Phenylmereuric Acetate and Phosphate Control of Light-Dependent Shrinkage and Reactions in Chloroplasts

An investigation of the action of phenylmereuric acetate (PMA) and phosphate on light-induced shrinkage (measured by light scattering and Coulter Counter techniques) and on photosynthetic reactions in spinach chloroplasts led to the following conclusions:

• 1) PMA stimulated light-induced shrinkage (under conditions of cyclic and non-cyclic electron flow) at concentrations which completely inhibited cyclic and non-cyclic photophosphorylation and nicotinamide adenine dinucleotide phosphate (NADP) reduction, though ferricyanide reduction was activated. Although PMA inhibited NADP reduction (probably because this sulfhydryl reagent interfered with the ferredoxin-NADP rednetase) it ean also be considered an uncoiipler (when ferricyanide is the electron acceptor).
• 2) Phosphate maximized light-induced shrinkage (under conditions of cyclic and non cyclic electron flow) at concentrations which did not affect ferricyanide reduction but caused a 40 to 50 per cent inhibition of NADP reduction.
• 3) The pattern of the light scattering response to these two compounds was quite different. In the presence of PMA, the forward (light on) and hack (light off) reactions went to completion rapidly. In the presence of phosphate, the back reaction was rapid but, in the light-induced reaction, three phases were discernible.
• 4) Compared with uncouplers such as NH₄Cl, carbonyl cyanide m-chlorophenyl-hydrazone, pentachlorophenol, and dicoumarol, all of which inhibited both photophosphorylation and conformational changes in chloroplasts, PMA (like quinacrine) had a specific action since it inhibited photophosphorylation while shrinkage was stimulated.
• 5) It appeared that PMA acted at a site beyond the formation of high energy inter-mediates and that, in the absence of photophosphorylation, more energy was diverted to mechanical work (shrinkage). It would seem that, in a cyclic electron flow system, in which ATP synthesis is blocked at a late step (e.g. by PMA), shrinkage may be an indirect method for measuring electron flow.

     

Photosynthetic Reactions by Lysed Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

Reactions of photosynthetic electron transport and photophosphorylation were studied in preparations from the blue-green alga, Phormidium luridum. Osmotic lysis of protoplasts proved to be a superior technique for the production of cell-free preparations with high enzymatic activity. Such lysed protoplasts sustain high rates of photophosphorylation coupled to the photo-reduction of NADP⁺ or ferricyanide. P/2e⁻ ratios close to unity were routinely observed. The same preparations, and also those prepared by grinding the cells in solutions containing sucrose or ethylene glycol, are active in cyclic photophosphorylation mediated by phenazine methosulfate or dichloro-phenolindophenol. The particles prepared by grinding the cells are, however, inactive in non-cyclic photophosphorylation.

Extensive washing of the membranes with solutions containing sucrose removes the majority of the residual soluble fraction of the algal cell which includes cytochromes C₅₅₄ and C₅₄₉ and phycocyanin. Cyclic photophosphorylation activity is unimpaired by this treatment, but is abolished when the membranes are washed with very dilute buffers. This activity is restored by the addition of a soluble protein which is not a known redox constituent such as cytochrome C₅₅₄ or plastocyanin, and may be a coupling factor.

Analysis of the well-washed membranes by low temperature (77°K) difference spectrophotometry reveals the presence of cytochrome b₆ and a bound form of cytochrome C₅₅₄ in proportions similar to that found in higher plant chloroplasts. The concentration of the membrane-bound cytochrome C₅₅₄, relative to cytochrome b₆ is not altered by extensive washing, sonication or treatment with 1% digitonin. This indicates that this cytochrome is an integral component of the cytoplasmic lamellae and we suggest that it is of functional significance. The soluble form of cytochrome C₅₅₄, which is present in concentrations about 3-fold higher than the bound form, depending upon growth conditions, is not essential for cyclic photophosphorylation. The concentration of cytochrome b₆: chlorophyll a was found to be 1:500.

Under the conditions employed, we were unable to detect a bound form of the low potential cytochrome C₅₄₉.

     

Inhibition of chloroplast photochemical reactions with 2-chloromercuri 4,6-dinitrophenol.

2-Chloromercuri 4,6-dinitrophenol inhibited photosystem I mediated photochemical reactions of Euphorbia hirta chloroplasts. The compound inhibited cyclic photophosphorylation and NADP reduction (in the presence of dichlorophenol indophenol and ascorbate couple) at concentrations as low as 10^?6m. At higher concentrations (above 10^?4m), however, it affected all NADP reductions but still showed negligible effect on ferricyanide reduction or noncyclic photophosphorylation. The compound may be used as an inhibitor of cyclic photophosphorylation.     

