Characterization of Chloroplasts Isolated from Triazine-Susceptible and Triazine-Resistant Biotypes of Brassica campestris L

Chloroplasts isolated from triazine-susceptible and triazine-resistant biotypes of Brassica campestris L. were analyzed for lipid composition, ultrastructure, and relative quantum requirements of photosynthesis. In general, phospholipids, but not glycolipids in chloroplasts from the triazine-resistant biotype had a higher linolenic acid concentration and lower levels of oleic and linoleic fatty acids, than chloroplasts from triazine-susceptible plants. Chloroplasts from the triazine-resistant biotype had a 1.6-fold higher concentration of t-Δ3-hexadecenoic acid with a concomitantly lower palmitic acid concentration in phosphatidylglycerol. Phosphatidylglycerol previously has been hypothesized to be a boundary lipid for photosystem II. Chloroplasts from the triazine-resistant biotype had a lower chlorophyll a/b ratio and exhibited increased grana stacking. Light-saturation curves revealed that the relative quantum requirement for whole chain electron transport at limiting light intensities was lower for the susceptible biotype than for the triazine-resistant biotype. Although the level of the chlorophyll a/b light-harvesting complex associated with photosystem II was greater in resistant biotypes, the increased levels of the light-harvesting complex did not increase the photosynthetic efficiency enough to overcome the rate limitation that is inherited concomitantly with the modification of the Striazine binding site.     

Manganese binding to sodium cyanide-treated chloroplasts: Effects of light and redox-potentials on the binding

Mn²⁺-binding to Mn-depleted chloroplasts by the treatment withcyanide was inhibited by light. Quantitative study indicatesthat only the binding with a high affinity constant was inhibitedby light. This photoinhibition required electron transport activityand disappeared on addition of 2,6-dichlorophenolindophenol.The binding of Mn²⁺ was also inhibited by addition of reductantin the dark. The high affinity binding at various redox potentialsindicates the participation of a two electrons-transfer componenthaving a midpoint potential of 336 mV. Mn²⁺-binding was decreasedon treatment of chloroplasts with proteolytic enzyme in thedark but not in the light. We propose that the high affinitybinding site is concealed in the membrane matrix of chloroplastsat low oxidation-reduction potential. (Received December 14, 1976; )     

Photosynthetic Properties of Green Barley Leaves after Treatment with Pyridazinone Herbicides--Comparison with the Effects of Diuron

Photosynthetic characteristics of detached green barley leavesafter 72 h of treatment with 0·2 mol m^–3 of thepyridazinone herbicides SAN 6706, SAN 9785 and SAN 9789 werestudied. For comparison, the effects of 0·01 mol m^–3diuron were also investigated. Pyridazinone herbicides causedonly a slight reduction of the total carotene content of thebarley leaves. The total chlorophyll content, as well as thelinolenic to linoleic acid ratio of chloroplast glycerolipids,however, remained unchanged. Diuron treatment caused total inhibitionof electron transport, as revealed by fast fluorescence inductionof leaves and the Hill reaction activity of chloroplasts. The¹⁴CO₂-nxation by the leaves and the light-induced fluorescencequenching were also completely inhibited in vivo by diuron.Pyridazinone herbicides left 20–40% of the ¹⁴CO₂-fixationfound in the control, in spite of the fact that their fast fluorescenceinduction tracings showed inhibition in the electron transport.Chloroplasts isolated from the leaves treated with pyridazinoneswere found to be highly active in mediating the ferricyanide-dependentHill reaction. In order to test the ability of pyridazinonesto inhibit photosynthetic electron transport in vivo, their‘prompt’ effect on fluorescence was also investigated.It is concluded that pyridazinone herbicides can readily andrapidly enter the chloroplasts and inhibit the photosyntheticelectron transport in vivo. The differences between the long-termeffects of pyridazinones and those of diuron suggest differencesin the inhibitory effectiveness on the various photosyntheticparameters between the two herbicide groups. It is suggestedthat pyridazinones can leave the chloroplasts during isolationowing to the loose binding onto the thylakoid membranes. Key words: Pyridazinone herbicides, electron transport, fluorescence induction     

Lipids of Dark-Treated Oat (Avena sativa) Leaf Chloroplast Thylakoids

The lipid content of thylakoids from dark-treated oat plantswas studied. The levels of chlorophyll, monogalactosyl diacylglycerol²,digalactosyl diacylglycerol, and the phospholipids all declined.The lipids declined more rapidly than the chlorophyll. As thequantity of the galactolipids declined, there was little changein the relative concentration of linolenic acid esterified tothem. In contrast, the relative concentration of linolenic acidesterified to the phospholipids increased as the quantity oflipid declined. The total fatty acid composition and the incorporationof label into thylakoid lipids dropped following the dark treatment.     

