Enhancement studies on algae and isolated chloroplasts. Part II. Enhancement of oxygen evolution in intact chloroplasts.

Intact isolated chloroplasts were shown to exhibit a characteristic three-phase pattern of development of oxygen evolution activity. The first phase, Phase I, appeared to be an equilibration phase in which the isolated chloroplasts adapted to the conditions on the electrode surface. It was characterised by a rapidly increasing rate of oxygen evolution accompanied by decreasing enhancement signals. The second phase, Phase II, was an intermediate phase in which the rate of oxygen evolution was maximal and no enhancement was observed. In the last phase, Phase III, the rate of oxygen fell again, normal enhancement was still missing, but the samples appeared to undergo slow adaptive changes closely related to the State I-State II changes previously reported for whole cell systems. The concentrations of Mg2+ within the chloroplast were shown to play an important role in the control of the development of both the oxygen evolution and enhancement signals. It was shown how these signals could be explained in terms of a model that was consistent with that developed in Part I of this investigation to account for the variability of enhancement of the alga Chlorella pyrenoidosa.     

Factors influencing photosynthetic enhancement in isolated chloroplasts

Photosynthetic enhancement of oxygen evolution (linked to CO₂ assimilation) in isolated chloroplasts was found to be governed by the supply of ATP. The addition of ATP (but not AMP) abolished enhancement that consistently occurred without added ATP. Enhancement in the H₂O → NADP reaction by chloroplasts was investigated in the light of one recent report that the phenomenon occurs when pure ferredoxin is replaced by a crude preparation (PPNR) and another report that the phenomenon is governed by Mg⁺⁺ concentration. Fractionation of PPNR led to the isolation of a protein factor which when added to pure ferredoxin induced enhancement. However, the rate of NADP reduction with pure ferredoxin and without enhancement was greater than the maximum rate of NADP reduction with enhancement induced by either the protein factor of PPNR. The report that Mg⁺⁺ concentration governs enhancement was not confirmed.     

Membrane structure and function. II. Alterations in the photo-induced absorption changes after treatment of isolated chloroplasts with large pulses of the ruby laser

Treatment of isolated chloroplasts with high-energy pulses of the ruby laser causes graded structural changes in the chloroplast membranes and is here correlated with the biochemical changes produced. The laser treatment caused decreases in the photoinducible absorption changes of cytochromes b₅₅₉, b₅₆₃, and P₅₂₀ (the carotenoid shift), but smaller decreases in cytochrome f. The decreases correlated with the quantum efficiency alterations produced by the laser treatment. Ferricyanide photoreduction and O₂ evolution was only slightly affected by the laser treatment. The slow phase of the dark recovery kinetics of P₅₂₀ was increased maximally by the lowest laser input energies and NADP⁺ photoreduction induced by carbonylcyanide-P-trifluoromethoxyphenylhydrazone (FCCP) was decreased maximally by the lowest energies, suggesting that uncoupling of the chloroplasts was the most sensitive parameter. This was corroborated by our previous observation (5) that chloroplast membrane bound surface particles (coupling factor) was the ultrastructural change most sensitive to the laser pulses. Electron flow from photosystem II to photosystem I was not altered by the laser treatment. The laser treatments did not cause a detectable decrease in total chlorophyll in the chloroplasts, however, approximately 10% of the total chlorophyll was present in the solution phase after the treatment, whereas no detectable cytochromes were present in the solution phase.     

The effects of digitonin on photochemical activities of isolated chloroplasts

The addition of digitonin to chloroplasts stimulated the rate of oxygen evolution followed by a gradual inhibition. The effect of digitonin was dependent on the digitonin to chlorophyll ratio and on temperature and time. The initial stimulation of oxygen evolution appeared to be a result of uncoupling as digitonin did not stimulate oxygen evolution by uncoupled chloroplasts. The stimulatory effect occurred more rapidly at high digitonin to chlorophyll ratios but the extent of stimulation was low and inhibition occurred soon after addition of the detergent. The inhibition of electron flow by digitonin was due to a site of action near photosystem II which resembled the inhibition reported for tris buffer and resulted in photobleaching. However, digitonin inhibition could not be recovered by washing with reducing agents and was only partially recovered by the addition of artificial electron donors to photosystem II. Electron flow mediated by photosystem I was unaffected by the addition of digitonin but was decreased when the chloroplasts were separated by subsequent centrifuging. This suggested that digitonin solubilizes photosystem I components which remain active in the soluble form.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

The relation of the alteration of photosystem. II. Activity to the inhibition of energy transfer and the proton pump in tris-washed chloroplasts

