V. D. Samuilov

Conversion of biomembrane-produced energy into electric form. III. Chromatophores of Rhodospirillum rubrum

Abstract The mechanisms of energy coupling and ion transport in Rhodospirillum rubrum chromatophores have been studied. Photoreduction of NAD + and photophosphorylation have been measured under anaerobic conditions in the presence of N,N,N ′,N′-tetramethyl- p -phenylenediamine (TMPD), ascorbate and antimycin A. The uncoupler p -trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP), as well as ADP + phosphate, has been found to inhibit the photoreduction of NAD + in this system. Addition of an electron acceptor, such as methylviologen, fumarate or O 2 , to antimycin-treated chromatophores initiates the process of photophosphorylation. Chromatophores of R. rubrum accumulate the penetrating anions, phenyl dicarboundecaborane and tetraphenyl boron, as well as iodide, if the I − carrier, di-(pentafluorophenyl) mercury, is added. The anion accumulation can be supported by light-induced cyclic electron flow (NADH → O 2 , succinate → ferricyanide), by hydrolysis of ATP or inorganic pyrophosphate, as well as by reversal of the energy-requiring transhydrogenase reaction (NADPH → NAD + ). The type of energy source influences only the extent of the anion accumulation process. Cessation of the energy supply ( e.g. by exhaustion of the energy source or poisoning of the system by specific inhibitors or an uncoupler) brings about an efflux of the accumulated anions. Uptake of anions is accompanied by alkalinization of the outer solution; release of anions is accompanied by acidification. It is concluded that there is an energy-dependent charge-specific mechanism for anion accumulation in the chromatophore membrane resembling that found in the membrane of submitochondrial particles. It is stated that the electric field (the “ plus ” inside the chromatophore) is the motive force for ion transfer through the chromatophore membrane against a concentration gradient. The data on NAD + photoreduction, noncyclic photophosphorylation and energy-dependent anion transport are summarized as the concept of four sites of energy coupling in the chromatophore redox chain localized at the same steps as in animal mitochondria (NADPH → NAD + , NADH → cytochrome b , cytochrome b → cytochrome c , and the region after cytochrome c ). Each of these coupling sites can provide energy for generation of a membrane potential.

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.

Generation of electric potential by reaction center complexes from Rhodospirillum rubrum

Several lines of indirect evidence indicate that the electrochemical H’ potential is produced by the lightdependent cyclic electron transfer in chromatophores and intact bacterial cells [l-4] , as was originally postulated by Mitchell [ 51. In this paper, data on the direct measurement of electric current generation by bacteriochlorophyll reaction center complexes, isolated from R. rubrum chromatophores, is reported. A method of reconstitution of the reaction center complex-containing proteoliposomes and their association with planar phospholipid membranes was elaborated. Formation of light-induced electric potential difference by the proteoliposomes was demonstrated by the conventional voltmeter techniques as well as by a phenyldicarbaundecaborane (PCB-) probe. The photoelectric effect was shown to increase on addition of TMPD or cytochrome c in combination with CoQ or vitamin Ka , and to decrease on addition of ferricyanide, o-phenanthroline and a protonophorous uncoupler.

Participation of Chloroplasts in Plant Apoptosis

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN−-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN−-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN− effect. The light-dependent activation of CN−-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

Involvement of Chloroplasts in the Programmed Death of Plant Cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (α-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Qo site of the chloroplast cytochrome b6f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.

Chitosan-induced programmed cell death in plants

Chitosan, CN−, or H2O2 caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H2O2, or chitosan + H2O2 on EC. H2O2 formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN− and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN−-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN−-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H2O2 on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H2O2 as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Energy problems in life evolution.

Evolutionary aspects of bioenergetics are considered. These include the origin of the first organisms, UV-protection and the beginnings of anoxygenic photosynthesis, the electron donor problem of life and the appearance of oxygenic photosynthesis, oxygen danger and strategies of defense, and the role of oxygen in programmed cell death.

A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria.

Generation of membrane potential (delta psi) and transmembrane pH difference (delta pH) was studied in PPi-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport. The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of delta psi, while an atebrin response is an indicator of delta pH. The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by delta psi and delta pH.

H2O2-induced inhibition of photosynthetic O2 evolution by Anabaena variabilis cells.

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN–, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ∼20% of the total manganese pool. This content remained unchanged upon the addition of CN– and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophylla and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.

Role of chloroplast photosystems II and I in apoptosis of pea guard cells.

We investigated the CN–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones QA and QB in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue YD in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.