Photosynthetic apparatus in chilling-sensitive plants

The levels of both tightly and loosely bound Mn in chloroplasts from fresh, cold and dark stored as well as illuminated leaves of Lycopersicon esculentum Mill. were studied in relation to Hill reaction activity. The tightly bound Mn pool represents one third of the total Mn content in chloroplasts isolated from the fresh leaves, and its level does not change following cold storage and illumination of leaves. Upon cold and dark storage of leaves the loss from the chloroplasts of about 40%–50% of the total amount of Mn is accompanied by an almost complete inactivation of the Hill reactions, as studied with water as an electron donor, as well as by the appearance of an EPR signal characteristic of free Mn²⁺ ions. Following illumination of such leaves, the restroration of Hill reaction activity is accompanied by an increase in the total Mn content in chloroplasts of up to 70%–80% of the Mn level measured in the fresh leaves and by disappearance of the EPR signal. In contrast, aging in the cold of isolated chloroplasts does not affect their Mn content. The addition of manganese does not result in the restoration of Hill reaction activity in chloroplasts from cold stored leaves but causes a restoration of this activity inhibited by linolenic acid. The data suggest that the loosely bound Mn pool (extractable with Tris) can be differentiated into two fractions: (1) one functionally inactive in electron transport and (2) one responsible for restoration of Hill reaction activity. Mn of the latter fraction (about 45% of the total Mn content) probably originates from the free Mn ions present in the interior of the chloroplasts following the cold and dark storage of leaves and from Mn reincorporated into chloroplasts from the cytoplasm. Incorporation of Mn from both these sources into thylakoid membrane to form a functionally active, loosely bound Mn pool proceeds during the illumination of leaves and results in the restoration of Hill reaction activity inhibited following the storage of leaves in dark and cold.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - Diquat 1,1-ethylene 2,2-dipiridylium dibromide - EPR electron paramagnetic resonance - FFA free fatty acid - MV methyl viologen, N,N-dimethyl-4,4 dipyridyldihydrochloride - Tris tris-(hydroxymethyl) aminomethane     

The effects of 2, 4-dichIorophenoxyacetk acid altered chloroplast development on photosynthesis

We studied the changes in function and physical properties of isolated radish ( Raphonus sativus L. cv. Sparkler) lamellar membranes 48 h after chloroplast development was altered by 2, 4-(dichlorophenoxy)acet, tc acid. The number of chlorophyll molecules attendant to each electron transport chain was approximately 25% less in the chloroplasts from 2, 4-(dichlorophenoxy)acetic acid-treated plants than in chloroplasts from untreated plants. The maximal turnover rate of Photosystem I] in the treated chloroplasts was slightly less than half the turnover rate in normal chloroplasts. The efficiency of coupling between electron flux and ATP formation was not significantly different in the two chloroplast types. This hight efficiency of photophosphorylation in addition to normal membrane conductance to hydrogen ions indicates that the herbicide has not brought about a general deterioration of the membrane. A dramatic increase in the proton binding capacity of the lamellar membrane was observed in the treated chloroplasts. This increase in hydrogen ion buffering groups was largely accounted for by extrinsic membrane proteins bound to the exterior surface of the lamellar membrane. Although the addition of 2, 4-(dichloro-phenoxy) acetic acid to chloroplasts isolated from untreated plants caused concurrent uncoupling of ATP formation and inhibition of electron transport, our data show that these direct effects of the compound have little to do with its herbicidal action.     

