Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO₂-fixation was initiated by oxygen concentrations as low as about 10 μM. Once CO₂ fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO₂ reduction.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Electron acceptors in isolated intact spinach chloroplasts act hierarchically to prevent over-reduction and competition for electrons

Electron fluxes in isolated intact spinach chloroplasts were analyzed under saturating light and under optimal CO₂ and P_i supply. When CO₂ assimilation was the only ATP- and NADPH-consuming reaction, the ΔpH decreased and the chloroplasts showed clear evidence of over-reduction. This suggested that additional electron flow is required in order to maintain the ΔpH and the stromal NADPH/ATP ratio. The additional electron flow may be cyclic electron transport around Photosystem I and linear electron transport towards either oxaloacetate or O₂. The contributions of, and the interrelationships between, these three electron transfer pathways were analyzed by following the reactions of chloroplasts in their presence or absence, and by monitoring to what extent they were able to compensate for each other. Inhibition of cyclic electron flow by antimycin A caused strong over-reduction and decreased the ΔpH. Only oxaloacetate, but not O₂, was able to restore photosynthesis. In the presence of H₂O₂, there was a rapid build-up of a high ΔpH, and the reduction of any other electron acceptor was prevented. It is concluded that the different electron acceptors in the stroma are organized in a hierarchical manner; this allows electron flux towards CO₂ and nitrite reduction to proceed without any competition for electrons, and any excess electrons to be taken by these additional non-assimilatory pathways. Hence, the ΔpH is maintained at the required level and over-reduction of the electron transport chain and the stromal redox components is avoided. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO2-fixation was initiated by oxygen concentrations as low as about 10 microM. Once CO2 fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO2 reduction.     

Photorespiration is More Effective than the Mehler Reaction in Protecting the Photosynthetic Apparatus against Photoinhibition

I. Isolated intact chloroplasts: Photosystem II, but not photosystem I, of the electron transport chain is rapidly photoinactivated even by very low intensities of red light when no large proton gradient can be formed and the electron transport chain becomes over-reduced in the absence of oxygen and other reducable substrates. Electron acceptors including oxygen provide protection against photoinactivation. Nevertheless, photosystem II is rapidly, and photosystem I more slowly, photoinactivated by high intensities of red light when oxygen is the only electron acceptor available. Increased damage is observed at increased oxygen concentrations although catalase is added to destroy H₂O₂ formed during oxygen reduction in the Mehler reaction. Photoinactivation can be decreased, but not prevented by ascorbate which reduces hydrogen peroxide inside the chloroplasts and increases coupled electron flow. II. Leaves: Simple measurements of chlorophyll fluorescence permit assessment of damage to photosystem II after exposure of leaves to high intensity illumination. In contrast to isolated chloroplasts, chloroplasts suffer more damage in situ at reduced than at elevated oxygen concentrations. The difference in the responses is due to photorespiration which is active in leaves, but not in isolated chloroplasts. After photosynthesis and photorespiration are inhibited by feeding glyceraldehyde to leaves, photoinactivation is markedly increased, although oxygen reduction in the Mehler reaction is not affected by glyceraldehyde. In the presence of reduced CO₂ levels, photorespiratory reactions, but not the Mehler reaction, can prevent the overreduction of the electron transport chain. Over-reduction indicates ineffective control of photosystem II activity. Effective control is needed for protection of the electron transport chain against photoinactivation. It is suggested to be made possible by coupled cyclic electron flow around photosystem I which is facilitated by the redox poising resulting from the interplay between photorespiratory carbohydrate oxidation and the refixation of evolved CO₂.     

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping

The role of cyclic electron transport has been re-examined in leaves of C₃ plants because the bioenergetics of chloroplasts (H⁺/e = 3 in the presence of a Q-cycle; H⁺/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C₃ photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700⁺ in the dark. At low CO₂ and O₂ molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in F_v/F_m brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO₂-dependent O₂ evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease F_v/F_m. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H⁺/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999     

Nitratabhängige nichtcyclische Photophosphorylierung bei Ankistrodesmus braunii in Abwesenheit von CO2 und O2

