Suppression of Carotenoid Photobleaching by Kaempferol in Isolated Chloroplasts

The effects of kaempferol on carotenoid photobleaching wereexamined using chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone(CCCP). Kaempferol suppressed carotenoid photobleaching withoutaffecting electron transfer reactions. Half-maximal suppressionwas observed at about 10 µM. Kaempferol was photooxidizedby CCCP-poisoned chloroplasts, as observed by its bleachingat 380 nm. Ascorbate inhibited the oxidation of kaempferol.Under anaerobic conditions, kaempferol did not affect the photobleachingof carotenoid. Other fiavonols, quercetin and its glycosides,also suppressed the carotenoid photobleaching. The results suggestthat flavonols act as antioxidants in illuminated chloroplastsunder aerobic conditions. (Received February 22, 1982; Accepted May 14, 1982)     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.     

Oxidation of Quercetin by Carotenoid Radical Generated in Illuminated Spinach Chloroplasts: The Effect of Ascorbate on Quercetin Oxidation

Carotenoid photobleaching in the presence of carbonylcyanidem-chlorophenylhydrazone (CCCP) was suppressed by quercetin,but not by ascorbate. When quercetin suppressed carotenoid photobleaching,quercetin was oxidized. The oxidation of quercetin was inhibitedby ascorbate with half-inhibition at about 10 µM. Ascorbatewas oxidized by CCCP-poisoned chloroplasts upon illumination.The rate of ascorbate oxidation in the presence of both ascorbateand quercetin was lower than that in the presence of ascorbatealone. Based on the present results, the physiological significanceof quercetin as an antioxidant and the redox reaction betweenascorbate and oxidized quercetin are discussed. (Received March 9, 1984; Accepted July 12, 1984)     

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl₂ at a molar ratio of HgCl₂ to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ? was similarly sensitive towards HgCl₂, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ? and light-induced increase in fluorescence yield were enhanced by the HgCl₂ treatment of chloroplasts.     

Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of O2- with electron transfer components

Chemiluminescence of luminol (CLL) was induced by illuminatedspinach chloroplast fragments. CLL was diminished by superoxidedismutase or under anaerobic conditions and increased by anautoxidizable electron acceptor, methyl viologen. The optimumpH for CLL was 10.0-10.5. Ferredoxin and cytochrome c reducing substance (CRS) did notaffect the intensity of CLL, but accelerated the dark decayin the absence of methyl viologen. In the presence of methylviologen, ferredoxin and CRS lowered the intensity and acceleratedthe dark decay. 3-(4-Chlorophenyl)-1,1-dimethylurea diminishedCLL. Carbonylcyanide m-chlorophenylhydrazone accelerated theinitial rate of CLL increase at low concentration and inhibitedit at high concentration. Half-decay time of CLL after the cessationof light was shortened by inhibiting electron transfer on theoxidizing side of photosystem II. We conclude that most of the CLL observed in illuminated chloroplastsis dependent on O₂^–. The results also suggest that O₂^–is reduced by reduced ferredoxin or CRS and oxidized on theoxidizing side of photosystem II. The half life of O₂^–in illuminated chloroplasts was estimated from the half-decaytime of CLL to be a few sec. ¹ Present address: Kyushu Dental College, Department of Biology,Kitakyushu 803, Japan. (Received May 30, 1977; )     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Gas-liquid Chromatographic Separation of Amine Enantiomers as Diastereoisomeric Chrysanthemoylamide Derivatives

An immobilized chloroplast film, prepared by immobilizing spinach chloroplasts in 2 wt% agar gel, was attached to a SnO₂ optically transparent electrode to obtain the immobilized chloroplast film electrode. The immobilized chloroplast film electrode worked as a photoanode under illumination in the presence of methyl viologen, which was an electron carrier from chloroplasts to the SnO₂ optically transparent electrode. Water photolysis for producing hydrogen by a photoelectrochemical cell using the immobilized chloroplasts film electrode was successfully achieved. A smooth platinum electrode was used as a cathode to produce hydrogen. The pH and temperature of the anolyte were kept at 7.8 and 25°C. Optimizations of the concentrations of methyl viologen and chlorophyll in the immobilized chloroplast film were studied. The optimum thickness for the immobilized chloroplast film was about 0.8 mm. The immobilized chloroplasts had higher storage stability than that of isolated chloroplasts and they retained more than 50% of the initial activities of photosystem I and photosystem II after 10 days when they were stored at 4°C in the dark. It was conceived from the relationship between the photocurrent and the photosystem I and II activities that the main cause for the decrease in the photocurrent was the photochemical inactivation of photosystem II.     

