Influence of antimycin a and uncouplers on anaerobic photosynthesis in isolated chloroplasts

Anaerobiosis depresses the light- and bicarbonate-saturated rates of O(2) evolution in intact spinach (Spinacia oleracea) chloroplasts by as much as 3-fold from those observed under aerobic conditions. These lower rates are accelerated 2-fold or more by the addition of 1 mum antimycin A or by low concentrations of the uncouplers 0.3 mm NH(4)Cl or 0.25 mum carbonyl cyanide m-chlorophenylhydrazone. Oxaloacetate and glycerate 3-phosphate reduction rates are also increased by antimycin A or an uncoupler under anaerobic conditions. At intermediate light intensities, the rate accelerations by either antimycin A or uncoupler are inversely proportional to the adenosine 5'-triphosphate demand of the reduction process for the acceptors HCO(3) (-), glycerate 3-phosphate, and oxaloacetate. The acceleration of bicarbonate-supported O(2) evolution may also be produced by adding an adenosine 5'-triphosphate sink (ribose 5-phosphate) to anaerobic chloroplasts. The above results suggest that a proton gradient back pressure resulting from antimycin A-sensitive cyclic electron flow is responsible for the depression of light-saturated photosynthesis under anaerobiosis.     

Effects of uncouplers on Mg2+-dependent fluorescence quenching in isolated chloroplasts

Uncoupling concentrations (about 1 mol l^-1) of desaspidin or carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone reverse the slow light-induced, Mg²⁺-dependent quenching of fluorescence of chlorophyll a in isolated (intact and broken) spinach chloroplasts. Likewise, uncoupling inhibits the light-induced increase of the Mg²⁺ concentration in the stroma of intact chloroplasts, as determined with Eriochrome Blue SE. Addition of higher amounts of the uncouplers to the chloroplasts leads to a slow, light-dependent fluorescence lowering which appears to be promoted by high light intensities and is not reversed in the dark. The reversal of the fluorescence quenching by uncoupling is interpreted to reflect exchange of protons for Mg²⁺ ions at negative sites of the inner thylakoid face, caused by the collapse of the proton gradient across the membrane. The secondary fluorescence lowering caused by high levels of the uncouplers and high light intensities is suggested to be related to an inhibition of non-cyclic photosynthetic electron transport.Abbreviation FCCP carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone     

Stimulation of microsecond-delayed fluorescence from spinach chloroplasts by uncouplers and by phosphorylation

Delayed fluorescence, as measured with a laser phosphoroscope, is stimulated not inhibited by uncouplers during the first 100 μs after the light is turned off. This is true only wen uncouplers cause an increase in the rate of electron transport. When ADP and P_i cause an increase in the electron transport rate, microsecond-delayed fluorescence is also increased. Indeed, there is a complex quantitative relationship between the rate of electron transport and the initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P_i also increase the rate of decay of delayed fluorescence so that after about 150 μs they become inhibitory, as already reported by many authors.

Microsecond-delayed fluorescence continues to rise with rising light intensities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport both with the intensity of the 5–100 μs-delayed fluorescence and with the rate of decay in the intensity of delayed fluorescence. The data imply that the decrease in intensity of millisecond-delayed fluorescence which has often been noted with uncouplers is probably not due to the elimination of a membrane potential. It seems more likely that the decrease in millisecond-delayed fluorescence is a reflection of the rate of disappearance of some other electron transport-generated condition, a condition which is uncoupler-insensitive. Certainly stimulations of microsecond-delayed fluorescence by electron transport which has been uncoupled by gramicidin suggest that ion gradients are not an essential component of the conditions responsible for delayed fluorescence.     

Sucrose as a product of photosynthesis in isolated spinach chloroplasts

Sucrose has been detected as a seasonal photosynthetic product in spinach chloroplast preparations. Sucrose when present accounted for up to 30% of the CO₂ fixed. Experiments in which sucrose was formed have been compared with experiments in which it was not formed, and a possible control mechanism for sucrose synthesis is discussed.     

Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.Abbreviations FITC fluorescein isothiocyanate - IEP Pinner-envelope protein - OE outer-envelope protein - pSSU precursor form of the small subunit of ribulose bisphosphate carboxylaseoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank P. Å. Albertsson, Lund, Sweden, for introducing one of us (S. E.) to the technique of phase partitioning. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246) and Fonds der Chemischen Industrie.     

Choline, tetraalkylammonium and aminoalcohol as preserves of photosynthetic activities in isolated chloroplasts

Choline chloride, tetraalkylammonium chloride and aminoalcoholshow preservative effects on photosynthetic activities in spinachchloroplasts against deterioration after isolation, when oneof them is present in the medium used for isolating and storingchloroplasts within the concentration range between 0.1 and0.5 M. Any of these chemicals cause uncoupling to some extentif present in the reaction medium. (Received August 23, 1973; )     

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented:

1. (1) MgCl₂ (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl₂ addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg²⁺.

2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.

3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl₂, the values of rate constants of Photosystem II deactivation were calculated.

4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl₂ addition. When this transfer is decreased by MgCl₂ the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.

A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl₂ decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.     

Starch degradation in isolated spinach chloroplasts

A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with ¹⁴C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.