Washing of spinach chloroplasts with high concentrations of Tris³ induces pH-dependent changes in chloroplast reactions. At high pH (8.4) Tris washing causes the inhibition of Photosystem 2 activity which can be prevented by the maintenance of reducing conditions during washing. Washing at low pH (7.2) causes an enhancement of oxygen evolution and increased rate of ferricyanide photoreduction which is not influenced by the presence of reducing conditions. The increased rate of electron flow is accompanied by the inhibition of light mediated phosphorylating activity, acid-induced ATP synthesis, light-induced proton uptake and light triggered Mg²⁺ ATPase activity. Tris treatment at low pH also causes a sensitization of Photosystem 2 activity such that oxygen evolution is inhibited by low concentrations of tris at high pH. This inhibition of the stimulated electron flow is not accompanied by a reconstitution of the photophosphorylation activity. A detailed analysis of the effect of tris treatment on Photosystem II activity and membrane dependent energy conversion shows that the treatment of chloroplasts causes an inhibition of the energy conversion process which is independent of the effect on oxygen evolution. Determination of the presence of coupling factor (as determined by ATPase activity) and membrane osmotic properties reveal normal levels of enzyme activity and osmotic response in treated chloroplasts. The inhibition of the energy conversion process is accompanied by reduced capacity to maintain a proton gradient. Kinetic analysis of the proton uptake reaction reveals that Tris treatment renders the grana membranes more permeable to protons.     

Comparison of photosynthetic activities of spinach chloroplasts with those of corn mesophyll and corn bundle sheath tissue

Bundle sheath and mesophyll chloroplasts from Zea mays showed comparable rates of O₂ evolution, which amounted to about half of the rate observed in spinach (Spinacia oleracea) chloroplasts.

Ratios of 4.5, 4.6, and 6.2 Mn²⁺ atoms per 400 chlorophylls were observed in mesophyll, bundle sheath, and spinach chloroplasts, respectively. These ratios roughly correspond to the observed O₂ evolution rates.

Rates of electron transport from water to methylviologen (photosystem I and II) in both types of corn chloroplasts were about one-third that in spinach. Compared to spinach, transport rates from reduced diaminodurene to methylviologen (photosystem I) were about one-third and greater than one-half in mesophyll and bundle sheath material, respectively.

In both types of corn chloroplasts, electron flow from photosystem II to P700 was abnormal. This observation, together with the low rates of all activities, suggests that damage occurred during isolation. Such damage may limit the quantitative significance of observations made with these materials (including the following data).

Measurements of flash yields of O₂ evolution or O₂ uptake showed that the size of the photosynthetic unit was the same in photosystems I and II and in all three types of chloroplasts (about 400 chlorophylls per equivalent).

Similarity of the photochemical cross-section of the two photosystems in the three preparations was also found in optical experiments: that is the half-times of the fluorescence rise in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (photosystem II) and of the photooxidation of P700 (photosystem I).

The ratio of P700 to chlorophyll appeared to be about 2-fold higher in bundle sheath chloroplasts than in the other materials (1/200 versus 1/400).

     

Electron spin resonance and photoreaction of Mn(II) in lettuce chloroplasts.

Electron spin resonance (esr) of lettuce chloroplasts yields three types of signals: (i) a broad (~900 G) signal around g = 2.22 (apparently due to Cu²⁺ complexes); (ii) an Mn²⁺ spectrum around g = 2.003 consisting of six hyperfine lines (A = 94.5 G) of ~30 G width; and (iii) a sharp signal at g = 2.00 due to photosignals I and II. The present work is concerned with the Mn²⁺ signal and its relation to the photosynthetic process. Intensity measurements were performed by comparing the intensities of the Mn²⁺ signals of two identical chloroplast preparations, one of which was slightly acidified. The integrated intensity of the signal in the normal preparation was approximately one-fourth of that in the acidified sample, suggesting that only the?$\text{1}\text{2}\text{?}\text{1}\text{2}$ fine structure band is observed in untreated chloroplasts. This indicates that the manganese in the chloroplasts is bound in an asymmetric environment, apparently in protein complexes. The Mn²⁺ signal is light sensitive, decreasing on illumination and reappearing in the dark. Typical values for the half-lives of the light and dark processes in normal chloroplasts are 0.25 and 2.1s, respectively. The effect is interpreted in terms of the photooxidation of Mn²⁺ to higher oxidation states which are invisible to esr spectroscopy. In order to determine whether this process is related to photosynthesis the effect of certain reagents and treatments that are known to affect the photosynthetic system was studied. It was found that the oxygen evolution inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) as well as the electron donors, phenylenediamine and sodium ascorbate, reduce or completely eliminate the light effect on the Mn²⁺ signal. Heat treatment and Tris washing caused deceleration of both the light and dark reactions. These effects indicate that the photooxidation of the Mn²⁺ is related to the photosynthetic cycle, the most probable site being the water splitting apparatus of photosystem II.     

