The role of monovalent cations for photosynthesis of isolated intact chloroplasts

The role of monovalent cations in the photosynthesis of isolated intact spinach chloroplasts was investigated. When intact chloroplasts were assayed in a medium containing only low concentrations of mono- and divalent cations (about 3 mval l^-1), CO₂-fixation was strongly inhibited although the intactness of chloroplasts remained unchanged. Addition of K⁺, Rb⁺, or Na⁺ (50–100 mM) fully restored photosynthesis. Both the degree of inhibition and restoration varied with the plant material and the storage time of the chloroplasts in low-salt medium. In most experiments the various monovalent cations showed a different effectiveness in restoring photosynthesis of low-salt chloroplasts (K⁺>Rb⁺>Na⁺). Of the divalent cations tested, Mg²⁺ also restored photosynthesis, but to a lesser extent than the monovalent cations.In contrast to CO₂-fixation, reduction of 3-phosphoglycerate was not ihibited under low-salt conditions. In the dark, CO₂-fixation of lysed chloroplasts supplied with ATP, NADPH, and 3-phosphoglycerate strictly required the presence of Mg²⁺ but was independent of monovalent cations. This finding excludes a direct inactivation of Calvin cycle enzymes as a possible basis for the inhibition of photosynthesis under low-salt conditions.Light-induced alkalization of the stroma and an increase in the concentration of freely exchangeable Mg²⁺ in the stroma, which can be observed in normal chloroplasts, did not occur under low-salt conditions but were strongly enhanced after addition of monovalent cations (50–100 mM) or Mg²⁺ (20–50 mM).The relevance of a light-triggered K⁺/H⁺ exchange at the chloroplast envelope is discussed with regard to the light-induced increase in the pH and the Mg²⁺ concentration in the stroma, which are thought to be obligatory for light activation of Calvincycle enzymes.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Two types of PS II centres as manifested by light saturation of delayed fluorescence from DCMU-treated chloroplasts

Intensity of 2 s delayed fluorescence (DF) as a function of steady-state actinic light intensity was investigated in pea chloroplasts in the presence of 10 M DCMU. The light saturation curve of DF was approximated by a sum of two hyperbolic components which differ by an order of magnitude in the half-saturating incident light intensity. The relative contribution of the amplitudes of the components was practically independent of cation (Na⁺ and Mg²⁺) concentration and a short-term heating of the chloroplasts at 45°C. The component saturating at low incident light intensity was selectively suppressed by 100 M DCMU or by 1 mol g^-1 Chl oleic acid. DF intensity following excitation by a single saturating 15 s flash was equal to the intensity of the component saturating at a low incident light intensity. Upon flash excitation, the maximum steady-state DF level was found to be attained only after a series of saturating flashes. It is concluded that the two components of the DF light saturation curves are related to PS II centres heterogeneity in quantum yield of stabilization of the reduced primary quinone acceptor.Abbreviations DF Delayed fluorescence - L₁- and L₂-components DF components saturating at low and high incident light intensity, respectively - I incident light intensity - L DF intensity - P₆₈₀ reaction centre chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively     

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA₈₂₀) in the microsecond time range. The extent of ΔA₈₂₀ induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg²⁺ free medium (plus 15 mM K⁺) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg²⁺ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.     

Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts

A highly purified light-harvesting pigment-protein complex (LHC) was obtained by fractionation of cation-depleted chloroplast membranes using the nonionic detergent, Triton X-100. The isolated LHC had a chlorophyll $\text{a}\text{b}$ ratio of 1.2 and exhibited no photochemical activity. SDS-polyacrylamide gel electrophoresis of the LHC revealed three polypeptides in the molecular weight classes of 23, 25, and 30 × 10³. Antibodies were prepared against the LHC and their specificity was established. The effect of the α-LHC (antibodies to LHC) on salt-mediated changes in PS I and PS II photochemistry, Chl α fluorescence inductions, and 77 °K fluorescence emission spectra was investigated. The results show that: (i) The Mg²⁺-induced 20% decrease in photosystem I (PS I) quantum yield observed in control chloroplasts was blocked by the presence of the α-LHC antibody, (ii) The Mg²⁺-induced 70% increase in photosystem II (PS II) quantum yield of control chloroplasts was reduced 35% for plastids in the presence of α-LHC antibody, (iii) The Mg²⁺-induced increase in room-temperature variable fluorescence was reduced 60% by α-LHC antibody, (iv) The Mg²⁺-induced increase in the $\text{F685}\text{F730}$ emission peak ratio at 77 °K was inhibited 50% in the presence of α-LHC antibody. These results provide direct evidence for the involvement of the light-harvesting complex in cation regulation of energy redistribution between the photosystems. The fact that the α-LHC antibody does not fully block Mg²⁺-induced PS II increases or chlorophyll fluorescence increases supports the concept that Mg²⁺ has two mechanisms of action: one effect on energy distribution and a second direct effect on photosystem II centers.     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

