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; )     

Comparative study of the reactivation of oxygen evolution in chloroplasts inhibited by various treatments

Reactivation of photosynthetic oxygen-evolution was investigatedwith chloroplasts inhibited by 0.8 M Tris-, 0.8 M Tris-20% acetone-,0.8 M KCl-, 0.5 M NaClO₄- or 1 mM NH₂OH-washing, and with heat-treatedor aged chloroplasts. These chloroplasts restored oxygen evolvingactivity by two successive treatments; incubation of chloroplastswith reduced DPIP, then with Mn^2$, Ca^2$, dithiothreitol andbovine serum albumin under weak illumination (light-reactivation). Some factors required for light-reactivation could be omitteddepending on the inhibition treatment. For example, Mn^2$, Ca^2$and dithiothreitol were not necessary for (1 mM NH₂OH-STN (pH7.0)-washed)-DPIP-treated chloroplasts, and dithiothreitol for(Tris-acetone (pH 8.4)-washed)-DPIP-treated chloroplasts. Uncouplers, such as atebrin, CCCP, DCCD and NH₄Cl, inhibitedthe lightreactivation. The Mn and Ca contents of the chloroplasts were determined withinhibited and DPIP-treated chloroplasts. The Mn content of thechloroplasts tended to decrease with increasing pH of the washingmedium for inhibition. The Ca content decreased when chloroplastswere washed with 0.8 M KCl. (Received November 22, 1974; )     

Photo-induced H+ Transport between Chloroplasts and the Cytoplasm in a Protoplasmic Droplet of Characeae

Svintitskikh, V. A., Andrianov, V. K. and Bulychev, A. A. 1985.Photo-induced H⁺ transport between chloroplasts and the cytoplasmin a protoplasmic droplet of Characeae.—J. exp. Bot. 36:1414–1429. The effects of light on the membrane potential and cytoplasmicpH of isolated droplets of protoplasm from Nitella have beenstudied using microcapillary electrodes and pH-sensitive antimonymicro-electrodes. Illumination of chloroplast-containing dropletscaused a change of the membrane potential with a concomitantacidification of both the cytoplasm and the outer medium, butit had no effect on the electrical resistance of the surfacemembrane. Treatment of protoplasmic droplets with uncouplers(NH₄Cl and CCCP) resulted in a complete inhibition of the light-inducedacidification of the cytoplasm, whereas the energy transferinhibitor DCCD had no effect. A correlation between the formationof a pH gradient across the thylakoid membrane and the acidificationof the cytoplasm was explicable in terms of the assumption ofrestricted spatial communication between the intra-thylakoidvolume and the cytoplasm in intact chloroplast. The photo-inducedacidification of the boundary layer of an external medium wasmarkedly stimulated under the action of inhibitors of H⁺-ATPaseDCCD and DES. These findings suggest that the active extrusionof H⁺ from the cytoplasm into the external medium is not drivenby an ATPase, although H+-conducting channels of membrane ATPaseprovide a pathway for a passive diffusion of protons from outsideinto the cytoplasm Key words: Transport of protons, protoplasmic droplet, intact chloroplasts, Characeae     

The Generation of Non-Spherical Oat Protoplasts Indicates that Hyperpolarization is both Necessary and Sufficient for Stimulating Membrane Incorporation into the Plasma Membrane

We have devised conditions which produced isolated protoplastsof non-spherical shape and which, therefore, affected the mechanismsthat control the exchange of membrane material between the plasmamembrane and an intracellular membrane reservoir. Non-sphericalprotoplasts of Avena sativa were obtained if protoplasts weretreated with hypertonic shock in the presence of 1.0 mol m^–3LaCl₃ at pH 8.3. This indicated that their ability to removeplasma membrane material via endocytotic vesiculation was suppressed.Non-spherical protoplasts were obtained under isotonic conditionsif protoplasts were incubated with 1.0 mol m^-3 LaCl₃ at pH 8.3and the proton carrier CCCP (12 mmol m^–3) was added. Thenon-spherical protoplasts had intact membranes as judged bystaining with fluorescein diacetate. The loss of the sphericalshape was reversible. On addition of EDTA protoplasts resphericulatedimmediately. Incubation in isotonic solution at pH 8.3 containingeither only 1.0 mol m^–3 LaCl₃ or only CCCP did not influencethe protoplast shape. We conclude that the membrane hyperpolarizationinduced by CCCP at high pH acted to stimulate the incorporationof membrane material into the plasma membrane and, subsequently,produced nonspherical protoplasts if the removal of membranematerial was simultaneously suppressed. This demonstrates thatmembrane incorporation and removal are two largely independentprocesses.     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Comparison between Electron Transfers through Plastocyanin in Spinach Chloroplasts and Cytocbrome C2 in Rhodopseudomonas sphaeroides