Observation of enhancement and state transitions in isolated intact chloroplasts

Enhancement of photosynthesis by supplemental photosystem 1-enriched (707nm) light has been investigated in intact spinach chloroplasts by the simultaneous measurement of the rate of oxygen evolution, yield of chlorophyll fluorescence and quenching of 9-aminoacridine fluorescence. Chloroplasts reducing CO₂ showed a 75% increase in the rate of O₂ evolution after the addition of 707nm light, whereas if nitrite was used as substrate, an enhancement of only 20% was observed. Reduction of glycerate-3-phosphate was associated with a 40% enhancement by 707nm light. There appears to be a correlation between the degree of enhancement and the requirement for ATP in addition to reducing power. Prolonged illumination in 707nm light resulted in an elevation of enhancement whereas illumination with 650nm light caused a loss of enhancement, demonstrating the operation of state transitions in intact isolated chloroplasts.     

Computer simulation study of pH-dependent regulation of electron transport in chloroplasts

A mathematical model is presented that describes the key steps of photosynthetic electron transport and transmembrane proton transfer in chloroplasts. Numerical modeling has been performed with due regard for regulatory processes at the donor and acceptor parts of photosystem (PS) I. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the light-induced redox transients of P₇₀₀, plastoquinone, and NADP as well as on the changes in intrathylakoid pH and ATP level is examined. It is demonstrated that pH-dependent regulatory processes alter the distribution of electron fluxes on the acceptor side of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle leads to significant enhancement of the electron flow from PS I to NADP⁺ and attenuation of the electron flow to molecular oxygen.     

Photo-induced pH Changes in the Vicinity of Isolated Peperomia metallica Chloroplasts

Photo-induced pH changes of the external medium in a regionimmediately adjacent to the surface of individual isolated Peperomiametallica chloroplasts have been measured using antimony pH-micro-electrodcs.The pH changes induced by continuous illumination were composedof two phases: an initial alkalization (phase I) and a subsequentacidification (phase II) of the medium. Both phases were severelysuppressed by DCMU and protonophorous uncouplers but they showeddifferent sensitivity towards DCCD₁ NH₄CI and some other agents.Phase I was selectively inhibited by DCCD and was partiallyrestored upon addition of ATP to DCCD-poisoned chloroplasts.Phase II was inhibited by 1.0 mol m^–3NH₄CI. It appearsfrom these data that phases I and II of light-induced pH changesare determined by different processes. It is suggested thatthe pH increase (phase I) is due to a photosynthetic CO₂ fixationand the pH decrease (phase II) is caused by a light-dependentextrusion of protons from intact chloroplasts. Key words: Transport of protons, intact chloroplasts, Peperomia metallica     

Reversible abolition of enhancement in isolated spinach chloroplasts

The photosynthetic evolution of oxygen by isolated chloroplasts of Spinacia oleracea L. was studied using a modulated oxygen electrode. The enhancement effect, measured as the increase in the relative quantum yield of 650-nanometer light due to the presence of 710-nanometer light, was profoundly influenced by the concentration of inorganic cations in the bathing medium. Chloroplast fragments immersed in a solution containing a very low concentration of MgCl₂ or KCl, did not display enhancement but could be made to do so by raising the concentration of MgCl₂ to 3 mm, or that of KCl to 35 mm. This change in the enhancement properties was completely reversible. The maximum value of enhancement in a MgCl₂ solution appeared to occur at a concentration between 15 and 30 mm, while in KCl, the enhancement effect increased almost linearly up to a concentration of 100 mm.     

Oxygen evolution and the permeability of the outer envelope of isolated whole chloroplasts

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.

Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.

A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.

     

Photosynthetic carbon metabolism of isolated corn chloroplasts

Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO₂ per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of ¹⁴C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO₂ at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.     

