Orientation and linear dichroism of chloroplasts and sub-chloroplast fragments oriented in an electric field

Whole or broken spinach chloroplasts, bacterial chromatosphores and CPI chlorophyll · protein complexes in aqueous suspensions at room temperature can be oriented in externally applied electric fields. The orientation is observed by monitoring the electric field induced linear dichroism (LD). With whole chloroplasts a detectable LD signal is observed using voltages as low as 2–3 V (50 Hz alternating voltage) across an 0.3 cm electrode gap, and nearly complete orientation is observed at fields of 30 V · cm^?1. The wavelength dependence of the LD signals using either orienting electric fields ($\text{E}$) alone, or magnetic fields ($\text{B}$) alone, are similar but opposite in sign with $\text{E}$ and $\text{B}$ pointing in the same direction. The chloroplasts tend to orient in such a way that the membrane planes are parallel to $\text{E}$. The CPI complexes and bacterial chromatophores require much higher electric fields for orientation than whole chloroplasts (for CPI complexes E > 2000 V · cm^?1); rectangular, millisecond duration, voltage pulses are utilized for the observation of electric field induced LD spectra in these cases. Oriented CPI complexes exhibit LD maxima of the same sign at 685 and at 440 nm. The oriented chromatophores exhibit an LD spectrum of either positive or negative sign, depending on the wavelength. The mechanisms of the orientation are discussed.     

Heat-induced reversible changes in Photosystem 1 absorption cross-section of pea chloroplasts and sub-chloroplast preparations

Reversible changes in the room temperature fluorescence quenching at 685 nm and light scattering level at 577 nm, indicating about 15% of granal unstacking, induced by high temperature treatment (40°C, for 5 min) of pea chloroplasts were shown. Analysis of the low temperature excitation fluorescence spectra of the 735 nm Photosystem 1 (PS 1) band (F735), in the 635–725 nm region, has revealed the involvement of light-harvesting (LHC 2, maxima at 650 and 676 nm) and the proximal Photosystem 2 antenna (maxima 668, 687 nm) in heat-induced enhancement of the PS 1 long wavelength antenna absorption cross-section. It was found that the two PS 1 sub-chloroplast preparations, achieved by the digitonin method, possessed different characteristics of this enhancement. For the heavier fraction (100 000 g) the additional absorption cross-section was formed mostly at the expense of PS 2 antennas (apparently spillover), but for the lighter PS 1 fraction (145 000 g) the changes have indicated an -transfer mechanism, i.e., participation of only LHC 2 in the energy transfer towards PS 1. This may indicate the heterogeneous character of the temperature-induced energy redistribution across the PS 1-containing chloroplast membrane compartments. The model of heat-induced changes in the pigment-protein complex arrangement is discussed in terms of domain organisation of the thylakoid membrane.Abbreviations Chl a/b ratio between chlorophyll a and chlorophyll b concentrations - CP43 and CP47 proximal Photosystem 2 antenna complexes - D1/D2 complex Photosystem 2 reaction centre complex - EDTA ethylenediaminetetraacetic acid - F685 and F696 Photosystem 2 low temperature fluorescence bands - F735 Photosystem 1 low temperature fluorescence band - Fp free pigment band in green gel electrophoresis - LHC 2 light-harvesting chlorophyll a/b complex - LHCP I, II and III light-harvesting bands in green gel electrophoresis - Cp1 and Cpa bands in green gel electrophoresis which are associated with Photosystem 1 and 2 reaction centre complexes with internal antennas - P700 Photosystem 1 reaction centre - PPC pigment-protein complex - PS 1 and Photosystem 1 alpha and Photosystem 1 beta - PS 2 and Photosystem 2 alpha and Photosystem 2 beta - RC reaction centre - SDS-PAGE sodiumdodecylsulphate-polyacrylamide gel electrophoresis - St1-St2 state-1-state-2 transitions     

Intramembranous particles and chlorophyll complexes in chloroplasts.

The size and population density of large and small particles from freeze-fractured chloroplasts of three wild-type algae and of normal spinach were determined. Computer analyses of low-temperature absorption spectra of chloroplast preparations from these species were performed, and a possible correlation between the occurrence of seven chlorophyll complexes and the aforementioned properties of the intramembranous particles was studies. It was found that only single-sized particles occur in a species containing neither chlorophyll b nor chlorophyll a-685 complexes. The three remaining species carry particles of two sizes, termed large and small particles. However, from quantitative considerations it is concluded that the chlorophyll content of none of the various pigment complexes is related to the size and the population density of the studied particles. If such a relationship exists, it seems likely to be due to the carrier moiety of the chorophyll b-chlorophyll a-685 complex.     

