Ageing of chloroplasts in vitro II. Changes in absorption spectra and the DCPIP Hill reaction

Changes in absorption characteristics and in Hill activity werestudied during ageing of isolated chloroplasts in the dark andlight. Ageing of chloroplasts brings about significant reductionin the peak absorption in the blue and red bands and resultsin a blue shift in the red band. Incubation of chloroplastsin the dark prevented the early appearance of a light-inducedblue shift at pH 7 and pH 8 but not at pH 6 of the storage media.Addition of BSA or DTT to chloroplast suspensions of pH 6 andpH 7 significantly delayed the blue shift both in the lightand dark, but the chemicals had no effect at pH 8. Ageing-induced loss in DCPIP reduction is restored considerablyby the addition of exogenous electron donors such as DPC orMn²⁺, which suggests loss of the oxygen evolution system. Mn²⁺loses its ability to donate electrons for DCPIP photoreductionduring ageing faster than DPC. This indicates that there aretwo sites between water and the PS II reaction centre for theentry of electrons from these two donors and that ageing causessequential loss of these sites. The loss in DPC supported DCPIP photoreduction coincides, intime, with the ageing-induced blue shift of the red absorptionband and thus suggests structure and function relations of theplastid membrane. (Received October 30, 1978; )     

Ageing of chloroplasts in vitro — III. Comparison of the effects of ageing in vitro and senescence on the red absorption band and primary photochemical reactions of sunflower chloroplasts

A comparison of changes in absorption properties and electron transport activities of chloroplasts ageing in vivo and in vitro is made. Chloroplasts from sunflower leaves senescing in vivo during 7 days in dark do not show a blue shift of the red absorption band; in contrast, the shift becomes apparent within 24 h of in vitro ageing of isolated organelles. Photosynthetic activity by chloroplasts is lost much faster during in vitro than in vivo ageing. During in vitro ageing, the rate of degradation of thylakoid membranes as characterised by the shift in the red absorption band and loss in Hill reaction is further accelerated in chloroplasts isolated from dark-induced senescing leaves, suggesting the influence of the in vivo status of the chloroplasts on their in vitro stability.Abbreviations DCPIP 2,6-dichlorophenol indophenol - PSI Photosystem I - Chl+ Chlorophyll     

Ageing of chloroplasts in vitro — III. Comparison of the effects of ageing in vitro and senescence on the red absorption band and primary photochemical reactions of sunflower chloroplasts

A comparison of changes in absorption properties and electron transport activities of chloroplasts ageing in vivo and in vitro is made. Chloroplasts from sunflower leaves senescing in vivo during 7 days in dark do not show a blue shift of the red absorption band; in contrast, the shift becomes apparent within 24 h of in vitro ageing of isolated organelles. Photosynthetic activity by chloroplasts is lost much faster during in vitro than in vivo ageing. During in vitro ageing, the rate of degradation of thylakoid membranes as characterised by the shift in the red absorption band and loss in Hill reaction is further accelerated in chloroplasts isolated from dark-induced senescing leaves, suggesting the influence of the in vivo status of the chloroplasts on their in vitro stability.Abbreviations DCPIP 2,6-dichlorophenol indophenol - PSI Photosystem I - Chl Chlorophyll     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

Absorption and Transfer of Light and Photoreduction Activities of Spinach Chloroplasts under Calcium Deficiency: Promotion by Cerium

Chloroplasts were isolated from spinach cultured in calcium-deficient, cerium-chloride-administered calcium-present Hoagland’s media or that of calcium-deficient Hoagland’s media and demonstrated the effects of cerium on distribution of light energy between photosystems II and I and photochemical activities of spinach chloroplast grown in calcium-deficient media. It was observed that calcium deprivation significantly inhibited light absorption, energy transfer from LHCII to photosystemII, excitation energy distribution from PSI to PSII, and transformation from light energy to electron energy and oxygen evolution of chloroplasts. However, cerium treatment to calcium-deficient chloroplasts could obviously improve light absorption and excitation energy distribution from photosystem I to photosystem II and increase activity of whole chain electron transport, photosystems II and I DCPIP photoreduction, and oxygen evolution of chloroplasts. The results suggested that cerium under calcium deficiency condition could substitute for calcium in chloroplasts, maintain the stability of chloroplast membrane, and improve photosynthesis of spinach chloroplast, but the mechanisms still need further study.     

