Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring pl... more Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring plant cells. Researchers routinely characterize the mobility of the green-fluorescent protein (GFP) and GFP fusions through Pd by calculating the proportion of sites in bombarded leaves which show fluorescence in multiple cell clusters (% movement). Here, the Arrhenius equation was used to describe the temperature dependence of GFP and GFP-TGBp1 (potato virus X triple gene block protein1) movement, using % movement values, and to calculate the activation energy for protein transport. The resulting low activation energy indicates GFP and GFP-TGBp1 movement are diffusion driven. Furthermore, GFP movement is inversely proportional to the leaf surface area of expanding leaves. The increase in leaf area results mainly from cell expansion during the sink-source transition. The increasing cell size results in lower Pd density, which decreases the probability that a GFP attains an open Pd by diffusion. The decline in GFP movement as leaf area expands indicates that, in addition to GFP diffusion through Pd, attaining an open Pd by undirected diffusion might be limiting for Pd transport. In summary, this report provides a new quantitative method for studying Pd conductivity.
Leaves of the C3 plant Brassica oleracea were illuminated with red and/or far-red light of differ... more Leaves of the C3 plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO2 and/or oxygen. In the absence of CO2, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P515 signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO2 inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO2 levels were decreased to or below the CO2 compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F0 fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor QA in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.
Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some phot... more Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some photosynthetic intermediates were measured in illuminated leaves. Oxygen and CO2 concentrations in the gas phase were varied in order to obtain information on control of Photosystem II activity under conditions such as produced by water stress, when stomatal closure restricts access of CO2 to the photosynthetic apparatus. Light scattering and energy-dependent fluorescence quenching indicated a high level of chloroplast energization under high intensity illumination even when linear electron transport was curtailed in CO2-free air or in 1% oxygen with 35 μll(-1) CO2. Calculations of the phosphorylation potential based on measurements of phosphoglycerate, dihydroxyacetone phosphate and NADP revealed ratios of intrathylakoid to extrathylakoid proton concentrations, which were only somewhat higher in air containing 35 μl l(-1) CO2 than in CO2-free air or 1% oxygen/35 μl l(-1) CO2. Anaerobic conditions prevented appreciable chloroplast energization. Acceptor-limitation of electron flow resulted in a high reduction level of the electron transport chain, which is characterized by decreased oxidation of P700, not only under anaerobic conditions, but also in air, when CO2 was absent, and in 1% oxygen, when the CO2 concentration was reduced to 35 μll(-1). Efficient control of electron transport was indicated by the photoaccumulation of P700 (+) at or close to the CO2 compensation point in air. It is proposed to require the interplay between photorespiratory and photosynthetic electron flows, electron flow to oxygen and cyclic electron flow. The field-indicating electrochromic shift (P515) measured as a rapid absorption decrease on switching the light off followed closely the extent of photoaccumulation of P700 (+) in the light.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1995
Under conditions (0.2% CO_2; 1% O_2) that allow high rates of photosynthesis, chlorophyll fluores... more Under conditions (0.2% CO_2; 1% O_2) that allow high rates of photosynthesis, chlorophyll fluorescence was measured simultaneously with carbon assimilation at various light intensities in spinach (Spinacia oleracea) leaves. Using a stoichiometry of 3 ATP/CO_2 and the known relationship between ATP synthesis rate and driving force (Delta pH), we calculated the light-dependent pH gradient (Delta pH) across the thylakoid membrane in intact leaves. These Delta pH values were correlated with the photochemical (q_P) and nonphotochemical (q_N) quenching of chlorophyll fluorescence and with the quantum yield of photosystem II (PhiPSII). At Delta pH > 2.1 all three parameters (q_P, q_N, and PhiPSII) changed very steeply with increasing Delta pH (decreasing pH in the thylakoid). The observed pH dependences followed hexacooperative titration curves with slightly different pK_a values. The significance of the steep pH dependences with slightly different pK_a values is discussed in relation to the regulation of photosynthetic electron transport in intact leaves.
We investigated the proton conductance of the channel portion of chloroplast ATP synthase (CFa). ... more We investigated the proton conductance of the channel portion of chloroplast ATP synthase (CFa). Thylakoids were CFi-depleted by EDTA treatment. Proton pumps were stimulated by short flashes of light. Proton flux through CFo was measured spectrophotometrically in three different ways: as proton efflux from the lumen (via neutral red), charge flow across the membrane (via electrochromism) and proton influx into the medium (via phenol red). Hence we completely tracked the protons on their way from the lumen through CF,, into the medium. A first treatment with EDTA removed up to 12% of total CFi without increasing the proton permeability of the membranes. A second treatment removed a further 20% of CFi and increased the proton permeability of membranes by 3 orders of magnitude. The electric potential difference and the pH transients, in both the lumen and medium, decayed with a relaxation time of 7 ms indicating electrically driven proton flow through CFo. If the electric driving force was shunted (e.g. by added gramicidin) both pH transients decayed at 85 ms compared with 2&60 s in control thylakoids. The longer relaxation time under chemical driving force was attributable to larger chemical than electrical capacitance of thylakoids. We calculated a lower limit of the unit conductance of CFO under the assumption that all exposed CFo were proton conducting. The value was 10 fS, corresponding to the passage of 6200 protons/s per CFa (at 100 mV electric driving force) and by orders of magnitude higher than so far reported for any F. channel.
Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring pl... more Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring plant cells. Researchers routinely characterize the mobility of the green-fluorescent protein (GFP) and GFP fusions through Pd by calculating the proportion of sites in bombarded leaves which show fluorescence in multiple cell clusters (% movement). Here, the Arrhenius equation was used to describe the temperature dependence of GFP and GFP-TGBp1 (potato virus X triple gene block protein1) movement, using % movement values, and to calculate the activation energy for protein transport. The resulting low activation energy indicates GFP and GFP-TGBp1 movement are diffusion driven. Furthermore, GFP movement is inversely proportional to the leaf surface area of expanding leaves. The increase in leaf area results mainly from cell expansion during the sink-source transition. The increasing cell size results in lower Pd density, which decreases the probability that a GFP attains an open Pd by diffusion. The decline in GFP movement as leaf area expands indicates that, in addition to GFP diffusion through Pd, attaining an open Pd by undirected diffusion might be limiting for Pd transport. In summary, this report provides a new quantitative method for studying Pd conductivity.
Leaves of the C3 plant Brassica oleracea were illuminated with red and/or far-red light of differ... more Leaves of the C3 plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO2 and/or oxygen. In the absence of CO2, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P515 signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO2 inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO2 levels were decreased to or below the CO2 compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F0 fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor QA in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.
Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some phot... more Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some photosynthetic intermediates were measured in illuminated leaves. Oxygen and CO2 concentrations in the gas phase were varied in order to obtain information on control of Photosystem II activity under conditions such as produced by water stress, when stomatal closure restricts access of CO2 to the photosynthetic apparatus. Light scattering and energy-dependent fluorescence quenching indicated a high level of chloroplast energization under high intensity illumination even when linear electron transport was curtailed in CO2-free air or in 1% oxygen with 35 μll(-1) CO2. Calculations of the phosphorylation potential based on measurements of phosphoglycerate, dihydroxyacetone phosphate and NADP revealed ratios of intrathylakoid to extrathylakoid proton concentrations, which were only somewhat higher in air containing 35 μl l(-1) CO2 than in CO2-free air or 1% oxygen/35 μl l(-1) CO2. Anaerobic conditions prevented appreciable chloroplast energization. Acceptor-limitation of electron flow resulted in a high reduction level of the electron transport chain, which is characterized by decreased oxidation of P700, not only under anaerobic conditions, but also in air, when CO2 was absent, and in 1% oxygen, when the CO2 concentration was reduced to 35 μll(-1). Efficient control of electron transport was indicated by the photoaccumulation of P700 (+) at or close to the CO2 compensation point in air. It is proposed to require the interplay between photorespiratory and photosynthetic electron flows, electron flow to oxygen and cyclic electron flow. The field-indicating electrochromic shift (P515) measured as a rapid absorption decrease on switching the light off followed closely the extent of photoaccumulation of P700 (+) in the light.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1995
Under conditions (0.2% CO_2; 1% O_2) that allow high rates of photosynthesis, chlorophyll fluores... more Under conditions (0.2% CO_2; 1% O_2) that allow high rates of photosynthesis, chlorophyll fluorescence was measured simultaneously with carbon assimilation at various light intensities in spinach (Spinacia oleracea) leaves. Using a stoichiometry of 3 ATP/CO_2 and the known relationship between ATP synthesis rate and driving force (Delta pH), we calculated the light-dependent pH gradient (Delta pH) across the thylakoid membrane in intact leaves. These Delta pH values were correlated with the photochemical (q_P) and nonphotochemical (q_N) quenching of chlorophyll fluorescence and with the quantum yield of photosystem II (PhiPSII). At Delta pH > 2.1 all three parameters (q_P, q_N, and PhiPSII) changed very steeply with increasing Delta pH (decreasing pH in the thylakoid). The observed pH dependences followed hexacooperative titration curves with slightly different pK_a values. The significance of the steep pH dependences with slightly different pK_a values is discussed in relation to the regulation of photosynthetic electron transport in intact leaves.
We investigated the proton conductance of the channel portion of chloroplast ATP synthase (CFa). ... more We investigated the proton conductance of the channel portion of chloroplast ATP synthase (CFa). Thylakoids were CFi-depleted by EDTA treatment. Proton pumps were stimulated by short flashes of light. Proton flux through CFo was measured spectrophotometrically in three different ways: as proton efflux from the lumen (via neutral red), charge flow across the membrane (via electrochromism) and proton influx into the medium (via phenol red). Hence we completely tracked the protons on their way from the lumen through CF,, into the medium. A first treatment with EDTA removed up to 12% of total CFi without increasing the proton permeability of the membranes. A second treatment removed a further 20% of CFi and increased the proton permeability of membranes by 3 orders of magnitude. The electric potential difference and the pH transients, in both the lumen and medium, decayed with a relaxation time of 7 ms indicating electrically driven proton flow through CFo. If the electric driving force was shunted (e.g. by added gramicidin) both pH transients decayed at 85 ms compared with 2&60 s in control thylakoids. The longer relaxation time under chemical driving force was attributable to larger chemical than electrical capacitance of thylakoids. We calculated a lower limit of the unit conductance of CFO under the assumption that all exposed CFo were proton conducting. The value was 10 fS, corresponding to the passage of 6200 protons/s per CFa (at 100 mV electric driving force) and by orders of magnitude higher than so far reported for any F. channel.
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Papers by Gerald Schönknecht