves is described.

Cross-Diffusion in a Water-in-Oil Microemulsion Loaded with Malonic Acid or Ferroin. Taylor Dispersion Method for Four-Component Systems

We describe an improved Taylor dispersion method for four-component systems, which we apply to measure the main- and cross-diffusion coefficients in an Aerosol OT water-in-oil microemulsion loaded with one of the reactants of the Belousov-Zhabotinsky (BZ) reaction, water(1)/AOT(2)/R(3)/octane(4) system, where R is malonic acid or ferroin. With [H(2)O]/[AOT] = 11.8 and volume droplet fraction phi d = 0.18, when the microemulsion is below the percolation transition, the cross-diffusion coefficients D(13) and D(23) are large and positive ( D(13)/ D(33) congruent with 14, D(23)/ D(33) congruent with 3) for malonic acid and large and negative for ferroin ( D(13)/ D(33) congruent with -112, D(23)/ D(33) congruent with -30) while coefficients D(31) and D(32) are small and negative for malonic acid ( D(31)/ D(33) congruent with -0.01, D(32)/ D(33) congruent with -0.14) and small and positive for ferroin ( D(31)/ D(33) congruent with 5 x 10(-4), D(32)/ D(33) congruent with 8 x 10(-3)). These data represent the first direct determination of cross-diffusion effects in a pattern-forming system and of the full matrix of diffusion coefficients for a four-component system. The results should provide a basis for modeling pattern formation in the BZ-AOT system.

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand Fmand the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H+extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO2in acid and alkaline regions. The acid zones are enriched with CO2that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO–3is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.

Dependence of chlorophyll P700 redox transients during the induction period on the transmembrane distribution of protons in chloroplasts of pea leaves

Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H+ conductance in thylakoid membranes) and nigericin (a K+/H+ antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curves of chlorophyll fluorescence in pea (Pisum sativum L., cv. Premium) leaves. The treatment of leaves with DCCD strongly modified the kinetics of P700+ absorbance changes (ΔA810) by promoting rapid photooxidation of P700. These characteristic changes in ΔA810 induction kinetics and P700+ accumulation did not appear when the leaves were treated with DCCD in the presence of nigericin. In addition to opposite modifications of ΔA810 kinetics evoked by permeability-modifying agents, the fluorescence induction curves differed conspicuously depending on leaf incubation in DCCD solutions with or without nigericin. The observed modifications of fluorescence induction curves and ΔA810 indicate that DCCD suppresses electron transport from photosystem II (PSII) to P700, whereas this inhibition is removed by nigericin. The results suggest that slowing down of the electron transport rate in the presence of DCCD was caused by elevation of ΔpH in thylakoids. The prevention of pH gradient formation in the presence of protonophore lowered also the steady-state P700+ level in far-red irradiated leaves and accelerated the subsequent dark reduction of P700. These findings indicate that PSI-driven cyclic electron flow is accelerated after the removal of the pH gradient.

Distribution of acid and alkaline zones on the cell surface of Chara corallina under stationary and local illumination

A scanning microprobe technique was used to study pH distribution near the cell surface of Chara corallina Klein ex Willd. under variations of light intensity, dark–light transitions, and local illumination of various cell parts. In darkness, the H+-transporting activity of plasmalemma was distributed homogeneously over the cell surface. However, after exposing the cell to weak light (irradiance 0.2–0.5 W/m2), individual alkaline peaks with ΔpH of 1–2 units were observed in the longitudinal pH profile. The peaks in the longitudinal pH profile became more numerous with the increase in light intensity. The plot of ΔpH as a function of light intensity included a steep transition from zero to its maximum amplitude. In strong light (100 W/m2), the pH bands alternated along the cell length with a periodic length of about 7 mm. It is shown that the light-induced formation of ring-shaped bands with H+-exporting and H+ sink activities is preceded by the appearance of irregularly located spots (patches). When small cell parts were illuminated and the light spot was suddenly shifted along the cell to another position, the alkaline bands reorganized in two ways. In some cases, this treatment was followed by a gradual shift in the band position (without attenuation in the peak height) toward the illuminated area. In other cases, the initial band disappeared after such treatment, and a new alkaline band emerged in the vicinity of the illuminated area. Despite the apparent similarity of regular bands in the longitudinal pH profiles, the properties of individual bands (variable sensitivity to light intensity changes, the ability of bands to move along the cell under external treatment) differ substantially. It is supposed that the light-induced formation of the pH profile is based on primary fluctuations of photosynthetic and transport activities in the chloroplast layer and the plasmalemma, as well as on further rearrangements of membrane domains that stabilize band locations.

Temporal suppression of periodic pH profile along a charophyte cell after the generation of action potential.