Metal-induced inhibition of photosynthetic electron transport chain of the cyanobacterium Nostoc muscorum

Abstract: This study presents information on the mechanism of inhibition of the photosynthetic electron transport of Nostoc muscorum by chromium (Cr) and lead (Pb). Photosystem II (PS II) was found to be more sensitive both to low and high concentrations of test metals used. A considerable inhibition of photosystem I (PS I) was, however, observed at high concentrations only. Although Cr-induced inhibition of DCPIP photoreduction and lowering of chlorophyll a (Chl a ) fluorescence intensity ( F ₆₈₅) could not be reversed by artificial electron donors (diphenyl-carbazide (DPC), NH₂OH, MnCl₂ and benzidine) of PS II, these electron donors did substantially reverse the Pb-induced inhibition of DCPIP photoreduction as well as the lowering of Chl a fluorescence. Nevertheless, an increase in Chl a fluorescence at high concentrations of Pb suggested that this metal also arrests electron flow on the reducing side of the PS II reaction centre. Besides this, the suppression of fluorescence intensity of phycocyanin at low concentrations of both metals points to the involvement of phycobilisomes in the inhibition of PS II activity. The present study demonstrates that the modes of action of Cr and Pb on PS II are quite different.     

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.     

IAA及KCN在黄瓜子叶离体叶绿体氧化还原作用过程中的相互作用

ＩＡＡ对于不同时期的叶绿体ＤＣＰＩＰ光还原均有促进效应。而对叶绿体的放氧效率的促进则随子叶叶岭的增长而下降至零。ＣＮ＾－（１０＾－３ｍｏｌ／Ｌ）存在时不同程度地抑制叶绿体的ＤＣＰＩＰ光还原和氧化放氧。而ＩＡＡ（１０＾－５,１０＾－４ｍｏｌ／Ｌ）则能消除ＣＮ＾－的抑制作用而仅表现ＬＡＡ原有的促进作用。同时,ＩＡＡ可以释放照光和不照光条件下ＫＣＮ对离体叶绿体分解外源Ｈ２Ｏ２的抑制。叶绿体光系统的光还原     

Effects of Acetazolamide (Diamox), Ethoxzolamide and High Levels of CO2 on Carbonic Anhydrase, Photosystem Activity, and Oxygen Evolution in Euglena gracilis

Investigations using steady-state culture conditions indicate that carbonic anhydrase activity is correlated to the photosynthetic rate in Euglena in some but not all circumstances. When cultures grown with 5% CO₂ were changed to air growth, the photosynthetic rate was independent of the carbonic anhydrase activity. While experiments using the inhibitor acetazolamide indicated a close correlation between photosynthetic capacity and carbonic anhydrase activity, the inhibitor was found to be nonspecific. Acetazolamide altered photosystem activities directly as measured by the photoreduction of DCPIP in chloroplast preparations, whole-cell fluorescence transients of chlorophyll a, and by whole chain photoelectron flow. Ethoxzolamide, another inhibitor of carbonic anhydrase, was also found to inhibit photosystem activities, i.e., the photoreduction of DCPIP, and in vivo photoelectron flow, at high concentrations. Cells grown in 5% CO₂ were less sensitive to the effects of acetazolamide than cells exposed to air. The rate of electron flow in chloroplasts from cells grown with 5% CO₂ and exposed to 10 mM acetazolamide was 2.5-fold faster than that of chloroplasts from air-grown cells exposed to the same concentration of inhibitor. The whole cell chlorophyll a fluorescence transients of cultures grown with high CO₂ were completely different from those of air-grown cells and also showed fewer effects on exposure to acetazolamide. These results suggest a reevaluation of the hypothesis that carbonic anhydrase activity regulates photosynthesis. It is also apparent that results from air-grown and 5% CO₂-grown cultures cannot be directly compared in such studies.     

Inhibition of electron flow around photosystem I in chloroplasts of Cd-treated maize plants is due to Cd-induced iron deficiency

Photosystem I activity of chloroplasts isolated from 21 days old maize seedlings ( Zea mays L. cv. Hidosil) cultivated in a nutrient solution containing different concentrations of Cd (10,20,30μM) was investigated. Cd markedly decreased ferredoxin(Fd)-dependent NADP⁺ photoreduction, while it had no effect on electron transport from 2. 6-dichlorophenolindophenol to methyl viologen, indicating that the metal interferred with electron transport on the reducing side of photosystem I. The decrease in electron transport correlated with a low Fd content, which in turn was correlated with a low Fe concentration, suggesting Cd-induced Fe deficiency. In in vitro experiments direct Cd inhibition of Fd-dependent NADP⁺ photoreduction required much higher Cd concentrations than those observed in Cd-treated plants.     