Uncoupling of photosynthetic phosphorylation by benzophenanthridine alkaloids

Sanguinarine, chelerythrine and chelidonine, benzophenanthridine alkaloids, inhibited both photosynthetic phosphorylation associated with ferricyanide reduction and cyclic photophosphorylation catalyzed by phenazine methosulphate. They did not affect electron transport in the presence of ADP and P_i and stimulated it in their absence. The inhibition of O₂ evolution by energy transfer inhibitors was reversed by the alkaloids. It is concluded that these alkaloids are uncouplers with the same efficiency in cyclic and non-cyclic photophosphorylation. This property might have some bearing in the physiological role of the alkaloids.     

Photosynthetic electron transport: Emergence of a concept, 1949–59

Historically, two main concepts guided research into possible mechanisms of light-induced atomic rearrangements in oxygenic photosynthesis: Photodecomposition of CO₂ and photodecomposition of water. Both concepts envisioned photoinduced transfers of cumbersome whole atoms and not, as is currently held, photoinduced electron transfers. Early proposals for light-induced electron transfers were relegated to obscurity because they were speculative ideas, not supported by meaningful experimental findings and tied to hypothetical and ephemeral schemes. The concept of photoinduced rearrangements of whole atoms rather than electrons was so well entrenched that it was even invoked to explain their findings by the discoverers of the Hill reaction and cyclic photophosphorylation. The light-induced electron flow concept gained acceptance in photosynthesis research only with the discovery of non-cyclic photophosphorylation in which ATP formation is coupled with electron transport to ferredoxin/NADP⁺ or to artificial substitutes like ferricyanide.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Photophosphorylation during Chloroplast Development in Red Kidney Bean: II. Photophosphorylation and Photoreduction Appear Concomitantly but Initially are Uncoupled

Cyclic phosphorylation with phenazine methosulfate and noncyclic phosphorylation and reduction with ferricyanide were detected in isolated chloroplasts from greening bean leaves after 3 to 4 hours of illumination. Activity commenced when rapid synthesis of chlorophyll was initiated. Rates of photophosphorylation were comparable to mature levels by 15 to 18 hours of development. Photoreduction of ferricyanide attained a peak value by 12 hours of illumination and subsequently fell to normal levels by 15 to 18 hours. With ferricyanide, the P/e₂ ratios were initially less than 0.1 but were close to 1.0 after 18 hours of illumination. The data suggested that photosystems I and II appeared concomitantly in the chloroplast but were not fully operative until later in development. Proplastids and immature chloroplasts exhibited high capacities to reduce ferricyanide in the dark. The rates of dark reduction rapidly diminished to low levels by 15 hours of illumination when normal rates of photochemical activity were observed. After a 2-to 3-day lag, a rapid increase in leaf fresh weight was noted at the time total chlorophyll content reached steady state values on a fresh weight basis. With fresh weight as an index of growth, primary leaves completed their development after 6 to 7 days of illumination.     

Changes in the Ability of Photophosphorylation and Activities of Surface-Bound Adenosine Triphosphatase and Ribulose Diphosphate Carboxylase of Chloroplasts Isolated from the Barley Leaves Senescing in Darkness

Dark-induced aging of detached primary leaves of 11-day-old barley seedlings brings about a significant decline in the rates of ferricyanide [Fe(CN)₆]^3? reduction and photophosphorylations of isolated chloroplasts. Ferricyanide-supported noncyclic photophosphorylation is somewhat more susceptible to leaf aging than phenazine methosulfate (PMS)-supported cyclic phosphorylation. Non-latent membrane-bound adenosine triphosphatase (ATPase) and ribulosediphosphate carboxylase (RuDPCase) lose about half of their initial activities after 24 h, while during this period the electron transport and photophosphorylation activities are much less affected. Also, the loss of RuDPCase is almost complete, while chloroplasts still exhibit a significant level of [Fe(CN)₆]^3? reduction and photophosphorylations after 7 days of dark incubation. This would suggest that the enzymatic dark reactions are more sensitive to aging stress than the primary photochemical reactions of chloroplasts. Studies on the effect of divalent cations such as Mg²⁺ and Ca²⁺ on non-latent ATPase activity revealed that the dark stressed aging of detached leaves brings about a time dependent alteration in the response of this enzyme to Mg²⁺, but not to Ca²⁺. The former showed inhibitory as well as stimulatory response, whereas the latter always caused the usual stimulation. Addition of kinetin (50 μM) ensured retention of [Fe(CN)₆]^3? reduction, photophosphorylations and RuDPCase activity in chloroplasts during leaf aging, but it failed to preserve the initial loss in the activity of the non-activated membrane-bound ATPase.     