Selective photobleaching of PSI-related chlorophylls in heat-stressed pea chloroplasts

Measurements of electron transport activity point to the occurrence of major changes in the organisation of the photosynthetic apparatus of heat-stressed chloroplasts. One of the consequences of these changes is shown to be a greatly increased susceptibility of chlorophyll to photobleaching. Despite the fact that the threshold temperature for this photobleaching coincides closely with that for the inhibition of PSII activity, the bleached components were found to be specifically associated with PSI. This increased susceptibility of PSI pigments to photobleaching is shown to be a direct consequence of an interruption of the flow of reductants from PSII to PSI that would normally protect PSI from photooxidation.Abbreviations PSI photosystem I - PSII photosystem II - chl a chlorophyll a - chl b chlorophyll b - LHCP chlorophyll a/b light-harvesting protein - CP₁ P700-chlorophyll a protein - DCMU 3-(34 dichlorophenyl)-11-dimethylurea - DCPIP dichlorophenolindophenol - Fecy potassium ferricyanide - MV methyl viologen Biochemistry Department, King's College (KQC), University of London     

Effects of electron donor and acceptors, electron transfer mediators, and superoxide dismutase on lipid peroxidation in illuminated chloroplast fragments

Effects of artificial electron donor and acceptors, electrontransfer mediators, and superoxide dismutase on lipid peroxidationin illuminated chloroplast fragments were studied. An indicator of lipid peroxidation, malondialdehyde (MDA) formation,was stimulated by 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU).The DCMU stimulated MDA formation was inhibited about 90% byreduced 2,6-dichloroindophenol (DCIP). In photosystem I-enrichedparticles, MDA formation was larger than that in normal chloroplastfragments on the chlorophyll basis. Benzyl viologen and ferredoxinstimulated DMA formation. Superoxide dismutase inhibited MDAformation strongly in the presence of benzyl viologen and weaklyin its absence; the enzyme sometimes stimulated MDA formationin the presence of ferredoxin. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated MDA formation and maximal stimulation wasattained at about 20 µM CCCP.Phenazine methosulfate, DCIPand benzoquinone inhibited MDA formation in the presence andabsence of CCCP. From the above results, we confirmed our previous conclusionthat most of the singlet molecular oxygen formed in illuminatedchloroplasts is generated by electron transfer from O_2^–to oxidized electron transfer components located on the oxidizingsides of photosystems I and II. (Received September 3, 1975; )     

Discrimination and characterization of photosystem I- and photosystem II-induced 515-nm absorbance change in chloroplasts I. Dissipation of electric field and related processes

Distinctive characteristics of the photosystem I-induced 515-nmabsorbance change and the photosystem II-induced change wereanalyzed in spinach chloroplasts in the absence of added salt.Two types of changes were distinguished by 3-(3,4-dichloro-phenyl)-1,1-dimethylurea(DCMU), carbonylcyanide m-chlorophenylhydrazone (CCCP) and illuminationwith red or far-red light. Half-recovery time of the photosystem I-induced absorbance changewas shorter than that of over-all absorbance change and wasinsensitive to a low concentration (<0.50 µM) of CCCP. In the presence of DCMU, the 515-nm absorbance change decayedin parallel with the rapid protonation of reduced 2,6-dichloroindophenol(DCIP) or methyl viologen. This indicates that the photosystemI-induced local field is dissipated in the electron transferfrom photosystem I to an electron acceptor. Thus the mechanismin dissipation of electric field formed by photosystem I isdifferent from that induced by photosystem II where rapid protonationof plastosemiquinone anion may be directly involved in fielddissipation (Yamamoto, Y. and M. Nishimura: Plant & CellPhysiol. 18: 293–301 (1977)). (Received December 9, 1977; )     

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O₂ evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O₂ uptake with methyl viologen as acceptor.

Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H₂O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.

The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O₂ evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O₂ evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

     

Direct Spectrophotometric Measurement of Photosystem I and Photosystem II Activities of Photosynthetic Membrane Preparations from Cyanophora paradoxa, Phormidium laminosum, and Spinach

Vesicles prepared with the French press from membranes of cyanelles of Cyanophora paradoxa retain O₂ evolution activity with rates up to 500 micromoles 2,6-dichlorophenolindophenol reduced per hour per milligram chlorophyll. This activity is immediately lost when the vesicles are transferred from the sucrose-phosphate-citrate preparation buffer into dilute phosphate buffer. Similar preparations from Phormidium laminosum, a thermophilic cyanobacterium retain activity under such conditions. Photosystem I activities of both cyanobacterial vesicle preparations were determined by direct spectrophotometric measurement of N,N,N′,N′-tetramethyl-p-phenylenediamine photooxidation in the presence of anthraquinone-2-sulfonate. The rates so determined were compared with rates of O₂ taken up in the presence of methyl viologen or anthraquinone-2-sulfonate as electron acceptors. The predicted stoichiometry of two was observed for moles of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidized per mole of oxygen taken up. Anthraquinone-2-sulfonate was the better electron acceptor, and maximal rates of 943 micromoles per hour per milligram chlorophyll for O₂ uptake were observed for Phormidium laminosum preparations in the presence of superoxide dismutase. For purposes of comparison, spinach chloroplasts were assayed for similar activities. All preparations were readily assayed for photosystem I activity by the direct spectrophotometric method, which has advantages of simplicity and freedom from errors introduced by photoxidation of other substrates by photosystem I when O₂ uptake is measured.     

Photosynthetic activities of cadmium-treated tomato plants

Tomato plants (Lycopersicum esculentum Mill. cv. Moneymaker) grown on nutrient medium containing cadmium exhibit reduced net photosynthesis and reduced contents of chlorophyll and accessory pigments. In chloroplasts isolated from cadmiumtreated plants photosystem II activity, as measured by 2,6-dichlorophenolindophenol photoreduction, and photosystem II + I activity (H₂O → methyl viologen) were both inhibited to about 60%. When 1,5-diphenylcarbazide was used as artificial electron donor, no significant cadmium effect was observed. Photosystem I activity was not affected by cadmium. The fine structure of chloroplasts in cadmium-treated plants was degenerated, similarly to senescence response. The principal symptom of cadmium action was the occurrence of large plastoglobules and a disorganization of the lamellar structure, mainly grana stacks. Transfer of cadmium-treated plants into a medium with increased manganese level caused grana stacking and restoration of photosystem II activity.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

The Response of Antioxidant Enzymes in Cellular Organelles in Cucumber (Cucumis sativus L.) Leaves to Methyl Viologen-induced Photo-oxidative Stress

In order to clarify the response of antioxidant systems in various cellular organelles to photo-oxidative stress, the activities of superoxide dismutase (SOD) and enzymes of the ascorbate–glutathione (AsA-GSH) cycle were investigated in chloroplasts, mitochondria and cytosol of cucumber leaves subjected to methyl viologen (MV) treatment. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate (Pn) and increases in the ratio of the quantum efficiency of photosystem II (PSII), Φ_PSII to that of the quantum efficiency of CO₂ fixation (ΦCO₂), followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) except that the content of ascorbate (AsA) in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase (AO) in these organelles was independent of the MV treatment while the activity of l-galactono-1,4- lactone dehydrogenase (GLDH) in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA.     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

Facilitated permeation of methyl viologen into chloroplasts in situ during electric pulse generation in excitable plant cell membranes

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV²⁺), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV²⁺ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV²⁺ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV²⁺ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA ₈₁₀ signals). These findings suggest that MV²⁺ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA ₈₁₀ signals, light-induced changes in membrane potential do not depend on the presence of MV²⁺ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV²⁺ at the expense of non-cyclic photosynthetic electron transport redirected to the MV²⁺ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV²⁺ in membrane barriers of the “plasmalemma-chloroplast envelope” system.