Modifications of the functioning of Photosystem II induced in the dark by alkaline pH in the presence of cations

Submission of chloroplasts to alkaline pH, in the range pH 7.5–9.5, leads to changes in their oxygen-evolving capacities. These changes are enhanced by the addition of divalent cations and also monovalent cations at high concentrations. (1) Dark incubation of chloroplasts at pH ? 9 gives rise to a time-dependent inactivation of electron transport from water to 2,6-dichlorophenolindophenol measured at neutral pH. The rate of inactivation is increased by adding cations. (2) The variable fluorescence is decreased with a dependence on incubation time and concentration of cations similar to that of the Hill reaction. Addition of the electron donor NH₂OH removes most of the fluorescence quenching, (3) EPR measurements indicate that the inactivations are accompanied by loss of Mn²⁺ and the appearance of signal II fast. (4) At lower pH (7.5) the oscillations of oxygen evolved per flash during a sequence of flashes show an increase in damping when 20 mM MgCl₂ is present instead of 100 mM KCI. These changes are not seen at pH 6. (5) None of these Mg²⁺-induced modifications are prevented by glutaraldehyde fixation. We conclude that the effects of alkaline pH and MgCl₂ do not involve major protein structural changes, and that both act on the manganese-containing protein of the oxygen-evolving site.     

Effect of cations on the reconstitution of heavy subchloroplast fractions (grana) in disorganized low-salt agranal chloroplasts

The disorganization of grana in spinach chloroplasts and their reconstitution has been studied by varying their ionic environment. Dissociation in low-salt media and reconstitution by added cations (monovalent or divalent) was correlated with the formation in high yield of light or heavy subchloroplast membrane fractions, respectively, produced after digitonin treatment of chloroplasts. The formation of heavy subchloroplast fractions was dependent on cation concentration and reached a plateau at 0.1 m monovalent cation or 0.002 m divalent cation. The cation reconstituted fractions recovered the composition and activities of the respective fractions obtained from control chloroplasts. Cation addition to light subchloroplast fractions isolated from low-salt agranal chloroplasts after digitonin disruption also produced heavy fractions. Divalent cations were more effective than monovalent. The heavy fractions produced were enriched in Chlorophyll b and photosystem II activity while the light fractions were enriched in Chlorophyll a and photosystem I activity. The mechanism by which cations induce formation of heavy subchloroplast fractions is not osmotic. Upon reconstitution, stacking of thylakoids seems to occur at specific membrane binding sites.     

Kinetic analysis of P-700 photoconversion: Effect of secondary electron donation and plastocyanin inhibition

The kinetics of P-700 photoconversion under weak continuous actinic illumination were quantitatively analyzed to provide information on the relative absorption cross-section σ_PSI of the light-harvesting pigments associated with photosystem I and on the number of electrons stored between the two photosystems in dark-adapted chloroplasts. The theory of chemical kinetics for a system of monomolecular consecutive first-order reactions is reviewed briefly to provide support for the experimental approach taken. A complete inhibition of plastocyanin by cyanide eliminated all secondary electron donation to P-700⁺ and allowed the registration of the exponential (monomolecular) P-700 photoconversion at room temperature. The rate constant K_p-700 of the exponential kinetics was independent of the ionic (± Mg²⁺) and osmotic (± sucrose) strength of the chloroplast suspension medium, and of the oxidation-reduction state of photosystem II. The extent of plastocyanin inhibition in partially inhibited samples was greater under low ionic and low osmotic conditions. In dark-adapted chloroplast samples that were not cyanide treated, the number of electrons stored between the two photosystems was 3.9 ± 0.2 and independent of divalent cations. It is concluded that plastocyanin inhibition by cyanide is favored under low ionic and low osmotic conditions. The Mg²⁺ ion and redox state of photosystem II-independent photoconversion of P-700 does not support significant changes in the spillover of excitation from photosystem II to photosystem I in isolated chloroplasts.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: Accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in F_m relative to F_o, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of Q_B-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of Q_B-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

Studies on (Na++K+)-activated ATPase: XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg2+ and inorganic phosphate