Photosynthetic electron transfers through the water-solubleperipheral membrane proteins of plastocyanin and cytochromec₂, were studied in spinach chloroplasts and the photosyntheticbacterium Rhodopseudomonas sphaeroides. In spinach chloroplasts,the rate of flash-induced oxidation of cytochrome f was highlydependent on the salt concentration in the suspending medium.The maximum rate with a half time of 200 µs was observedin the presence of 50 mas KCl or 5 mM MgCl₂. The salt effectwas similar to that on the reaction rate between P700 in thylakoidfragments and externally added plastocyanin. On the other hand,in intact cells of R. sphaeroides, in which cytochrome c₂ islocated in the periplasmic space exposed to the outer ionicenvironment, the rate of cytochrome c₁ oxidation via cytochromec₂ was almost independent of salt concentration. This independencewas a contrast to the strong dependence on salt concentrationof reactions between isolated reaction centers and cytochromec₂ These results suggest that plastocyanin reacts collisionallywith the photosystem I reaction center and cytochrome b₆f complexin a manner that is controlled by the surface electrostaticpotential. Cytochrome c₂, on the other hand, reacts with thebacterial reaction center and cytochrome bc₁ complex probablyby forming a complex prior to activation of the reaction center. ¹ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan.     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Radial Hydraulic Conductivity of Individual Root Tissues of Opuntia ficus-indica (L.) Miller as Soil Moisture Varies

The constraints on water uptake imposed by individual root tissueswere examined forOpuntia ficus-indicaunder wet, drying, andrewetted soil conditions. Root hydraulic conductivity (L_P) andaxial conductance (K_h) were measured for intact root segmentsfrom the distal region with an endodermis and from midroot witha periderm;L_Pwas then measured for each segment with successivetissues removed by dissection. Radial conductivity (L_R) wascalculated fromL_PandK_hfor the intact segment and for the individualtissues by considering the tissue conductivities in series.Under wet conditions,L_Rfor intact distal root segments was lowestfor the cortex; at midroot, where cortical cells are dead,L_Rforthe cortex was higher and no single tissue was the predominantlimiter ofL_R.L_Rfor the endodermis and the periderm were similarunder wet conditions. During 30d of soil drying,L_Rfor the distalcortex increased almost three-fold due to the death of corticalcells, whereasL_Rfor the midroot cortex was unaffected;L_Rforthe endodermis and the periderm decreased by 40 and 90%, respectively,during drying. For both root regions under wet conditions, thevascular cylinder had the highestL_R, which decreased by 50–70%during 30d of soil drying. After 3d of rewetting, new lateralroots emerged, increasingL_Rfor the tissues outside the vascularcylinder as well as increasing uptake of an apoplastic tracerinto the xylem of both the roots and the shoot. The averageL_Rforintact root segments was similar under wet and rewetted conditions,but the conductivity of the tissues outside the vascular cylinderincreased after rewetting, as did the contribution of the apoplasticpathway to water uptake. Opuntia ficus-indica; prickly pear; root hydraulic conductivity; endodermis; periderm; apoplast; lateral root emergence     

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; )     

The Effect of Various Nitrogen Sources upon the Production of Extracellular Polysaccharide by the Blue-Green Alga Anabaena flos-aquae A-37

Anabaena flos-aquae A-37 was grown in bacteria-free cultureon a variety of nitrogen sources. Extracellular polysaccharideproduction was found to be a part of the normal metabolic processescausing the liberation of excess carbohydrates into the surroundingmedium rather than the result of cellular lysis. Non-adaptedcells supplied with NH₄Cl produced abundant polysaccharide.However, the population did not increase. Cells adapted to NH₄Clproduced smaller quantities of polysaccharide, but the populationdid increase. Polysaccharide production per cell was similaron Mg(NO₃)₂, KNO₃, NaN0₃, NH₄NO₃, and NH₄Cl. It was concludedthat an adequately utilized nitrogen source does not affectpolysaccharide production in A. flos-aquae A-37.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

Embryogenic Response and Mitotic Instability in Callus Cultures Derived from Maize Inbred Lines Differing in Heterochromatic Knob Content of Chromosomes

Four families of sister inbred lines derived from a tropicalmaize variety have been evaluated for their ability to formcallus cultures with a morphogenetic response. Lines were homozygousfor heterochromatic knobs at 6L₂, 6L₃, 7L and 8L₁but differedfor the presence or absence of K2L, K3L, K7S and K9S. Cleardifferences in embryogenic response were observed between thefamilies of inbreds. Only one family formed friable, highlyembryogenic type II calli; the other families formed slow growingnon-embryogenic or poorly embryogenic cultures. All the genotypesscreened showed a similar response to the two culture mediatested, suggesting that genetic factors are responsible forthe major differences among the families. Mitotic abnormalitieswere investigated in Feulgen preparations of most cultures.Anaphase bridges resulting from delayed chromatid separation,typical bridges and fragments were observed. In a previous study,delayed chromatids were shown to be held together at heterochromaticknob sites, while typical bridges would be formed by dicentricchromatids arising from breakage-fusion-bridge cycles initiatedby chromosome arms broken during the primary event. In the presentstudy, the frequency of both types of bridges was not strictlycorrelated with the knob content of the genotypes analysed.This suggests that knobs may undergo alterations in cultureleading to mitotic disturbance, and that this response may begenotype dependent.Copyright 1998 Annals of Botany Company Zea maysL., maize, plant tissue culture, somatic embryogenesis, somaclonal variation, heterochromatin, C-banding, breakage-fusion-bridge cycle.     