Spatial heterogeneity of the ion composition of the cytoplasm and the properties of the plasma membrane play an important role in the responses of plant cells induced by external stimuli. Ample data has been accumulated indicating that local intracellular Ca 2+ gradients play a key role in growth, morphogenesis, and responses to various stresses [1]. Although pH and pCa values in the cytoplasm are thought to correlate with one another [4], much less is known about the role of the intracellular and extracellular pH gradients [2, 3]. Local pH gradients are generated as a result of spatially heterogeneous activation of membrane ç + -pumps and ç + -conducting channels over the cell surface [5, 6]. These are the factors that explain the appearance of periodic distribution of pH near the cell surface in charophyte algae ( uf8ffç o ) during transition from dark to light conditions [6]. Alternation of acid and alkaline zones along the cell length reflects the existence of the domains differing in conductivity [5], membrane potential [7], and photosynthetic activity [8].

One-year recombinant growth hormone therapy does not improve hemoglobin state and morphology of erythrocytes in growth hormone deficient children

An increase in growth rates of children suffering from growth hormone deficiency (GHD) subjected to recombinant growth hormone treatment (rGHT) was shown to be accompanied by acceleration of metabolic processes that may stimulate oxygen consumption in various organs and tissues. Therefore, oxygen-transporting properties of RBC should undergo considerable changes during the rGHT. The aim of this study was to examine the effects of rGHT on erythrocyte shape and hemoglobin state in GHD children. The level of oxyhemoglobin (Oxy-Hb) in RBC was analyzed by Raman spectroscopy. The RBC count, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and other parameters were calculated. The blood of eleven treatment-naive prepubertal children with GHD (aged 3-9, median 5.7 years) was examined and compared with control group (aged 5-7; median 6.0 years) at three time points: 0, 3 and 12 months of rGHT. Before rGHT, the MI in GHD children was higher (median 0.48 vs 0.14 p=0.0018) and the RBC count was lower (median 4.20 vs 4.96 1012 cells/L p=0.0022) than in control group. After the treatment, cell count in GHD patients did not differ significantly from the control group, but Oxy-Hb level became higher (median 0.64 vs 0.41 p=0.0075). During rGHT, MCV decreased (median 80.3 vs 83.2μm3 p=0.0231). Morphological and functional characteristics of erythrocytes in GHD children were shown to differ significantly from the healthy control group. A twelve-month rGHT partially improved some of the studied parameters but Oxy-Hb level and echinocyte count remained high.

Longitudinal profiles of the vacuolar pH in internally perfused cells of characean alga

Activities of ion pumps and H+-conducting channels in the plasmalemma of illuminated characean algae are distributed inhomogeneously along the internode, which accounts for the shifts of surface pH up to 3.5 units between various cells regions. Spatial variations in cytoplasmic properties provide the basis for uneven distribution of photosynthetic activity along the cell length and might affect the operation of H+-transporting systems at the tonoplast. In order to visualize the longitudinal distribution of the vacuolar pH in Chara corallina internodal cells, the pH microelectrode was inserted into the vacuole and the cell sap was gradually displaced along the cell during intracellular perfusion with an artificial medium. Fluorescein was added to the perfusion medium as a fluorescent marker to detect the arrival of the replacing medium into the area of pH and fluorescence measurements. In light-adapted cells, nonuniform longitudinal pH profiles were observed, with pH shifts as large as 2–2.5 units. In dark-adapted cells, the pH shifts in longitudinal profiles did not exceed 0.5 pH units. The occurrence of large pH changes within the vacuole of individual internodes indicates the possibility of nonuniform distribution of the tonoplast H+-transporting systems in different regions of the illuminated cell.

Outward photocurrent component in chloroplasts of Peperomia metallica and its assignment to the 'closed thylakoid' recording configuration

The photoinduced electrical events at energy-conserving chloroplast membranes can be studied in whole plastids using suction electrodes. In chloroplasts of Peperomia metallica the kinetic profile of photocurrent contains a minor outward component that occurs prior to and differs in polarity from the main component. The origin of this outward current was analyzed using single-turnover flashes in combination with prolonged light exposures and differential physicochemical treatments of tip-located (internal) and the exposed parts of a chloroplast. The outward current signal was higher after 10- to 20-s preillumination and gradually reduced in darkness. The relative amplitude of the outward peak current was enhanced when photosystem II (PS II) was excited by flashes given in the presence of far-red background light (lambda = 712 nm). The outward current was small or absent under conditions promoting activity of photosystem I (cyclic electron transport supported by artificial redox mediators in the presence of diuron) and was particularly high in the presence of PS II electron acceptors (e.g., p-phenylenediamine). This indicates the predominant association of the outward current with activity of PS II. The external application of diuron strongly inhibited the inward current, giving rise to a temporal increase in the outward current. On the contrary, when diuron was added into the suction pipette, the outward current was inhibited soon after sealing. The data suggest that the outward current originated in the tip-located portions of the thylakoid membrane that have orientation opposite to the exposed part of whole thylakoid. These tip-located membrane portions are least accessible for inhibitors added into the outer medium and are highly sensitive to inhibitors (diuron), ionophores (gramicidin D), and detergents (Triton X-100) added into the pipette. Differential involvement of two photosystems in generation of the outward current may be caused by uneven structural distribution of photosystems I and II between appressed (granal) and nonappressed (stromal) thylakoids and by different recording configurations for these thylakoids.