Dissociation of the (Na⁺+K⁺)-ATPase ouabain complex, formed presence of Mg²⁺ and inorganic phosphate (Complex II), is inhibited by Mg²⁺ (21–45%) and the alkali cations Na⁺ (25–59%) and K⁺ (27–75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0–11% by Na⁺ and 11–19% by K⁺. The degree of inhibition is not directly related to the size of the dissociation rate constant ($\text{k}{}^{\mathrm{?}}$) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues.Inhibition curves for Na⁺ and K⁺ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30–40 mM Na⁺ and 6–10 mM K⁺, and the maximally inhibitory concentrations are 50–150 and 15–20 mM, respectively. Maximal inhibition by Na⁺ or K⁺ for these tissues is the same. For guinea pig kidney cortex Na⁺ and K⁺ are almost equally effective, but 150 mM K⁺ or 200 mM Na⁺ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations.The inhibition curve for Mg²⁺ is not sigmoidal. In the kidney preparations Mg²⁺ inhibits half-maximally at 0.4-0.5 mM, maximally at 1–3 mM. Maximal inhibition by Mg²⁺ is higher than by Na⁺ or K⁺ for rabbit cortex and lower for guinea pig kidney cortex.There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg²⁺ and the alkali cations.Complex II differs in stability, in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na⁺ and K⁺, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.     

Calcium and magnesium effect on divalent cation deficientHydrilla verticillata thylakoid electron transport activity

The role of divalent cations like magnesium (Mg²⁺) and calcium (Ca²⁺) was irrvestigated on energy distribution process ofHydrilla verticillata thylakoids. Effect of these cations was tested on relative quantum yield of photosystem (PS) II catalyzed electron transport activity, room and liquid nitrogen temperature fluorescence emission properties and thylakoid light scattering characteristics. The electron transport activity was found to be stimulated in the presence of these cations in a light intensity independent manner. The concentration of cation required for maximum stimulation was nearly 10–12 mM. Comparatively, Ca²⁺ was more effective than Mg²⁺. Cation induced stimulation in electron transport activity was not accompanied by increase in chlorophylla fluorescence intensity either at room (25°C) or liquid nitrogen (77°K) temperatures. Furthermore, 540 nm absorption and 90° light scattering properties of thylakoids remained insensitive towards divalent cations. These facts together suggest that divalent cations inHydrilla thylakoids are not effective in supporting the excitation distribution between the interacting photosystem complexes.     

Flash Inactivation of Oxygen Evolution: IDENTIFICATION OF S(2) AS THE TARGET OF INACTIVATION BY TRIS

Brief saturating light flashes were used to probe the mechanism of inactivation of O₂ evolution by Tris in chloroplasts. Maximum inactivation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting O₂-evolving catalyst of photosystem II (S₂-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequilibration caused a three-flash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S₂-states were similar; and (c) reagents accelerating S₂ deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II_s, as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH₃ was shown to be a competitive inhibitor of the Tris-induced inactivation. S₂-dependent inactivation was inhibited by glutaraldehyde fixation of chloroplasts, possibly suggesting that inactivation proceeds via conformational changes of the S₂-state.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

Correlated influence of cation concentration and excitation intensity on PS II activity-II. Comparative study between green plant and brown-alga chloroplasts

A preparation of photochemically active chloroplasts of Fucus was added to a low-salt medium with high osmolarity (HEPES AMPD buffer, 1M sorbitol). The rate of DCIP reduction (DCIPr) and the variable fluorescence (Fv) of these phaeoplasts were measured and compared with the same activities in spinach chloroplasts. A study of the influence of mono- and divalent-cations showed that salt effects on PS II activity also exist in Fucus.

1. Mg⁺⁺ action on Fv is similar, although noticeably weaker in Fucus than in spinach chloroplasts.
2. Na⁺ has no effect on Fv of Fucus chloroplasts, but its influence on DCIPr is more pronounced than in spinach.
3. Mg⁺⁺ influence on DCIPr is largely dependent upon excitation energy. In subsaturating light (100\2-1000 W m^\t-2), Mg⁺⁺ stimulation increases up to 100 mM, almost doubling the level. In very low wight conditions (3Wm^\t02), however, this stimulation saturates at about 10 mM; higher concentrations are no longer effective but do not quench DCIPr noticeably, unlike the case in spinach.

Therefore, cations act through similar pathways on Fucus as on spinach isolated chloroplasts but the effects on PS II centers are preponderant in Fucus whereas the modifications in non-radiative decay or pigment array size are weaker.     