Effects of Cucumber Mosaic Virus Infection on Photosynthetic Activities of Tobacco Leaves and Chloroplasts

Changes in photosynthetic activities were studied with tobacco (Nicotiana tabacum L.) leaves and chloroplasts infected by cucumber mosaic virus (CMV) at the top, middle and bottom located leaves. Net photosynthetic rate was reduced at all three positioned leaves, with the maximum reduction occurring at the top leaves (31.9% of control). The infected chloroplasts showed a reduction in electron transport rates of the whole chain electron transport, photosystem Ⅱ (PSⅡ) and photosystem Ⅰ (PSⅠ). Since the decline in the whole chain electron transport (15.6% of control, H2O→MV) closely paralleled the decline in PSⅡ activity (20.9% of control, H2O→PBQ), the inhibition of the latter was probably responsible for the overall decrease. Chlorophyll a fluorescence measurements showed a variable reduced fluorescence yield (Fv/Fo) which indicated that PSⅡ was impaired and the CO2 assimilation was disturbed by CMV infection. Fluorescence emission spectra at 77 K indicated that energy distribution between PSⅡ and PSⅠ was affected. F686/F734 of infected leaves and chloroplasts increased and the greatest increase (331.1% of control ) was found in the top leaves. These data may conclude that the infection inhibited mainly the PSⅡ activity.     

Cyclic and noncyclic photophosphorylation during the ontogenesis of high-light and low-light leaves of Sinapis alba

Noncyclic electron transport to ferricyanide and photophosphorylation as well as the methylviologen mediated aerobic and anaerobic photophosphorylation with dichlorophenolindophenol-ascorbate as the electron donor of photosystem I were measured during the development of high-light and low-light adapted leaves of Sinapis alba. Anaerobic methylviologen-catalyzed phosphorylation is more than twice as high as aerobic phosphorylation. The difference between the rates of aerobic and anaerobic phosphorylation is sensitive to dibromothymoquinone. Thus, under anaerobic conditions, methylviologen mediates a cyclic phosphorylation including plastoquinone. All photochemical activities of high-light chloroplasts are about twice as high as that of low-light chloroplasts and show a permanent decline with increasing plant age. The lower activities of low-light chloroplasts correlate with a decrease of electron transport components, such as cytochrome f. This indicates that the number of electron transport chains is decreased under low-light conditions and more chlorophyll molecules interact with one electrontransport chain.Abbreviations Asc ascorbate - Chl chlorophyll a+b - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenolindophenol - HL high light - LL low light - MV methylviologen - PhAR photosynthetically active radiation - PS photosystem     

Vieillissement de l'appareil photosynth?tique II. Effet synergique de la lumi?re et du vieillissement in vitro sur les activit?es photochimiques de chloroplastes isol?s d'?pinard

The consequences of chloroplast ageing in vitro were furtherinvestigated, especially on the photochemical activities ofthese organelles. Ageing of chloroplasts in dark was accompanied by decreasesin activities for photohydrolysis and cyclic and non-cyclicsyntheses of ATP, photoreduction of NADP⁺ and O₂ evolution;but there was no decrease in ferricyanide photoreduction. Therates of decrease in these activities were comparable to therate of increase in chloroplast volume. Complete inhibitionswere reached when maximum chloroplast swelling had occurred,i.e. after 5 to 6 hr of incubation at 20?C in a Tris-NaCl (pH8) medium. Ageing in the light resulted in much accelerateddecreases in activities tested; the loss of capacity for light-inducedshrinkage was also accelerated by the light during ageing. Thus,light acts synergetically towards the ageing process. Moreover,light and, to a less extent, dark ageing, resulted in an uncouplingof chloroplast photophosphorylation and associated electronflow measured by ferricyanide photoreduction. The part of theelectron flow which is induced by coupling (+ ADP, Pi, MgCl₂,pH 8) or by uncoupling (+ NH₄C1, pH 7) was found to be verysensitive to light ageing. The NADP⁺ photoreduction loss wasrestored by addition of the ascorbate-DCPIP electron donor system,suggesting that ageing interferes with the integrity of photosystemII. In many respects, these effects of ageing are comparable withthe action of detergents and fatty acids on the structure andphotochemical activities of chloroplasts, especially in thatthey attack the energy transducing mechanism in chloroplasts. (Received May 24, 1969; )     

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     

The Photochemical Activity of Chloroplasts and Subchloroplast Particles Isolated from Etiolated Bean Leaves Exposed to Continuous Light

