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     

Induction Kinetics of Millisecond-Delayed Luminescence in Intact Bryopsis Chloroplasts

The induction curve of delayed luminescence emitted from 0.5to 2.5 ms after excitation of dark-adapted intact chloroplastsof the green alga, Bryopsis maxima, showed three transient peaks,L₁, L₂ and L₃ (in order of appearance), at about 0.1, 1 and5 s after theonset of intermittent illumination. Intact chloroplastswere needed for L₂ to appear, whereas L₁ and L₃ were presentin hypotonically treated chloroplasts. L₁ and L₂ are related to the electric field generated acrossthe thylakoid membranesbecause the two peaks parallelled theappearance of the first and second peaks of electrochromic absorptionchanges at 560 nm and they were totally abolished by valinomycinand CCCP. A smaller contribution to the L₁ and L₂ of the protonactivity gradient across the membranes, or of pH changes insideor outside the membranes, was suggested by the partial suppressionof the transient by NH₄CI. L₃ is related to the proton gradient or pH changes because thetransient was inhibited by NH₄CI and CCCP but enhanced by N,N'-dicyclohexylcarbodiimide.In the presenceof valinomycin, which somewhat lowered the peakheight of L₃, the kinetics of delayed luminescence parallelledthat of fluorescence. Electrogenic reactions which occur sequentiallyduring the dark to light transition of the photosynthetic machineryin intact chloroplasts is discussed in connection with transientchanges in delayed luminescence. (Received November 8, 1982; Accepted May 21, 1983)     

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; )     

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; )     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

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; )     

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; )     

Effect of Ammonia on Cellular pH of Anabaena cylindrica Determined with 31P NMR Spectroscopy

The pH changes in the blue-green alga (cyanobacterium) Anabaenacylindrica caused by addition of ammonia were investigated using³¹P NMR spectroscopy. A pH shift of 0.9 or more was observedwhen 30 nM NH₄OH was added to the cell suspension, but no significantcellular pH change was observed with 50 mM NH₄CI, a concentrationhigh enough to stimulate dark CO₂ fixation of this alga. Thechange in cellular pH does not seem to cause ammonia-inducedstimulation of dark CO₂ fixation. (Received June 22, 1985; Accepted January 10, 1986)     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

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.     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO{dot})by thylakoids, chloroplasts and leaves of Spinacia oleraceawas investigated using dimethylsulfoxide as HO{dot} trappingagent. Maximum rates of HO{dot} production by thylakoids asindicated by the formation of methane sulfinic acid were observedunder aerobic conditions in the absence of added electron acceptors.They were higher than 2 µmol (mg Chl h)^–1. Saturationof HO{dot} production occurred at the low photon flux densityof 100 µmol m^–2 s^–1. Trapping of HO{dot} bydimethylsulfoxide suppressed, but did not eliminate light-dependentinactivation of PSI and II suggesting that HO{dot} formationcontributed to the photosensitivity of isolated thylakoids.DCMU inhibited HO{dot} formation. Importantly, methylviologendecreased HO{dot} formation in the absence, but stimulated itin the presence of Fe³⁺. In intact chloroplasts, HO{dot} formation became appreciableonly after KCN had been added to inhibit effective H₂O₂ scavengingby ascorbate peroxidase. It was stimulated by ferrisulfate,but not by ferricyanide which does not penetrate the chloroplastenvelope. Infiltrated spinach leaves behaved similar in principleto intact chloroplasts in regard to HO{dot} formation but HO{dot}production was very slow if detectable at all by the formationof methylsulfinic acid indicating effective radical detoxification. HO{dot} formation is interpreted to be the result of a Fenton-typereaction which produces HO{dot} in chloroplasts from H₂O₂ andreduced ferredoxin, when O₂ is electron acceptor in the Mehlerreaction and radical detoxification reactions are inhibited. (Received November 13, 1996; Accepted April 23, 1996)     

Ammonium Triggers Uptake of NO-3 by Chenopodium rubrum Suspension Culture Cells and Remobilization of their Vacuolar Nitrate Pool