Ultrastructural Changes in Chloroplasts of Quercus rotundifolia Lam. in Response to Evernic Acid

The amount of total chlorophyll, chlorophylls a and b as wellas the ratio of a to b decreased in chloroplasts isolated fromQuercus rotundifolia leaves, kept for 17 d in a solution of35.5 µM evernic acid in 1 mM Na HCO₃, when compared withthe chloroplasts of control leaves (kept in NaHCO₃). The chloroplastsin the spongy parenchyma were smaller and the amount of starchand plastoglobuli lower. The number of grana per chloroplastsection, the number of thylakoids per grana and the height ofgrana stacks were also less in the chloroplasts of leaves treatedwith evernic acid. Quantitative ultrastructural differenceswere determined by means of electron microscopy and image analysistechniques. Quercus rotundifolia Lam., chloroplasts, ultrastructure, lichens, evernic acid     

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; )     

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     

Role of pulvinar chloroplasts in light-driven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.)

Laminar pulvini of bean (Phaseolus vulgaris L.) contain numerouschloroplasts in cells of their motor tissue. The quantitativerelationships of the chloroplast pigments, chlorophyll a andb, ß-carotene, lutein, neoxanthin as well as the xanthophyllcycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin)were similar to those of mesophyll chloroplasts from leafletlaminae. Exposure of pulvinules to light caused deepoxidationof violaxanthin to zeaxanthin, showing that the xanthophyllcycle is functioning. Chlorophyll fluorescence analysis of pulvinulesconfirmed that their chloroplasts are capable of both photosyntheticelectron transport and non-photochemical fluorescence quenching,showing that they build up a considerable transthylakoid protongradient in the light. Application of DCMU to excised pulvinulesand laminar discs, as well as to pulvinules of intact, attachedterminal leaflets blocked electron transport and fluorescencequenching. Application of the uncoupler CCCP to intact pulvinulesalso prevented non-photochemical fluorescence quenching. Therate of movement of the low-light-adapted terminal leaflet inresponse to exposure of its pulvinule to overhead red light(500 µmol m^–2 s^–1) was reduced when the pulvinulewas pretreated with DCMU. The pulvinar response to overheadblue light (50 µmol ^–2 s^–1), which is morepronounced than to red light, was not affected by similar pretreatmentwith DCMU. Pretreatment with CCCP caused a short lag in theresponse to red light, but did not affect its subsequent rate.The results suggest that the pulvinar response to red, but notto blue light, requires non-cyclic electron transport and theresulting generation of ATP Key words: Leaf movements, light, non-cyclic electron transport, Phaseolus, pulvinar chloroplasts     

Plasmalemma Transport of HCO-3 and OH- in Chara corallina: Inhibitory Effect of Ammonium Sulphate

The influence of (NH₄)₂SO₄ on ¹⁴C assimilation and cyclosisin internodal cells of Chara corallina was investigated. Severeinhibition of ¹⁴C assimilation was found at pH values above7·0, this inhibition being correlated with the exogenouslevel of NH3 rather than NH⁺₄. Cyclosis was also affected athigher concentrations of (NH₄)₂SO₄. This effect was similarlycorrelated with exogenous levels of NH₃. ¹⁴C assimilation was inhibited non-competitively by (NH₄)₂SO₄,the apparent Km being increased from 0·55 to 1·5mM. The results suggest that the site(s) of inhibition is locatedat the plasmalemma, rather than at the chloroplasts. (Evidencein support of in vivo uncoupling of photophosphorylation, bylow concentrations of (NH₄)₂SO₄, was not obtained). Significant perturbation of the OH^– efflux pattern wasobserved as the level of (NH₄)₂SO₄ was increased. Induced migrationof efflux sites indicates that NH₃ may interfere with the cellularmechanism that controls OH^– transport. Using a cell-segmentisolating chamber it was shown that (NH₄)₂SO₄ inhibited OH^–efflux rather than HCO^–₃ transport. This inhibitory effectwas readily reversible. These data are discussed in terms of a possible relationshipbetween the observe NH₄)²SO₄ stimulation of ³⁶Cl^– influxand the effect of this compound on ¹⁴C assimilation.