Summary Manometric measurements show that oxygen evolution proceeds in synchronised cells of Ankistrodesmus braunii even in an atmosphere of pure nitrogen. In this case the slow oxygen evolution is dependent on the presence of nitrate (Table 1). Light saturation is found at a low light intensity at pH 5.6, at a higher light intensity at pH 8.0 (Fig. 1). The light saturation curves are in good agreement with those of ³²P-labelling in Ankistrodesmus under the same conditions (Fig. 2).DCMU inhibition in N₂ of both O₂-evolution and ³²P-labelling begins only at a DCMU concentration of 5×10^-7M or more. Complete inhibition of O₂-evolution is reached only at 10^-5M (Fig.3). In ³²P-labelling a variable percentage is still left uninhibited at 10^-5 M DCMU (Fig. 4, Table 2), which is at least partly due to cyclic photophsphorylation. Nitrate starvation for several hours causes a considerable decrease in O₂-evolution and also in the sensitivity to those high concentrations of DCMU (Fig. 5), but it leads to a sensitivity to antimycin A not observed under normal conditions (Table 3). The effects of nitrate starvation thus become comparable to those of far-red light, under which noncyclic electron transport is slow or completely prevented.The inhibition by DCMU of electron transport in photosystem II is also estimated by measuring the increase in fluorescence at 684 nm in air containing additional CO₂. This fluorescence is saturated only at 10^-5M DCMU and shows that a certain percentage of photosystem II remains uninhibited at 5×10^-7M (Fig. 6), a concentration found to be almost ineffective in inhibiting O₂-evolution and ³²P-labelling in an N₂-atmosphere.The results indicate that in synchronised cells of Ankistrodesmus noncyclic electron flow and noncyclic photophosphorylation can proceed in an atmosphere of pure nitrogen if nitrate is available as the electron acceptor. In this case noncyclic photophosphorylation, inspite of its low rates, still dominates over cyclic photphosphorylation. At low pH, when nitrate reduction is slow, cyclic photophosphorylation accounts for a greater part of the total phosphorylation than at high pH. Thus in the absence of CO₂ and O₂ cyclic photophosphorylation can be regarded as the main process of ATP formation only after nitrate starvation, in far-red light or in the presence of high concentrations of DCMU.Inhibition by DCMU, though very efficient under conditions of high photosynthetic activity, becomes rate-limiting only if the electron transport is so far reduced by DCMU that the remaining rate is of the same order as the low rate of the control or less. Therefore high concentrations of DCMU are required for the inhibition of low rates of noncyclic photophosphorylation.     

Photosystem I-dependent cyclic electron flow in intact spinach chloroplasts: Occurrence,dependence on redox conditions and electron acceptors and inhibition by antimycin A

Photosystem I-dependent cyclic electron transport is shown to operate in intact spinach chloroplasts with oxaloacetate, but not with nitrite or methylviologen as electron acceptors. It is regulated by the redox state of the chloroplast NADP system. Inhibition of cyclic electron transport by antimycin A occurs immediately on addition of this antibiotic in the light. It is unrelated to a different function of antimycin A, inhibition of nonphotochemical quenching of chlorophyll fluorescence, which requires prior dissipation of the transthylakoid proton gradient before antimycin A can become effective.     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, Q_A, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark Q_A reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.     

Cyclic and Non-cyclic Photophosphorylation as Energy Sources for Active K Influx in Hydrodictyon africanum