Photophosphorylation in isolated maize bundle sheath chloroplasts and cells

Isolated maize bundle sheath chloroplasts showed substantial rates of noncyclic photophosphorylation. A typical rate of phosphorylation coupled to whole-chain electron transport (methylviologen or ferricyanide as acceptor) was 60 μmol per hour per milligram chlorophyll) with a coupling efficiency (P/e₂) of 0.6. Partial electron transport reactions driven by photosystem I or II supported phosphorylation with P/e₂ values of 0.2 to 0.3. Thus, two sites of phosphorylation seem to be associated with the photosynthetic chain in much the same way as in spinach chloroplasts.     

Localization of the reaction site of cytochrome 552 in chloroplasts from Euglena gracilis. Cytochrome content and photooxidation in different chloroplast preparations

Isolated Euglena chloroplasts retain up to 50% of cytochrome 552 on a chlorophyll basis compared to the content of cells. Cytochrome 563 is found in equal amount in chloroplasts and cells. The amount of cytochrome 552 retained depends on the isolation procedure of chloroplasts.Cytochrome 552 can be further liberated from chloroplasts by mechanical treatment or incubation with detergent. It is concluded that cytochrome 552 is not tightly bound in the membrane but rather trapped in the thylakoids of the chloroplasts.In photosynthetic electron flow, cytochrome 552 is functioning as donor for photosystem I, mediating electron flow from cytochrome 558 to P₇₀₀ under our conditions.Antimycin A stimulates the photooxidation of cytochrome 552 and of cytochrome 558.The rates of electron flow from water to NADP⁺ and of cyclic photophosphorylation mediated by phenazine methosulfate correlate with the content of endogenous cytochrome 552 in the chloroplasts. External readdition of cytochrome 552 to deficient chloroplasts causes reconstitution of NADP⁺ reduction but not of cyclic photophosphorylation. Mechanical treatment or other means of fragmentation of chloroplasts results in the exposure of originally buried reaction sites for external cytochrome 552.     

Partial Reactions of Photosynthesis in Briefly Sonicated Chlamydomonas: II. Photophosphorylation Activities

Briefly sonicated Chlamydomonas reinhardi cells are capable of both cyclic and noncyclic photophosphorylation and, in each case, the maximum rates approach those reported for higher plant chloroplasts. Photophosphorylation coupled to ferricyanide reduction occurs with a P/2e ratio approaching unity.     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O₂ evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Effects of Manganese Deficiency and Added Cerium on Photochemical Efficiency of Maize Chloroplasts

The mechanism of the fact that manganese deprivation and cerium addition affect the photochemical efficiency of plants is unclear. In this study, we investigated the improvement by cerium of the damage of the photochemical function of maize chloroplasts under manganese-deprived stress. Chlorophyll fluorescence induction measurements showed that the ratio of variable to maximum fluorescence (Fv/Fm) underwent great decreases under manganese deficiency, which was attributed to the reduction of intrinsic quantum efficiency of the photosystem II units. The electron flow between the two photosystems, activities of Mg²⁺–ATPase and Ca²⁺–ATPase, and rate of photophosphorylation on the thylakoid membrane of maize chloroplasts were reduced significantly by exposure to manganese deprivation. Furthermore, the inhibition of cyclic photophosphorylation was more severe than non-cyclic photophosphorylation under manganese deficiency. However, added cerium could relieve the inhibition of the photochemical reaction caused by manganese deprivation in maize chloroplasts. It implied that manganese deprivation could disturb photochemical reaction of chloroplasts strongly, which could be improved by cerium addition.     