Nano-Anatase Relieves the Inhibition of Electron Transport Caused by Linolenic Acid in Chloroplasts of Spinach

Linolenic acid is an inhibitor of electron transport in chloroplasts of higher plants. It has obvious effects on the structure and function of chloroplasts. In the present paper, we investigated the nano-anatase relieving the inhibition of photoreduction activity and oxygen evolution caused by linolenic acid in spinach chloroplasts. The results showed that linolenic acid in various concentrations could obviously reduce the whole chain electron transport and the photoreduction activity of two photosystems, especially on the oxidative reside and reduce reside of photosystem II (PS II). After adding nano-anatase to chloroplasts treated by linolenic acid, the whole chain electron transport rate, the photoreduction activity of two photosystems, and the oxygen evolution rate were increased significantly, indicating that nano-anatase could obviously decrease the inhibition of linolenic acid on the electron transport, photoreduction activity, and oxygen evolution of spinach chloroplasts.     

Fatty-acid-induced release of manganes from chloroplasts

The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio (w/w, 2:1–10:1). The efficiency of C₁₈ unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C_18:3 > C_18:2 > C_18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.     

Effect of water stress on the lipid and fatty acid composition of cotton (Gossypium hirsutum) chloroplasts

In order to investigate the effects of water stress on the photosynthetic compartment, analysis of chloroplast polar lipids and their fatty acid composition was made. Cotton ( Gossypium hirsutum L. cv. Reba) plants were submitted to water stress by witholding irrigation. Chloroplasts were purified on a Percoll gradient and three fractions were collected: intact, apparently intact and broken chloroplasts. The percentage of broken chloroplasts increased with a decrease in leaf water potential, indicating a greater weakness of the membrane. Galactolipid content (expressed as mg lipid per mg chlorophyll), particularly digalactosyldiglyceride, decreased with decreasing water potential. Phospholipid content decreased in the broken chloroplast fraction. The fatty acid composition of chloroplasts was also affected. The perecentage of linolenic acid (18:3), the major fatty acid of thylakoids, decreased, whereas that of linoleic acid(18:2) and oliec acid (18:1) increased. An accumulation of fatty acids having less than 16 carbon atoms was also observed. These changes in the lipid and fatty acid composition of cotton chloroplasts under water stress might affect the properties of the thylakoid membrane and thereby the photosynthetic activity and the compartmentation of the leaf cells.     

Effect of light on lipid composition of Ricinus cell cultures

The changes of lipid composition were determined in callus cultures from Ricinus communis endosperm upon transfer from darkness into light. Culture in light induced chlorophyll synthesis and formation of differentiated chloroplasts. In light-grown cultures the major lipid classes were phospho- and glycolipids, dark-grown cultures were rich in triacylglycerol. The major fatty acids were linolenic acid and palmitic acid in both cultures. In the green cultures linolenic acid was predominantly esterified in glycolipids whereas in the dark-grown cultures this fatty acid was the major component of phospholipids. Ricinoleic acid was not found.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - MGD monogalactosyldigylceride - DGD digalactosyldiglyceride - SE steryl esters - NL neutral lipids     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Role of pulvinar chloroplasts in light-driven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.)

Laminar pulvini of bean (Phaseolus vulgaris L.) contain numerouschloroplasts in cells of their motor tissue. The quantitativerelationships of the chloroplast pigments, chlorophyll a andb, ß-carotene, lutein, neoxanthin as well as the xanthophyllcycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin)were similar to those of mesophyll chloroplasts from leafletlaminae. Exposure of pulvinules to light caused deepoxidationof violaxanthin to zeaxanthin, showing that the xanthophyllcycle is functioning. Chlorophyll fluorescence analysis of pulvinulesconfirmed that their chloroplasts are capable of both photosyntheticelectron transport and non-photochemical fluorescence quenching,showing that they build up a considerable transthylakoid protongradient in the light. Application of DCMU to excised pulvinulesand laminar discs, as well as to pulvinules of intact, attachedterminal leaflets blocked electron transport and fluorescencequenching. Application of the uncoupler CCCP to intact pulvinulesalso prevented non-photochemical fluorescence quenching. Therate of movement of the low-light-adapted terminal leaflet inresponse to exposure of its pulvinule to overhead red light(500 µmol m^–2 s^–1) was reduced when the pulvinulewas pretreated with DCMU. The pulvinar response to overheadblue light (50 µmol ^–2 s^–1), which is morepronounced than to red light, was not affected by similar pretreatmentwith DCMU. Pretreatment with CCCP caused a short lag in theresponse to red light, but did not affect its subsequent rate.The results suggest that the pulvinar response to red, but notto blue light, requires non-cyclic electron transport and theresulting generation of ATP Key words: Leaf movements, light, non-cyclic electron transport, Phaseolus, pulvinar chloroplasts     

Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

Restoration by silicotungstic acid of DCMU-inhibited photoreactions in spinach chloroplasts

The restoration by silicotungstic acid of the reversible light-induced pH rise mediated by pyocyanine in EDTA-treated chloroplasts corresponds to an irreversible fixation of the acid. The proton uptake is linearly related to the amount of fixed acid (4 protons per molecule of acid) as long as the amount of silicotungstic acid does not exceed 200 nmoles/mg of chlorophyll.In the same conditions silicotungstic acid partly restores ferricyanide reduction and O₂ evolution in chloroplasts suspensions supplemented with DCMU. These photoreactions are observed only with chloroplasts and these chloroplasts must have an unimpaired water-splitting mechanism.Silicotungstic acid does not impair DCMU fixation on the specific sites. More likely in its presence the properties of the membrane change and ferricyanide can accept electrons from a part of the electron transport chain, between the Photosystem II reaction center and the block of the electron flow by DCMU.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Proteomic comparison of etioplast and chloroplast protein complexes

Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed.     

水杨酸对高温强光胁迫下小麦叶绿体D1蛋白磷酸化及光系统Ⅱ功能的影响

为了研究水杨酸（SA）对高温强光胁迫下小麦叶片类囊体膜D₁蛋白磷酸化和PSⅡ功能的影响,用0.5 mmol·L^-1 SA溶液预处理灌浆期小麦叶片,以水预处理为对照,然后将预处理植株进行高温强光（35 ℃,1 600 μmol·m^-2·s^-1）处理,测定胁迫处理过程中小麦旗叶光合电子传递速率、净光合速率、叶绿素荧光参数及D₁蛋白的变化.结果表明：SA预处理有效抑制了高温强光下D₁蛋白的净降解,保持了较高的D₁蛋白磷酸化水平、全链电子传递速率和PSⅡ电子传递速率,维持了较高的PSⅡ原初光化学效率（F_v/F_m）、实际光化学效率(Ф_PSⅡ)、光化学淬灭系数（q_P）和净光合速率（P_n）.表明外源SA通过调节小麦叶绿体D₁蛋白的周转,减轻了高温强光胁迫对叶片光合机构的损伤,有利于PSⅡ的正常运转.     

Photosynthetic apparatus in chilling-sensitive plants

The composition of free fatty acids (FFA) in relation to Hill reaction activity and photoperoxidation of lipids was studied in chloroplasts isolated from fresh, cold and dark-stored as well as illuminated leaves of Lycopersicon esculentum Mill., Phaseolus vulgaris L. and Cucumis sativus L. Following the cold and dark-storage of leaves the loss of Hill reaction activity is accompanied by approximately a 5-fold increase in the amount of FFA and by an increase in the percentage of unsaturated FFA, particularly that of linolenic acid. Illumination of the cold- and dark-stored leaves restores both Hill reaction activity and the content and composition of chloroplast FFA. Following the second and third cycles of cold storage and illumination of leaves the percentage of unsaturated fatty acids in chloroplasts increases while that of saturated ones decreases despite of the significant restoration of Hill reaction activity. Since the illumination of cold-stored leaves results in peroxidation of inhibitory fatty acids it seems likely that this phenomenon could, at least partially, be responsible for the restoration of Hill reaction activity. Inhibition of Hill reaction activity by exogenous linolenic acid in chloroplasts of fresh, cold-stored as well as cold-stored and illuminated leaves could be reversed following the incubation of chloroplast suspension with BSA, however only to a value measured in the absence of unsaturated fatty acid. All these results indicate that the inhibition of Hill reaction activity due to the cold and dark storage of leaves is caused by both inhibitory FFA released from chloroplast lipids as well as by damage to the thylakoid structure affecting the electron transport within photosystem II.Abbreviations BSA bovine serum albumin - DCIP 2,6-dichlorophenolindophenol - DGDG digalactosyl diglyceride - HEPES 2-(4(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - FFA free fatty acids - MDA malondialdehyde - MGDG monogalactosyldiglyceride - TBA thiobarbituric acid - Tris tris-(Hydroxymethyl)aminomethane     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)