Ammonium and nitrate uptake by Eucalyptus nitens: effects of pH and temperature

Ammonium and nitrate uptake by roots of Eucalyptus nitens was characterised with respect to pH and temperature. Uptake of ammonium and nitrate was measured as depletion from solutions by roots of intact 11 week old solution-cultured seedlings. Uptake rates of ammonium were consistently higher than those of nitrate in all experiments. Uptake rates for ammonium were 200% higher at pH 4 than at pH 6, but for nitrate were unchanged. Uptake rates of ammonium and nitrate were both reduced to a similar extent (70%) with a decrease in temperature from 20 °C to 10 °C. For ammonium uptake, there was rapid (<24 hr) adaptation to a reduction in root temperature. The apparent preference shown here for ammonium over nitrate could be indicative of E. nitens growing in cold, acidic forest soils where ammonium is commonly more available than nitrate. These results suggest that N uptake rates of E. nitens may be maximised under a wide variety of conditions if N is supplied predominantly in the ammonium form. This revised version was published online in June 2006 with corrections to the Cover Date.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Autodigestion in crude extracts of soybean leaves and isolated chloroplasts as a measure of proteolytic activity

Two methods of measuring protein breakdown resulting from self-digestion during incubation in extracts of soybean leaves were examined. The release of free α-amino-nitrogen was measured with ninhydrin, and the disappearance of the large subunit of ribulose bisphosphate carboxylase (RuBPcase) was followed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rates of protein breakdown were measured as a function of temperature, pH, and leaf developmental stage and in the presence of various proteinase inhibitors. These treatments had differential effects on apparent proteolysis, depending on the method used. Determination of the ratio of α-amino-nitrogen plus peptide bond-nitrogen to α-amino-nitrogen indicated that the ninhydrin method detected the activity of exopeptidases preferentially. The disappearance of the large subunit of RuBPCase as shown on gels was due primarily to the activity of endopeptidases. The sensitivity of the two types of proteolytic degradation to proteinase inhibitors differed.     

Temperature and pH interaction in NO3 uptake

Summary The interaction effects of temperature, pH and the presence of the poly-valent cation aluminium on nitrate uptake by excised barley roots have been studied. Results indicate that nitrate uptake increased with temperature at the two pH values studied. NO₃ ⁻ uptake was higher from pH6 than from pH4 at all temperatures in the absence of Al⁺³. The addition of Al⁺³ to the external solution modifies this effect. Different theories are given to explain these interactions.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Intramembranous particles and chlorophyll complexes in chloroplasts

The size and population density of large and small particles from freeze-fractured chloroplasts of three wild-type algae and of normal spinach were determined.Computer analyses of low-temperature absorption spectra of chloroplast preparations from these species were performed, and a possible correlation between the occurrence of seven chlorophyll complexes and the aforementioned properties of the intramembranous particles was studied.It was found that only single-sized particles occur in a species containing neither chlorophyll b nor chlorophyll a-685 complexes. The three remaining species carry particles of two sizes, termed large and small particles. However, from quantitative considerations it is concluded that the chlorophyll content of none of the various pigment complexes is related to the size and the population density of the studied particles. If such a relationship exists, it seems likely to be due to the carrier moiety of the chlorophyll b · chlorophyll a-685 complex.     

A method for measuring the internal pH in illuminated chloroplasts based on the stimulation of proton uptake by amines.

A simple method for measuring the internal pH of chloroplasts during steady-state illuminations based on the stimulation of proton uptake by monofunctional amines was developed. Predictions of a mathematical derivation concerning the dependence of the stimulation on the amine concentration, the internal volume, the pK of the amine and the external pH have been verified experimentally. To circumvent uncoupling and swelling due to large internal accumulation of amines extrapolation of the stimulation to low amine concentrations was suggested and shown to lead to valid values. Alternatively, swelling could be largely reduced in a medium containing potassium aspartate and valinomycin.     

Pyruvate uptake induced by a pH jump in mesophyll chloroplasts of maize and sorghum, NADP-malic enzyme type C4 species

A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP-malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD-malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient-collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP-malic enzyme type C4 species is primarily driven by the proton gradient across the envelope.     

Accumulation of bicarbonate in intact chloroplasts following a pH gradient

With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties:

1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved.

2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0.

3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range.

4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler.

5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake.

The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked.

From these results the following conclusions have been drawn:

1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma.

2. (b) The uptake of bicarbonate proceeds via diffusion of CO₂ across the inner membrane. There are no indications for a specific transport of bicarbonate.

3. (c) The CO₂ concentration in the chloroplasts may be equal to the CO₂ concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons.

A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO₂ affinity of CO₂ fixation is discussed.