Protein synthesis in isolated spinach chloroplasts: comparison of light-driven and ATP-driven synthesis

A comparison has been made of the optimal concentrations of Mg²⁺ and K⁺ ions necessary for both light-driven protein synthesis in intact spinach chloroplasts and for ATP-driven protein synthesis in broken chloroplasts, and the products of the two systems have been compared by polyacrylamide gel electrophoresis. Light-driven incorporation of amino acids into polypeptides in intact chloroplasts assayed in buffer systems containing sucrose or sorbitol as the osmoticum is inhibited by the addition of Mg²⁺, the effect being most marked at low concentrations (less than 40 mm) of KCl. On the other hand, chloroplasts suspended in 0.2 m KCl as osmoticum require Mg²⁺ (3 mm) for optimal light-driven protein-synthesizing activity. Incorporation of amino acids by broken chloroplasts in the dark, supplemented with ATP and GTP, requires 9 mm Mg²⁺ for maximum activity. A requirement for monovalent cations is best filled by K⁺ (approx 30 mm) in the case of the light-driven, intact chloroplast system whereas, in the ATP-driven, broken chloroplast system, NH_4⁺ (approx 80 mm) gave the highest activity.Autoradiographs of Na dodecyl sulfate-polyacrylamide gels of the products from both the light-driven, intact chloroplasts and from the ATP-driven, broken chloroplasts reveal qualitatively similar patterns. There are at least four radioactive polypeptides in the soluble protein fraction the dominant product being coincident with the large subunit of Fraction 1 protein. In the membrane fraction at least nine discrete products can be resolved.     

Sensitivity of photosynthesis by spinach chloroplast membranes to osmotic stress in vitro: Rapid inhibition of O2 evolution in presence of magnesium

Thylakoids prepared from spinach (Spinacea oleracea L.) chloroplasts were exposed to osmotic stress in vitro in the presence or absence of different inorganic salts. By an hour after incubation in 1.0 M sorbitol and 10 mM (or more) MgCl₂, the thylakoids lost approximately 80% of their photosystem (PS) II activity, but not PS I. The inhibition occurred only in presence of magnesium as indicated by the combinations of several cations/anions. The PS II activity was relatively insensitive to osmotic stress in the presence of diphenyl carbazide. We therefore conclude that under conditions of water stress in the presence of 10 mM or higher Mg²⁺, the oxygen evolving system in chloroplasts is rapidly inactivated.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - DPC diphenyl carbazide - MV methyl viologen - PS photosystem Part of this work was included in the thesis submitted by the first author of M.Phil.degree.     

Role of membrane organization in the Mg+2-mediated regulation of excitation energy transfer in barley chloroplasts

Photosystem II fluorescence of barley chloroplasts has been monitored to understand the role of membrane organization in the cation mediated regulation of excitation energy transfer from photosystem II to photosystem I. Membrane organization has been perturbed by adding 60 mM benzyl alcohol which is known to increase the membrane fluidity and decrease its thickness. An addition of 60 mM benzyl alcohol increases the fluorescence at 683 nm (excitation at 436 mn) by 43% whereas 5 mM Mg⁺² increased the fluorescence by 38%. An addition of 5 mM Mg⁺² to benzyl alcohol treated chloroplasts resulted in only a small increase in the fluorescence (6.5%). Circular dichroic measurements showed that 5 mM Mg⁺² decreased the circular dichroic signals suggesting an alteration in the orientation of the chromophores. However, the effect was insignificant on the benzyl alcohol treated chloroplast membranes. Benzyl alcohol itself had large effect on the circular dichroic signals. Based on these results, it appears that a change in the orientation of photosystem I and photosystem II, rather than their segregation, is responsible for the cation-induced increase in the photosystem II fluorescence.     

The stacking of chloroplast thylakoids: Evidence for segregation of charged groups into nonstacked regions

Small particles derived from the digitonin treatment of chloroplast thylakoid membranes in either the stacked (grana-containing) or unstacked condition, as determined by cation concentration, have been used to study the aggregation of thylakoid membranes. At pH values above 5, the small particles from stacked chloroplasts do not aggregate in the presence of Mg²⁺ or other screening cations at concentrations sufficient to cause the restacking of thylakoids from low-salt chloroplasts. However, the small particles from stacked chloroplasts are aggregated either by lowering the pH to 4.6 or adding the binding cation La³⁺. In contrast, the small particles obtained on digitonin treatment of unstacked chloroplasts were aggregated by cations at neutral pH. Large particles (mainly grana) derived from digitonin treatment of stacked chloroplasts could not be unstacked by transfer to media of low cation concentration. It is concluded that the nonappressed regions of the chloroplast thylakoid membranes under stacking conditions carry higher than average negative surface charge densities under physiological pH conditions. Transfer of chloroplasts to media of low cation concentration causes a time-dependent lateral redistribution of charge between the appressed and nonappressed regions, but this redistribution is prevented by prior digitonin treatment of stacked chloroplasts.