Comparative Studies on Plastoquinones. III. Distribution of Plastoquinones in Higher Plants

The distribution of plastoquinones A 45, B and C was studied in representatives from 34 different plant families beginning with liverworts and mosses to higher plants. All of these species, including many monocots and dicots, contained significant amounts of the 3 quinones. Two species of Aesculus contained plastoquinone A 20 in addition to plastoquinone A 45, B, and C. Many dicots, such as Aesculus, watermelon, tobacco and tomato accumulated increasing quantities of plastoquinones A and C₁-C₄ during the growing season. The concentrations of plastoquinones B and C₅-C₆ tended to remain at a constant low level during the season (<0.01 μmole per mg chlorophyll). Preliminary studies with bean plants (Vicia faba and Phaseolus sp.) indicate that the levels of quinones varied little under different growth conditions (day length and temp.) although Vicia faba tended to have higher PQ A values with increased temperature.     

Comparative Studies on Plastoquinones: V. Changes in Lipophilic Chloroplast Quinones during Development

Changes of lipophilic chloroplast quinones in corn, oats, peas, and Vicia faba are reported after 0, 4, 8, 12, 16, 20, 24, 48, 72, or 96 hours of exposure to light. There is a pronounced increase in plastoquinone A and chlorophyll levels and slight increase, in plastoquinone C_1-6, vitamin K₁, and α-tocopherylquinone content. Coenzyme Q levels, on the other hand, show little change upon exposure to light.     

Cyclic electron flow around photosystem I in unicellular green algae

Cyclic electron flow around PSI, or cyclic photophosphorylation, is the photosynthetic process which recycles the reducing equivalents produced by photosystem I in the stroma towards the plastoquinone pool. Through the activity of cytochrome b ₆ f, which also transfers protons across the membrane, it promotes the synthesis of ATP. The literature dealing with cyclic electron flow in unicellular algae is far less abundant than it is for plants. However, in the chloroplast of algae such as Chlorella or Chlamydomonas, an efficient carbohydrate catabolism renders the redox poise much more reducing than in plant chloroplasts. It is therefore worthwhile highlighting the specific properties of unicellular algae because cyclic electron flow is highly dependent upon the accumulation of these stromal reducing equivalents. Such an increase of reducing power in the stroma stimulates the reduction of plastoquinones, which is the limiting step of cyclic electron flow. In anaerobic conditions in the dark, this reaction can lead to a fully reduced plastoquinone pool and induce state transitions, the migration of 80% of light harvesting complexes II and 20% of cytochrome b ₆ f complex from the PSII-enriched grana to the PSI-enriched lamella. These ultrastructural changes have been proposed to further enhance cyclic electron flow by increasing PSI antenna size, and forming PSI-cyt b ₆ f supercomplexes. These hypotheses are discussed in light of recently published data.     

Plastoquinone B

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K₁ (10, 12), an unknown naphthoquinone (13) and α-, β- and γ-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R_F values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.     

Inorganic carbon acquisition by eukaryotic algae: four current questions

The phylogenetically and morphologically diverse eukaryotic algae are typically oxygenic photolithotrophs. They have a diversity of incompletely understood mechanisms of inorganic carbon acquisition: this article reviews four areas where investigations continue. The first topic is diffusive CO₂ entry. Most eukaryotic algae, like all cyanobacteria, have inorganic carbon concentrating mechanisms (CCMs). The ancestral condition was presumably the absence of a CCM, i.e. diffusive CO₂ entry, as found in a small minority of eukaryotic algae today; however, it is likely that, as is found in several cases, this condition is due to a loss of a CCM. There are a number of algae which are in various respects intermediate between diffusive CO₂ entry and occurrence of a CCM: further study is needed on this aspect. A second topic is the nature of cyanelles and their role in inorganic carbon assimilation. The cyanelles (plastids) of the euglyphid amoeba Paulinella have been acquired relatively recently by endosymbiosis with genetic integration of an α-cyanobacterium with a Form 1A Rubisco. The α-carboxysomes in the cyanelles are presumably involved in a CCM, but further investigation is needed.Also called cyanelles are the plastids of glaucocystophycean algae, but is it now clear that these were derived from the β-cyanobacterial ancestor of all plastids other than that of Paulinella. The resemblances of the central body of the cyanelles of glaucocystophycean algae to carboxysomes may not reflect derivation from cyanobacterial β-carboxysomes; although it is clear that these algae have CCMs but these are now well characterized. The other two topics concern CCMs in other eukaryotic algae; these CCMs arose polyphyletically and independently of the cyanobacterial CCMs. It is generally believed that eukaryotic algal, like cyanobacterial, CCMs are based on active transport of an inorganic carbon species and/or protons, and they have C₃ biochemistry. This is the case for the organism considered as the third topic, i.e. Chlamydomonas reinhardtii, the eukaryotic alga with the best understood CCM. This CCM involves HCO₃ ^? conversion to CO₂ in the thylakoid lumen so the external inorganic carbon must cross four membranes in series with a final CO₂ effux from the thylakoid. More remains to be investigated about this CCM. The final topic is that of the occurrence of C₄-like metabolism in the CCMs of marine diatoms. Different conclusions have been reached depending on the organism investigated and the techniques used, and several aspects require further study.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

Comparative studies of the photosynthesis of the submerged macrophyte Elodea canadensis and the filamentous algae Cladophora glomerata and Spirogyra sp.