Modification by gibberellin of the growth-temperature relationship in mutant and normal genotypes of several cereals

High-resolution growth measurements were conducted using a linear variable displacement transformer in conjunction with a temperature-programmed meristem-cooling collar. Chilling and rewarming profiles were determined for a range of Gramineae, in the presence and absence of varying concentrations of gibberellic acid (GA₃). In wheat (Triticum aestivum L.) seedlings, the growth-constraining temperature (P_e) was progressively lowered by increasing GA₃ concentration, with a difference of-4.8°C between controls and material treated with 10^–4 M GA₃. Dwarf-5 maize (Zea mays L.) seedlings had a higher P_e than tall segregates and the difference was markedly reduced by exposure to a saturating concentration of GA₃. A similar effect was observed with Tanginbozu dwarf rice (Oryza sativa L.). The growth ratetemperature responses of Rht3 gibberellin-insensitive dwarf wheat seedlings were unaffected by GA₃ and the P_e values for these segregates were around 5° C higher than for normals. Slender (s1) barley (Hordeum vulgare L.) genotypes had P_e values of-7° C, compared with +4° C for wild-type material, and did not show positive hysteresis for growth rate during the rewarming phase. These studies indicate that GA₃ modifies the thermal sensitivity of meristem function in Gramineae in a manner which enhances low-temperature growth.Abbreviations GA gibberellin - GA₃ gibberellic acid - LVDT linear variable displacement transducer     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of Photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg²⁺. The reaction is inhibited by (i) an antibody to ferredoxin-NADP⁺ reductase (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q.It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Photosynthetic Activity of Chloroplasts Isolated From Bermudagrass (Cynodon dactylon L.), a Species With a High Photosynthetic Capacity

Chloroplasts have been isolated from bermudagrass (Cynodon dactylon L.) leaves and assayed for photophosphorylation and electron transport activity. These chloroplasts actively synthesize adenosine triphosphate during cyclic electron flow with phenazine methosulfate and noncyclic electron flow concurrent with the reduction of such Hill oxidants as nicotinamide adenosine dinucleotide phosphate, cytochrome c, and ferricyanide. Apparent Km values for the cofactors of photophosphorylation have been determined to be 5 × 10⁻⁵ M for phosphate and 2.5 × 10⁻⁵ M for adenosine diphosphate. The influence of light intensity on photophosphorylation has been studied and the molar ratio of cyclic to noncyclic phosphorylation calculated. It is concluded that the high photosynthetic capacity of bermudagrass leaves probably could be supported by the photophosphorylation capacities indicated in these chloroplast studies and the anomalous lack of data in chlorolast studies on the production of sufficient reductant for CO₂ assimilation at high light intensities has been noted.     

Comparison of endogenous gibberellins and of the fate of applied radioactive gibberellin a(1) in a normal and a dwarf strain of Japanese morning glory

The effect of application of GA₃ on hypocotyl growth, the endogenous GAs, and the metabolism of applied ³H-GA₁ were investigated in relation to dwarfism and light-mediated growth inhibition in the normal (tall) strain Violet and the dwarf strain Kidachi of Japanese morning glory (Pharbitis nil). GA₃ applied in a wide concentration range (10⁻⁹ to 10⁻³m) to 4-day-old seedlings caused great extension of the hypocotyls in light-grown plants of both the normal and the dwarf strain. However, the dwarf strain did not attain the same length as the normal one at any given GA₃ concentration, even when saturation was reached. Dark-grown plants of the dwarf strain responded to GA₃, although relatively much less than light-grown ones; dark-grown plants of the normal strain showed no GA₃ response at all.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Inhibition of photosynthetic electron transport by DDT and DDE

The effect of DDT and DDE (a metabolite of DDT) on chloroplast electron transport was investigated. Photosynthetic electron transport in isolated spinach and barley chloroplasts as well as chloroplasts isolated from macroscopic green algae,Cdium fragile andChaetomorpha aerea, was inhibited by both compounds. Photoreduction and photophosphorylation measured in the presence of ferricyanide showed 50% inhibition at 2×10^–5 M DDT and DDE. P/2e ratios were 1·2–1·5, and remained constant in the presence of both inhibitors. The addition of uncouplers such as ammonium ion and carbonyl cyanide,m-chlorophenylhydrazone did not overcome the inhibition of the chlorinated hydrocarbons. Inhibition of phenazine methosulfate-catalyzed cyclic photophosphorylation by DDT and DDE was observed at low light intensities but was not seen at 2·5×10⁵ erg cm^–2sec^–1 and above. In the presence of DDT, a slow rise in measuring beam fluorescence was observed. The actinic beam fluorescence was slightly less than that in the control. Inhibition by DDT and DDE appears to be similar to that of DCMU. Brief sonication of the chloroplasts increases the sensitivity to DDT. The lack of penetration of DDT to terrestrial plant chloroplasts may be the reason why these are protected from this insecticide.