Chloroplastic activity in response to gibberellic acid treatment of dwarf and normal cultivars of pea (Pisum sativum L.)

Levels of ferricyanide reduction, cyclic and non-cyclic photophosphorylation were measured in chloroplasts of two cultivars of pea and a comparison of their P/2e⁺ ratios were made. No differences were observed in cyclic photophosphorylation or ferricyanide reduction but non-cyclic photophosphorylation was lower in chloroplasts from the dwarf than the normal cultivar. Thus the P/2e⁺ ratio of the dwarf was lower than the normal. Dwarf seedlings treated with gibberellic acid (GA₃) had similar rates of cyclic photophosphorylation as the untreated dwarf but non-cyclic photophosphorylation was lower as was ferricyanide reduction. This resulted in P/2e⁺ ratios that were higher in chloroplasts from the GA₃ treated dwarf seedlings than the untreated, and were the same as the untreated normal. Addition of GA₃ directly to the chloroplasts did not alter the activity in any way. Hence gibberellins do not directly affect changes in chloroplastic activity but may conceivably be involved in a feed-back control system.     

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.     

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.     

The relationship of cyclic and non-cyclic electron flow patterns with reduced indophenols to photophosphorylation

Optimal cyclic photophosphorylation with reduced indophenols under anaerobic conditions was shown to require a critical redox balance. Over-reduction inhibited this phosphorylation; addition of oxidizing agents like ferricyanide, air, ferredoxin or ferredoxin plus triphosphopyridine nucleotide relieved the inhibition.

When ascorbate and indophenol served as the electron donor couple for TPN⁺ reduction, only the amount of TPNH formed was dependent on the concentration of TPN⁺. The phosphorylation observed in this system was dependent only on the concentration of indophenol, and on the ability of reduced indophenol to mediate cyclic photophosphorylation. The cyclic electron flow with reduced indophenol was shown to operate simultaneously with the non-cyclic electron flow to TPN⁺. It was concluded that there was no phosphorylation site in the non-cyclic electron flow between ascorbate-indophenol and TPN⁺ and that the phosphorylation observed in this case was due only to cyclic photophosphorylation with the reduced indophenols.

In the light of these results, a working hypothesis with two different sites for cyclic and non-cyclic photophosphorylation is suggested.     

Regulation of photosynthetic electron transport and photophosphorylation in intact chloroplasts and leaves of Spinacia oleracea L.

Oxygen ist reduced by the electron transport chain of chloroplasts during CO₂ reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO₂-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO₂ reduction by electron flow to NADP and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.     

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM.     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

Specific inhibition of photosystem II activity in chloroplasts by fumigation of spinach leaves with SO2

The phytotoxic effects of sulfur dioxide (SO₂) were investigatedby fumigating spinach plants with SO₂. Inhibition of 2,6-dichloroindophenol(DCIP) photoreduction was observed in spinach chloroplasts isolatedfrom fumigated leaves. NADP and DCIP photoreductions were inhibitedto a similar extent by fumigation with 2.0 ppm SO₂ but electronflow from reduced DCIP to NADP was not affected. When electronflow from H₂O to NADP was inhibited by 36%, a 39% inhibitionof non-cyclic photophosphorylation was observed. However, phenazinemethosulfate(PMS)-catalyzed cyclic photophosphorylation wasas active as in the control chloroplasts. Moreover, in the presenceof PMS, no significant suppression was observed in the extentof light-induced H⁺ uptake or in the rate of H⁺ efflux in chloroplasts.From these results, it can be concluded that SO₂ inhibits theelectron flow driven by photosystem II when plants have beenfumigated with SO₂. In spinach leaves fumigated with SO₂, the rate of photosyntheticO₂ evolution was reduced under light-limited conditions, whilethe rate of respiratory O₂ uptake changed slightly. (Received February 8, 1979; )     

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.     

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.

     

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.     

Inhibition of photophosphorylation by kaempferol

Kaempferol, a naturally occurring flavonol, inhibited coupled electron transport and both cyclic and noncyclic photophosphorylation in isolated pea (Pisum sativum) chloroplasts. Over a concentration range which gave marked inhibition of ATP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids. It is suggested that kaempferol acts as an energy transfer inhibitor.     

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.

Photophosphorylation in Scenedesmus in vivo: O2 evolution, ATP pools and transients, and phosphate binding during photoreduction of NO3−, NO2− and CO2

The relation between light-induced electron transport with NO₃^?, NO₂^? or CO₂ as acceptors, ATP pools and transients in dark-light-dark transitions, and phosphate uptake was examined in phosphorus-starved cells of Scenedesmus obtusiusculus Chod. Net O₂ evolution at saturating light was around 6 μmol × (mg chlorophyll × h)^?1 in the absence of any acceptor, but reached average rates of 21, 65 and 145 μmol × (mg chlorophyll × h)^?1 upon additions of 5 mM KNO₃, KNO₂ and KHCO₃, respectively. The apparent rate of photophosphorylation in transition experiments was only a few percent of the rate calculated from CO₂-dependent O₂ evolution. Blocking non-cyclic electron transport with DCMU inhibited phosphate assimilation, but acceleration of non-cyclic electron flow by addition of NO₃^? or NO₂^? did not stimulate phosphate assimilation as compared to the situation without an acceptor. A functional non-cyclic system might primarily be needed for an efficient shuttle transfer of ATP from the chloroplast to the cytoplasm. An inhibition of the non-cyclic system due to lack of reducible substrates accelerates the cyclic system and thus indicates a regulation mechanism between the two systems.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the $\text{P}\text{2e}$ ratios remain unaltered.In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

Stimulation of photosynthesis by carbonyl compounds and chelators

A number of carbonyl compounds including bicarbonate, ethylene carbonate, dimethylcarbonate, propylene carbonate, bis-pentamethylene urea, and glycidol, and several chelators were tested for their effect on photosynthetic reactions in isolated spinach chloroplasts. It was found that carbonyl compounds inhibited the DCMU-insensitive silicomolybdate reduction by photosystem II but stimulated the O₂ evolution associated with ferricyanide reduction in presence of DBMIB and the H₂O→methylviologen reaction. Many chelators behaved in the same manner except 1,10-phenanthroline which shows the opposite effect. The carbonyl compounds did not uncouple because they stimulated the proton gradients associated with noncyclic photophosphorylation, whereas some chelators, such as bathocuproine or bathophenanthroline inhibited the proton gradients 100%. Electron transport in presence of ADP and inorganic phosphate showed a stimulation of rates beyond that obtained in presence of an uncoupler. The data are discussed in terms of inhibition of cyclic electron flow around PS II which leads to increased electron transport rates toward PS I.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation.

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

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.     

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.