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; )     

Chelator-sensitive in chloroplast electron transport

The effect of various chelators (orthophenanthroline, bathophen-anthroline, bathophenanthroline sulfonate and bathocuproine) on electron transport of spinach chloroplasts has been studied by means of various photosystem I and II reactions. It was found that photosystem II has at least 3 chelator-sensitive sites, photosystem I from 3–4. An uncoupler-affected site was found in each photosystem. In addition, photosystem I had a stimulator site and a soak site. The soak site was sensitive to chelators only after a period of incubation with the chelator.     

Mechanism of Linolenic Acid-induced Inhibition of Photosynthetic Electron Transport

The effect of linolenic acid on photosynthetic electron transport reactions in chloroplasts has been localized at a site on the donor side of photosystem I and at two functionally distinct sites in photosystem II.     

Effects of DDT on photosynthetic electron flow in Secale species

Following a survey of a range of varieties of rye, mainly Secale cereale, for reaction to DDT, the mode of action of the pesticide in a susceptible variety was studied. Two sites of interaction of DDT with the photosynthetic electron transport chain were demonstrated. The first site of inhibition was on the oxidizing side of photosystem 2, between the sites of electron donation from diphenylcarbazide at pH 6.0 and pH 8.0 in Tris-washed chloroplasts. The second site of DDT inhibition was in the intermediate electron transport chain, and was demonstrated by using dichlorophenol-indophenol and phenyldiamines as electron donors in chloroplasts where electron flow from photosystem 2 was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The sites are distinct from those characteristic of herbicides which affect photosynthetic electron flow.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

Inhibition of photosynthetic electron transport by diphenyl ether herbicides

The effects of the diphenyl ether herbicides HOE 29152 (methyl-2[4-(4-trifluoromethoxy) phenoxy] propanoate) and nitrofluorfen (2-chloro-1-[4-nitrophenoxy]-4-[trifluoromethyl]benzene) on photosynthetic electron transport have been examined with pea seedling and spinach chloroplasts. Linear electron transport (water to ferricyanide or methylviologen) is inhibited in treated chloroplasts, but neither photosystem II activity (water to dimethylquinone plus dibromothymoquinone) nor photosystem I activity (diaminodurene to methylviologen) is affected. Cyclic electron flow, cata-lyzed by either phenazine methosulfate or diaminodurene, is resistant to inhibition by nitrofluorfen. In diphenyl ether-treated chloroplasts the half-time for the dark reduction of cytochrome f is increased 5- to 15-fold. These data indicate that the site of inhibition for the diphenyl ethers is between the two photosystems in the plastoquinone-cytochrome f region.     

Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts

In isolated barley chloroplasts, the presence of 2 millimolar ZnSO₄ inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, O₂ evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCl₂. These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO₄ is observed. However, with concentrations of ZnSO₄ above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn²⁺-treated chloroplasts partially restores the O₂-evolving activity.     

On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

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.     

Inhibition of Chloroplasts by UV-Irradiation and Heat-Treatment

The site of inhibition in UV-irradiated and heat-treated chloroplasts was examined by using artificial electron donor compounds such as p-phenylenediamine and hydroquinone which donated electrons specifically to photosystem II. In both cases the electron donors restored the photoreduction of nicotinamide adenine dinucleotide phosphate and the restored activity was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. The fluorescence of variable yield was eliminated by both inhibitory treatments and was partially restored by the electron donors in the heat-treated but not the UV-irradiated chloroplasts. The results suggest that the sites of inhibition of UV-radiation and heat treatment are in the photosynthetic electron transport chain between water and photosystem II.     

Studies on cytochromes in photosynthetic electron transport system II. Participation of photosystem I in the light-induced oxidation-reduction of cytochrome b559 in spinach chloroplasts

The role of photosystem I in the photooxidation-reduction reactionsof cytochrome b₅₅₉ was elucidated by studying the effects ofelectron acceptors and inhibitors of photosystem I on reactionsof the cytochrome in spinach chloroplasts. We concluded thatthe cytochrome is photoreduced through photosystem I and, inthe presence of DCMU and ferrocyanide, is photooxidized by photosystemI. (Received December 20, 1972; )     

Photophosphorylation in isolated maize bundle sheath chloroplasts and cells

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

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Effects of oxygen on the electron transport chain of photosynthesis

Summary Oxygen was taken up by both intact and broken chloroplasts when catalase was posioned. In confirmation of other work we found that oxygen enters the electron transport chain of isolated chloroplasts by oxidizing the primary photoreductant of system I. In isolated intact chloroplasts this reaction proceeds in addition to oxygen evolution by PGA reduction. The reductant produced by photosystem II does not react with oxygen at a significant rate.In normal leaves oxygen depresses chlorophyll fluorescence. However, this depression does not take place in DCMU poisoned leaves or in a mutant having a nonfunctional photosystem II; furthermore, another mutant with a weakly functioning photosystem I gave only a very small fluorescence depression with oxygen. This shows that the site of interaction of oxygen is at the reducing end of the electron transport chain. This view is supported by the extent of the fluorescence depression in leaves as a function of oxygen concentration which is very similar to the oxygen dependence of oxygen uptake by isolated chloroplasts.An oxygen requirement of isolated intact chloroplasts reducing PGA and nitrate was indicated by lower reaction rates and faster decay of activity under nitrogen than under air.Dedicated to Prof. Harder on his eightieth birthday.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.     

Development of photosynthetic electron transport in Pinus silvestris

Photosynthetic electron transport activity has been measured in chloroplasts isolated from dark-grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark-grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark-grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark-grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness. Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark-grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark-grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Photosynthetic Responses of Different Varieties of Wheat to High Temperature: II. EFFECT OF HEAT STRESS ON PHOTOSYNTHETIC ELECTRON TRANSPORT

The effect of heat stress on photosynthetic electron transportwas investigated in thylakoids isolated from the wheat (Triticumaestivum L.) varieties APU (Finland) and K65 (India) grown underboth cool (13 °C day, 10 °C night) and warm (30 °Cday, 25 °C night) regimes which gave rise to varietal differencesin photosynthetic temperature acclimation. The responses ofthe uncoupled activities of both whole-chain electron transportand photosystem II to heat stress were similar. Both activitiesexhibited higher rates in thylakoids isolated from warm-grownplants and were more resistant to high temperature pretreatmentthan in those isolated from cool-grown plants, but varietaldifferences were not observed. Uncoupled photosystem I activity driven by either reduced 2,6-dichlorophenol indophenol (DCPIPH₂) or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPDH₂) showed a stimulation following high temperaturepretreatment which was more marked in thylakoids isolated fromwarm-grown plants, followed by inhibition at extreme high temperatures.This stimulation was due largely to an increase in V_max butdid not occur when reduced diaminodurene, which is highly lipophilic,was used as the electron donor. It appears that stimulationof PS I activity may involve increased accessibility of someartificial electron donors to the native acceptor sites withinthe thylakoid membrane in a process which is influenced by growthtemperature. Key words: Photosynthetic electron transport, heat stress, Triticum aestivum