Enhancement studies on algae and isolated chloroplasts. Part I. Variability of photosynthetic enhancement in Chlorella phyrenoidosa

Studies of the variability of enhancement in Chlorella pyrenoidosa confirm the existence of two types of variability: a very slow diurnal variation linked to the growth cycle and a much more rapid adaptive response to the immediate incident light conditions (State I–State II transitions). Measurements of the wavelength dependencies and relative contributions of these two types of variability suggest that they may be linked.A close examination of the enhancement signals associated with the State I–State II transition reveals that the transitions can take place in any one of three ways: by a change in Photosystem II efficiency alone, by a change in Photosystem I efficiency alone or by a simultaneous change in the efficiencies of both photosystems.Measurements of the rates of transition between State I, State II and the dark adapted state, Dark, suggest that the behaviour of State II and Dark are normally, but not always, identical. The transitions between the three states were found to be first order. For those samples exhibiting the same behaviour in Dark and State II, the rate of the State I–State II transition was found to be independent of the wavelength of Light II, suggesting that the return from State I to State II is essentially a dark process and that the driving force for the adaptive transition is the over-stimulation of Photosystem I.Finally, a model is proposed, involving an antagonistic control of the quantum yields of photochemistry of the two photosystems, that is capable of explaining the links between the two types of variability, their wavelength dependencies and the shapes of the individual enhancement signals.     

Photosynthetic reactions of chloroplasts with unusual structures

Photosynthetic reactions of whole leaves and isolated chloroplasts from various mutants of Nicotiana tabacum have been correlated to the lamellar structure seen in electron micrographs of the chloroplasts. In this way it could be established that a fully active photosystem I can be associated with single unfolded thylakoids. The complete photosynthetic electron transport system including the oxygen evolving apparatus of photosystem II, on the other hand, appears to require a close packing of at least 2 thylakoids. The unusual high capacity for photosynthesis observed earlier for leaves of certain aurea mutants is reflected by a correspondingly high activity of the isolated chloroplasts in the Hill reaction. These chloroplasts contain extended areas where 2 thylakoids touch by forming simple lamellar overlappings instead of the familiar stacks of lamellar discs.     

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

The retention of photosynthetic activity by senescing chloroplasts of oat leaves

The retention of photosystems I and II and or RuDP carboxylase activity in chloroplasts isolated from the first leaves of Victory oat (Avena sativa L.) seedlings was followed as the chloroplasts senesced in darkness. Both photosystems (PS) I and II retained their full activity after 3 days at 1°C, while even after 7 days at 1°C around 80% of the activity was still present. After 3 days at 25°C, PS I lost only 20% and PS II 50% of the initial activity. Acid pH increased the rate of decay of both systems, PS II falling almost to zero after 3 days at pH 3.5 (at 25°C). The preparations were almost bacteria-free, and addition of antibiotics not only did not improve their stability, but accelerated the rates of loss of photosynthetic activity. This is held to indicate that the enzymes are undergoing some turnover even in isolated chloroplasts. If the leaves were allowed to senesce in the dark first and the chloroplasts then isolated, their photosynthetic activities had greatly decreased, showing that senescence is more rapid in situ than in isolation. Under these conditions PS I decayed more rapidly than PS II. Ribulosediphosphate carboxylase, as measured by CO₂ fixation, declined more rapidly than the photosystems, though the addition of kinetin and indole-3-acetic acid somewhat decreased the rate of loss, at least for the first 24 h. When the intact (detached) leaves were held in the dark, the rate of oxygen evolution declined rapidly, but in monochromatic blue light (450 nm) at 25°C about 30% of the initial rate was retained after 72 h.Abbreviations BSA bovine serum albumin, chl, chlorophyll - DCPIP dichlorophenol-indophenol - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - PS photosystem - PVP soluble polyvinylpyrrolidine - RuDP Ribulose-1,5-diphosphate - TES N-tris-(hydroxymethyl)-methyl-2-amino-ethane sulfonic acid     

Oxygen activation by isolated chloroplasts from Euglena gracilis. Ferredoxin-dependent function of a fluorescent compound and photosynthetic electron transport close to photosystem.

Oxygen reduction by isolated chloroplast lamellae from spinach, yielding the superoxide free radical in the light, is stimulated by a fluorescent factor (“compound No. 4”, isolated from Euglena gracilis strain Z) in a ferredoxin-dependent reaction. This reaction is not observed with Euglena chloroplasts, although there is a stimulation by compound No. 4 of ferredoxin-dependent oxygen reduction at the expense of NADPH + H⁺ as electron donor in the dark. Evidence is provided that in Euglena chloroplasts in the absence of NADP as electron acceptor a cyclic electron transport is predominating, including photosystem I, ferredoxin, NADP-ferredoxin reductase, and cytochrome₅₅₂. Isolated spinach chloroplast lamellae show a similar “cyclic” electron transport after treatment with digitonin, depending on the addition of the above cofactors. This result might indicate that Euglena chloroplast lamellae show this cyclic electron transport only as an artifact due to the isolation procedure. The results furthermore indicate that the pteridine-like, fluorescent compound No. 4 is not active as the primary electron acceptor of photosystem I; it may however be involved in oxygen activation by Euglena gracilis chloroplasts.