Photoautotrophic cell suspension cultures of Chenopodium rubrumrequire high concentrations of nitrate and ammonium. Duringthe growth phase total NH₄⁺ and the greater portion of NH₃^–were consumed. During the stationary phase nitrate uptake continuedbut at a substantially smaller rate than during the growth phase.During growth the bulk of the absorbed N was incorporated intoprotein, the amount of which was then maintained constant untilsenescence. NH₃^– was accumulated upon transition betweenthe growth and the stationary phase. NH₃^–, like the freeamino acids, was deposited in the vacuole but, unlike thesecompounds, could not be remobilized upon transfer of the cellsinto N-free medium. Readdition of NH₄⁺ to the medium, however,resulted in a mobilization of the vacuolar NH₃^–-pool.Reutilization of both vacuolar N-storage pools must have beenaccomplished by recycling from the vacuole to the cytoplasmbecause N-metabolizing enzymes could not be detected in isolatedvacuoles. Transfer of the cells of the stationary phase intomedium containing NH₃^– and NH₄⁺ resulted in an inductionof nitrate uptake by the cells, but only after a lag phase of4–5 days. It is conceivable that NH₄⁺ induces NH₃^–-translocatingsystems in the plasmalemma and in the tonoplast. (Received December 19, 1988; Accepted March 2, 1989)     

Uptake of Methylamine by Ulva rigida: Transport of Cations and Diffusion of Free Base

Methylamine¹ is taken up rapidly by disks cut from fronds ofUlva rigida (‘Ulva lactuca’) and can be accumulatedat concentrations several hundred times greater than those inthe bathing medium. At pH 8.0 (the pH of sea-water) the relationshipbetween influx and concentration is normally linear up to 0.1–0.3mM, followed by a second, less steep linear phase, the slopeof which decreases or increases with decreasing or increasingexternal pH. When pH is greater than 9.0, however, net uptakesoon ceases. Methylamine influx is greatly reduced at low temperature,by low concentrations of ammonia and, depending on the lengthof storage of the material, by darkness. Influx is also greatlyreduced when disks are pretreated in solutions containing ammoniaand to a much lesser extent when they are pretreated in methylamine,imidazol or nitrate. Methylamine influx lowers intracellularK⁺, increases Cl^– and has no effect on Na⁺. We suggestthat the first linear phase of influx versus concentration reflectsthe operation of an amine cation porter that is rate-limitedby diffusion of CH₃NH₃⁺ through the external unstirred layer,and that the second phase is due to diffusion of CH₃NH₂ intothe tissue.     

Effect of Tribenzylphosphate on the Phosphate Translocator of Isolated Pea Chloroplasts

The effect of tribenzylphosphate on the activity of the phosphatetranslocator of intact pea chloroplasts was tested. The translocatoractivity was followed by O₂ evolution, ¹⁴CO₂ fixation and ³²Pback-exchange. The reagent inhibited 3-phosphoglycerate dependent-photosyntheticactivities probably through an interaction with the PGA translocator. (Received September 11, 1985; Accepted November 21, 1985)     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan     

Borate absorption in excised sugarcane leaves

Borate absorption in sugarcane consists of a rapid and reversibleinflux into the mesophyll cells of the leaf which is completedwithin 20 rains. (Phase I), followed by a slower and irreversibleaccumulatory phase (II). Phase II uptake represents the summationof 3 absorption mechanisms, each dependent upon the externalconcentration. Highly specific mechanisms 1 and 2 transportborate across the initial barrier into the cells, reaction 3carries the borate across the vacuolar membrane. Calcium isshown to be essential for maximum rates of borate absorption.All 3 reactions are inhibited by OH^– through a combinationof competitive inhibition and irreversible disruption of cellularfunction or structure. Temperature changes over the range of10–40 profoundly affect V_maz and K_m1, but have no effecton K_m2 and K_m3. Reactions 1 and 2 are unaffected by 50 mtl Cl^–,SO^–– or H2PO4^–, whereas each of these anionscompetes with H2BO3^– for site 3. Specific metabolic inhibitorswere used to delineate a linkage of mechanisms 1 and 2 to respiratoryelectron transport. Mechanism 3 is coupled to oxidative phosphorylation. ¹Published with the approval of the Director of the Hawaii AgriculturalExperiment Station as Technical Paper No. 954.     

Effect of Nitrate, Ammonium and Some Amino Acids on Growth and Nitrate Reductase Activity in Suspension Cultures of Atropa belladonna

Growth and nitrate reductase activity (NRA) of Atropa belladonnacells were studied in medium supplemented with NaNO₃, NH₄NO₃,and amino acid precursors to tropane alkaloids. Growth and NRAwere stimulated by NH₄⁺ and by proline, by proline plus ornithine,but not by glutamate, in NO₃^–-containing medium. Testedamino acids inhibited neither utilization of inorganic nitrogennor growth. (Received September 30, 1988; Accepted August 28, 1989)     

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; )