The photosynthetic and respiratory responses of Elodea canadensis Michx., Cladophora glomerata (L.) Kütz. and Spirogyra sp. to oxygen, temperature, HCO₃⁻¹ concentration, pH and irradiance were determined. Photosynthesis was inhibited by O₂ in all three species under all conditions and inhibition was greatert in E. canadensis. This inhibition was not caused solely by an accelerated rate of dark respiration and this suggests that photorespiration may be an important factor controlling productivity, particularly in E. canadensis.Photosynthetic performance was impaired much more in E. canadensis than in either of the filamentous algae conditions of high pH and low CO₂ concentrations. In the field photosynthesis of these algae may increase the pH and reduce the CO₂ content of the water sufficiently to exert deleterious effects on macrophyte photosynthesis. Such a plant-induced change in water quality could give C. glomerata a competitive advantage over E. canadensis and be a factor in the replacement of the vascular plant by the alga in some waters.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Nitric oxide represses photosystem II and NDH-1 in the cyanobacterium Synechocystis sp. PCC 6803

Photosynthetic electron transfer comprises a series of light-induced redox reactions catalysed by multiprotein machinery in the thylakoid. These protein complexes possess cofactors susceptible to redox modifications by reactive small molecules. The gaseous radical nitric oxide (NO), a key signalling molecule in green algae and plants, has earlier been shown to bind to Photosystem (PS) II and obstruct electron transfer in plants. The effects of NO on cyanobacterial bioenergetics however, have long remained obscure. In this study, we exposed the model cyanobacterium Synechocystis sp. PCC 6803 to NO under anoxic conditions and followed changes in whole-cell fluorescence and oxidoreduction of P700 in vivo. Our results demonstrate that NO blocks photosynthetic electron transfer in cells by repressing PSII, PSI, and likely the NDH dehydrogenase-like complex 1 (NDH-1). We propose that iron?sulfur clusters of NDH-1 complex may be affected by NO to such an extent that ferredoxin-derived electron injection to the plastoquinone pool, and thus cyclic electron transfer, may be inhibited. These findings reveal the profound effects of NO on Synechocystis cells and demonstrate the importance of controlled NO homeostasis in cyanobacteria.     

Requirement for plastoquinone a in the hill reaction of isolated chloroplasts

In order to avoid the problem of an uncoupling effect in stimulation of potential Hill reaction activity during extraction with heptane. Hill reaction activity was measured with uncoupled spinach chloroplasts. The reactions were also run under aerobic conditions. Under these conditions a correction for a 3-(p-chlorophenyl)-1, 1-dimethylurea insensitive, photostimulated O₂ uptake reaction is necessary, especially with extracted chloroplasts. The reaction displayed proper Hill reaction stoichiometry in untreated and restored systems if these corrections were made. Plastoquinone A always restored some activity to the extracted chloroplasts but never restored activity to the original level. Additional activity could be restored by the crude extract. It appears that there is a clear requirement for plastoquinone A in the Hill reaction but the restoration of maximum activity depends upon additional factors. The variability of response to plastoquinone A which has been reported in various laboratories may be determined by the degree of extraction of the other materials.     

H2 metabolism in photosynthetic organisms. II. Light-dependent H2 evolution by preparations from Chlamydomonas, Scenedesmus and spinach.

Light-dependent H₂ evolution from dithiothreitol as electron donor was observed with cell-free preparations of anaerobically adapted $\text{Chlamydomonas reinhardii, Scenedesmus obliquus}$ and from spinach chloroplasts mixed with $\text{Chlamydomonas}$ hydrogenase. NADH substituted for dithiothreitol as electron donor only in the $\text{Chlarmydomonas}$ preparation. Dibromothymoquinone, an antagonist of plastoquinone, selectively inhibited H₂ photoevolution from NADH. These results are interpreted as indicating that 3-(3,4-dichlorophenyl)-1,1-dimethyl urea insensitive H₂ photoevolution by algae containing hydrogenase is due to the capability of NADH to reduce plastoquinone in the electron transport chain, and to evolve H₂ by a low redox potential carrier of photosystem I.     

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn₄Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.     

Polyprenyl quinones and α-tocopherol in Calendula officinalis

In various cellular subfractions of Calendula officinalis leaves a study was made of the distribution of polyprenyl quinones and α-tocopherol and the dynamics of their labelling with ¹⁴CO₂ and acetate-[1-¹⁴C] and incorporation of mevalonate-[2-¹⁴C] after 3 hr. It was confirmed that plastoquinone occurs only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions. Phylloquinone was found in the chloroplast and mitochondrial fractions as well as in the post-mitochondrial supernatant. Studies of the dynamics of radioactive precursor incorporation indicated that α-tocopherol is metabolized more rapidly than the polyprenyl quinones studied; the incorporation of mevalonate-[2-¹⁴C] suggests that the side chain of plastoquinone can be synthesized in the cytoplasm and transported to the chloroplasts.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N₂-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with ³²P_i, and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Fermentative Metabolism of Chlamydomonas reinhardtii: II. Role of Plastoquinone

Evidence is presented to substantiate a chloroplastic respiratory pathway in the green alga, Chlamydomonas reinhardtii, whereby reducing equivalents generated during the degradation of starch enter the thylakoidal chain at the plastoquinone site catalyzed by NADH-plastoquinone reductase. In this formulation, the reduced plastoquinone is oxidized either by the photoevolution (photosystem I) of H₂ under anaerobic conditions or by O₂ during dark respiration.     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 (b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/b₆ complex and one, UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q_B.Q_B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q_B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.     

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P₇₀₀ ⁺ in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s