The photochemical activity of chloroplasts and subchloroplastparticles isolated from primary bean leaves between the 4thand 24th hour of illumination of etiolated seedlings is thesubject of this paper. The photosystem I activity (oxygen uptakein the presence of MV, DCIP, ascorbate and DCMU), expressedon a unit chlorophyll basis, decreased approximately 10-foldbetween 4 and 8 h of greening. At the same time the photosystemII activity (DCIP photoreduction in the presence of DPC) wasreduced to a half. The photosystem I activity also decreasedin all hitherto investigated fractions which were isolated fromthe digitonin-treated chloroplasts. However, at the initialphase of greening this decrease was the most significant inthe fraction containing heavy particles. After 24 h of greening DCMU, at concentrations higher than 10^–10M, limited the rate of ferricyanide photoreduction by isolatedchloroplasts, whereas after 6 h of greening this effect wasobservable even in the presence of 10^–12 M DCMU. The resultsobtained demonstrated that under those conditions both photosystemswere active after 4 h of greening and PS I activity developedmore rapidly than that of PS II. It also follows from the presenteddata that the water splitting reactions were delayed in developmentas compared to the other reactions investigated, and that PSII units may limit the electron flow in chloroplasts at earlierstages of leaf greening.     

Effect of temperature on senescing detached rice leaves: I. Photoelectron transport activity of chloroplasts

Detached rice(Oryza sativaL cv Mousouri)leaves were induced to senesce in darkness at 0°C(cold), 27°C(room temperature) and 40°C(heat). 2,6-dichlorophenol indophenol (DCPIP) Hill reaction activity of chloroplasts isolated from senescing leaves under all experimental temperatures with H₂O, Mn²⁺ or diphenyl carbazide (DPC) as electron donor declined during the period of incubation. Since DPC and Mn²⁺ augmented 2,6-dichlorophenol indophenol photoreduction by chloroplasts from senescing leaves, damage and/or change in the conformation of a site between H₂O and DCPIP in photosystem II (PS II) is suggested. Heat caused a faster decline of the Hill activity compared to cold or room temperature. However, cold treatment showed no significant effect on the photoelectron transport from H₂O to DCPIP compared with room temperature.     

Effect of exogenous histidine on restoration of electron transfer on the donor side of Photosystem II depleted of Mn

It is shown that restoration of photoinduced electron flow with added Mn²⁺ (measured by photoreduction of DCPIP and photoinduced change of chlorophyll fluorescence yield) in Mn-depleted Photosystem II (PS II) membrane fragments isolated from spinach chloroplasts, is considerably increased by exogenous histidine (His). The stimulating effect of His is not observed if other electron donors (NH₂OH or diphenylcarbazide) are used instead of Mn²⁺. His added alone does not induce electron transfer in Mn-depleted PS II preparations. Investigation of pH dependence of the stimulating effect of 2 mM His shows that the effect is observed only at pH > 5.0, it gives a 50% activation around pH 6.0 and saturates at pH 7.0–7.5. Nearly 200 μM His is required for a 50 effect at pH 7.0. It is suggested that the added His can be involved in stimulation of electron transfer on the donor side of PS II through direct interaction of Mn²⁺ with deprotonated form(s) of His resulting in formation of Mn–His complexes capable of efficient electron donation to PS II (though it is not excluded that His serves as a base that takes part in proton exchange coupled with redox reactions on the donor side of PS II or as an electron donor to the oxidized Mn).     

Effects of Lipoxygenase on the Activities of Photosynthetic Electron Transport in Cucumber Leaves

Photosynthesis and electron transport activity decreased with leaf aging, and however, lipoxygenase (Lox) activity increased correspondingly. Soybean Lox-1 inhibited significantly PSⅡ electron transport activity of chloroplasts isolated from cucumber (Cucumis sativus L. ) cotyledon. But the inhibition could be eliminated by the addition of propyl gallate (PG) or 3, 3, 4, 5, 7-pentahydroxyflavon (PF). The inhibition of PSⅠ activity by soybean Lox-1 was enhanced in the presence of 3, 4, dichlorophenyl-1, 1-dimethylurea (DCMU) or 2, 5-dibromothymoquinone (DBMIB), bfft could be restored to its original level when PG was added. Addition of 2, 2-diphenylcarbonic dihydrazide (DPC) to the mixture of isolated chloroplasts and Lox-1, PSⅡ activity resumed obviously. Chlorophyll a fluorescence study showed that Fm was decreased by Lox-1 and resumed slightly by DPC. Based on the above results, it was suggested that Lox might act at least on three sensitive sites located on Q, PQ and the oxidative side of PSⅠ . The bleaching of chlorophyll and carotenoid stimulated by Lox-l, and the inhibition of PSⅠ electron transport activity by active oxygen might be. one of the important reasons to explaine the effect of Lox on the function of photosynthetic membrane.     

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

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