Under anaerobic conditions in the light, active K influx inHydrodictyon africanum is supported by cyclic photophosphorylation.The use of selective inhibitors shows that, in the presenceof CO₂, a considerable portion of the ATP used by the K pumpis supplied by noncyclic photophosphorylation. The rest of theATP in these conditions comes from cyclic photophosphorylation.This is true under light-limiting as well as light-saturatedconditions. If non-cyclic photophosphorylation is inhibited (by removalof carbon dioxide, by the addition of cyanide which interfereswith the carboxylation reaction, or by inhibition of photosystemtwo with DCMU or supplying only far-red light), the K influxat low light intensities is stimulated, and its characteristicsbecome those of a process powered by cyclic photophosphorylationalone. These results are interpreted in terms of a competitionfor ATP between K influx and CO₂ fixation. Implicit in thisexplanation is a requirement for a switch of excitation energyabsorbed by photosystem one from cyclic photophosphorylationto non-cyclic photophosphorylation whenever conditions (presenceof CO₂and photosystem two activity) allow CO₂ fixation to occur. Further evidence for such a switch of excitation energy absorbedby photosystem one was obtained in experiments in which redand far-red light were applied separately and together. It wasfound that CO₂ fixation showed the Emerson enhancement effect,while K influx (in the presence of CO₂) shows a ‘de-enhancement’.This suggests that far-red light alone powers cyclic photophosphorylation;if red light is also present, some of the far-red quanta arediverted to non-cyclic photophosphorylation. The nature of the interaction between cyclic and non-cyclicphotophosphorylation is discussed in relation to these and otherpublished results.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O₂ in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b ₆-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b ₆-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and poises the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO₂ can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone - LHC light harvesting chlorophyll - LHCP II light harvesting chlorophyll protein of Photosystem II - PQ plastoquinone - PS I, II Photosystem I, II - SHAM salicyl hydroxamic acid - TBT Tri-n-butyltin CIW/DPB Publication No. 1146     

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+ reductases (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.     

Analysis of fast chlorophyll fluorescence rise (O‐K‐J‐I‐P) curves in green fruits indicates electron flow limitations at the donor side of PSII and the acceptor sides of both photosystems

Limited evidence up to now indicates low linear photosynthetic electron flow and CO₂ assimilation rates in non‐foliar chloroplasts. In this investigation, we used chlorophyll fluorescence techniques to locate possible limiting steps in photosystem function in exposed, non‐stressed green fruits (both pericarps and seeds) of three species, while corresponding leaves served as controls. Compared with leaves, fruit photosynthesis was characterized by less photon trapping and less quantum yields of electron flow, while the non‐photochemical quenching was higher and potentially linked to enhanced carotenoid/chlorophyll ratios. Analysis of fast chlorophyll fluorescence rise curves revealed possible limitations both in the donor (oxygen evolving complex) and the acceptor (Q_A^?→ intermediate carriers) sides of photosystem II (PSII) indicating innately low PSII photochemical activity. On the other hand, PSI was characterized by faster reduction of its final electron acceptors and their small pool sizes. We argue that the fast reductive saturation of final PSI electron acceptors may divert electrons back to intermediate carriers facilitating a cyclic flow around PSI, while the partial inactivation of linear flow precludes strong reduction of plastoquinone. As such, the photosynthetic attributes of fruit chloroplasts may act to replenish the ATP lost because of hypoxia usually encountered in sink organs with high diffusive resistance to gas exchange.     

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 redox state of the NADP system in illuminated chloroplasts

Pyridine nucleotide levels were measured in intact spinach chloroplasts. The NADPH/NADP ratio was close to unity in darkened chloroplasts. On illumination, chloroplast NADP levels decreased rapidly. The decrease was more prominent at low than at high light intensities. In the presence of bicarbonate, NADP subsequently increased to reach a steady-state level. The kinetics of the increase were related in general, but not in detail, to the lag phase of photosynthesis. In the steady state, chloroplast NADP was sometimes, particularly during photosynthesis at high light intensities, less reduced in the light than in the dark. In the dark-light transition, phosphoglycerate reduction is driven by increases in the ratios NADPH/NADP and ATP/ADP. When photosynthesis accelerates after the initial lag phase, the NADPH/NADP ratio decreases and a high ratio of phosphoglycerate to triose phosphate becomes an important factor in driving carbon reduction. Under photosynthetic flux conditions, the redox state of the chloroplast NADP system appeared to be governed largely by the chloroplast ratio of phosphoglycerate to dihydroxyacetone phosphate and by the phosphorylation potential [ATP]/[ADP] [P_i]. The inhibitor of cyclic electron transport, antimycin A, increased reduction of the chloroplast NADP system. Even when reduction was almost complete in the presence of 5 μM antimycin A, photosynthesis was still significant at low light intensities. Electrons appeared to be effectively distributed between the cyclic electron-transport pathway and the noncyclic route to NADP at NADPH/NADP ratios as low as about 1. When bicarbonate was absent, the NADP system remained largely reduced in the light. The energy-transfer inhibitor, Dio-9, and uncouplers and agents which interfered with pH regulation of the Calvin cycle increased reduction of the NADP system while decreasing photosynthesis.