Effects of dehydration on the electron transport of Chlorella. An in vivo fluorescence study.

Chlorella was used to study the effects of dehydration on photosynthetic activities. The use of unicellular green algae assured that the extent of dehydration was uniform throughout the whole cell population during the course of desiccation. Changes in the activities of the cells were monitored by measurements of fluorescence induction kinetics. It was found that inhibition of most of the photosynthetic activities started at a similar level of cellular water content. They included CO2 fixation, photochemical activity of Photosystem II and electron transport through Photosystem I. The blockage of electron flow through Photosystem I was complete and the whole transition occurred within a relative short time of dehydration. On the other hand, the suppression of Photosystem II activity was incomplete and the transition took a longer time of dehydration. Upon rehydration, the inhibition of Photosystem II activity was fully reversible when samples were in the middle of the transition, but was not thereafter. The electron transport through Photosystem I was also reversible during the transition, but was only partially afterward.

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction.

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level Fj are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.

Characterization of the photosystem II centers inactive in plastoquinone reduction by fluorescence induction.

In order to characterize the photosystem II (PS II) centers which are inactive in plastoquinone reduction, the initial variable fluorescence rise from the non-variable fluorescence level Fo to an intermediate plateau level Fi has been studied. We find that the initial fluorescence rise is a monophasic exponential function of time. Its rate constant is similar to the initial rate of the fastest phase (α-phase) of the fluorescence induction curve from DCMU-poisoned chloroplasts. In addition, the initial fluorescence rise and the α-phase have the following common properties: their rate constants vary linearly with excitation light intensity and their fluorescence yields are lowered by removal of Mg(++) from the suspension medium. We suggest that the inactive PS II centers, which give rise to the fluorescence rise from Fo to Fi, belong to the α-type PS II centers. However, since these inactive centers do not display sigmoidicity in fluorescence, they thus do not allow energy transfer between PS II units like PS IIα.

Toxic Effects of Copper on Photosystem II of Spinach Chloroplasts.

The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary charge separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat.

Functional size of photosynthetic electron transport chain determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H(2)O to methylviologen) was 623 +/- 37 kilodaltons; for photosystem II (H(2)O to dimethylquinone/ferricyanide), 174 +/- 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 +/- 11 kilodaltons. The difference between 364 +/- 22 (the sum of 174 +/- 11 and 190 +/- 11) kilodaltons and 623 +/- 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b(6)/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.

The two binding sites for DCMU in Photosystem II.

Measurements on the fluorescence induction of Triton X-100 extracted Photosystem II (PSII) particles confirmed the existence of the two sites of inhibition in PSII for the herbicide DCMU. The two sites were located on the reducing and oxidizing sides of PSII, respectively. The inhibition on the oxidizing side, unlike that on the reducing side which was of the "none or all" type, was found only to slow down the electron donation at low concentrations of DCMU. The results also suggested that the inhibitions of DCMU at these two sites were mutually exclusive, i.e., the binding on one site prevented the binding on the other site.

The high affinity binding site for calcium on the oxidizing side of photosystem II.

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.

Enhanced sensitivity to plastoquinone inhibitors of ferredoxin photoreduction by photosystem II in inside-out chloroplast vesicles.

New evidence is presented in support of the concept that reducing power for photosynthesis is generated solely by photosystem II (the oxygenic photosystem) when it transfers electrons from water to ferredoxin without the collaboration of photosystem I, the anoxygenic photosystem responsible for cyclic photophosphorylation. Membrane vesicles of opposite sidedness were prepared from spinach chloroplasts by the two-phase partition method: inside-out-vesicles greatly enriched in photosystem II and right-side-out vesicles containing both photosystems and having the same sidedness orientation as unfractionated chloroplast membranes. In both types of vesicles, plastoquinone analogues were used to inhibit light-induced electron transport from water to ferredoxin and from water to native photosystem I acceptors, the membrane-bound iron-sulfur centers A and B. In right-side-out vesicles the photoreduction of iron-sulfur centers A and B was more sensitive to plastoquinone inhibitors than the photoreduction of ferredoxin, whereas the converse was found in inside-out vesicles in which a greatly enhanced sensitivity of ferredoxin reduction to plastoquinone inhibitors was detected: the photoreduction of ferredoxin was about 80% inhibited at low concentrations of plastoquinone inhibitors that had practically no effect on the photoreduction of iron-sulfur centers A and B. These findings appear to exclude the possibility that these photosystem I contaminants were involved in the photoreduction of ferredoxin by the PSII-enriched inside-out vesicles.

Photosynthetic electron transport from water to NADP driven by photosystem II in inside-out chloroplast vesicles.

It is now widely held that the light-induced noncyclic (linear) electron transport from water to NADP(+) requires the collaboration in series of the two photosystems that operate in oxygen-evolving cells: photosystem II (PSII) photooxidizes water and transfers electrons to photosystem I (PSI); PSI photoreduces ferredoxin, which in turn reduces NADP(+) (the Z scheme). However, a recently described alternative scheme envisions that PSII drives the noncyclic electron transport from water to ferredoxin and NADP(+) without the collaboration of PSI, whose role is limited to cyclic electron transport [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1981) Proc. Natl. Acad. Sci. USA 78, 2942-2946]. Reported here are findings at variance with the Z scheme and consistent with the alternative scheme. Thylakoid membrane vesicles were isolated from spinach chloroplasts by the two-phase aqueous polymer partition method. Vesicles, originating mainly from appressed chloroplast membranes that are greatly enriched in PSII, were turned inside-out with respect to the original sidedness of the membrane. With added plastocyanin, ferredoxin, and ferredoxin-NADP(+) reductase, the inside-out vesicles enriched in PSII gave a significant photoreduction of NADP(+) with water as electron donor, under experimental conditions that appear to exclude the participation of PSI.