Two forms of the Photosystem II D1 protein alter energy dissipation and state transitions in the cyanobacterium Synechococcus sp. PCC 7942

Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. D1: 1 predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only D1:1 have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing D1:2. We show that when dark-adapted or under low to moderate light, cells with D1:1 have higher non-photochemical quenching of PS II fluorescence (higher q_N) than do cells with D1:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697–698 nm in cells containing D1:1 than in cells with D1:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing D1:1 than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D1:1 confers capacity for extreme State II which may be of benefit under low and variable light.Abbreviations D1 the atrazine-binding 32 kDa protein of the PS II reaction centre core - D1:1 the D1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942 - D1:2 an alternate form of the D1 protein induced under excess excitation in Synechococcus sp. PCC 7942 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Fo minimal fluorescence in the dark-adapted state - Fo minimal fluorescence in a light-adapted state - F_M maximum fluorescence with all quenching mechanisms at a minimum, measured in presence of DCMU - F_M maximal fluorescence in a light-adapted state, measured with a saturating flash - F_Mdark maximal fluorescence in the dark-adapted state - F_V variable fluorescence in a light-adapted state (F_M-Fo) - PAM pulse amplitude modulated fluorometer - q_N non-photochemical quenching of PS II fluorescence - q_N (dark) q_N in the dark adapted state - q_P photochemical quenching of fluorescence     

Proton translocation in intact cells of Thiobacillus denitrificans

Proton translocation assessed by the quinacrine fluorescence technique was compared with oxygen uptake during thiosulphate oxidation by cells of Thiobacillus denitrificans. The addition of thiosulphate to cell suspensions resulted in an outwardly directed proton translocation as reflected by an increased quinacrine fluorescence. Compared to the O₂ uptake activity, the proton translocating system was much more sensitive to proton conductors, other ionophores and inhibitors of electron transport. The results indicate that (a) the proton-translocation activity (membrane energization) is enhanced in aged cell suspensions, (b) intactness of the cytoplasmic membrane is essential for establishing a protonmotive force in cells, (c) the fluorescence increase and proton translocation are reversible processes, (d) inhibitors of electron transport may also act as proton conductors by altering the integrity of the cytoplasmic membrane.Abbreviations CCCP carbonyl cyanide m-chlorophenyl-hydrazone - DBP 2,4-dibromophenol - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - PCP pentachlorophenol - TPB tetraphenyl boron - TTFA 1-[thenoyl-(2)]-3,3,3-trifluoracetone     

Reactions of seven basic fluorochromes with unfixed cells obtained from the salivary glands of the dipteran fly Megaselia scalaris Loew (Phoridae)

Summary Seven basic fluorochromes with varying specificities were used to stain the large squamous epithelial cells isolated from the larval salivary glands of Megaselia scalaris (Phoridae). Although the EDTA-based method selected for isolating the cells produced permeabilization and a loss of viability of the cells, consistent results were obtained with the various fluorochromes. The classical pattern of green nuclear and red cytoplasmic fluorescence observed in cells stained with acridine orange could be changed to green cytoplasmic and red nuclear fluorescence by pretreatment with RNase. The predominantly cytoplasmic and nucleolar fluorescence obtained with pyronine Y could be changed to mainly nuclear fluorescence by RNase pretreatment. The other five fluorochromes tested were not affected appreciably by extraction with RNase. Quinacrine mustard, dicarbocyanine (DiOC₆(3)), and rhodamine 123 produced primarily cytoplasmic and nucleolar fluorescence, while nile red revealed mainly cytoplasmic lipid droplets. Phosphine 3R initially stained lipid droplets but very rapidly redistributed throughout the cytoplasm and nucleus. Because of their large size, flatness, and content of histochemically demonstrable components, the cells of Megaselia are especially appropriate for use as optical objects or controls in various studies. New methods of isolating the cells, however, will be needed to prevent permeabilization and loss of viability of the cells.     

Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Internal pH (pH_i) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pH_i and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pH_i was 7.29 (±0.11) for cells in the presence of 2 mM HCO ₃ ^– . In the absence of HCO ₃ ^– the pH_i was decreased by 0.8 pH unit. The importance of these changes in pH_i is considered in relation to inorganic-carbon uptake.Abbreviations AM acetoxymethylester - BCECF 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - pH_i intracellular pH     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

Functional analysis of the iron-stress induced CP 43′ polypeptide of PS II in the cyanobacterium Synechococcus sp. PCC 7942

Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43, which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43 apoprotein (designated isiA ^–) and (ii) flavodoxin (designated isiB ^–). The function of CP 43 was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA ^– and isiB ^– strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43 and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43 is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB ^– strain grown under iron-deficient conditions, CP 43 was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (F_o) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.Abbreviations Chl chlorophyll - CP 43, CP 47 and CP 43 Chl a binding protein complexes of indicated molecular mass - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_m and F_m fluorescence when all PS II reaction centers are dosed in dark- and light-acclimated cells, respectively - F_o fluorescence when all PS II reaction centers are open in dark acclimated cells - F_v variable fluorescence after dark acclimation (F_m–F_o)     

Metabolism of reduced pyridine nucleotides in ascites cell nuclei

Summary 1. The conditions are described under which the fluorescence due to reduced pyridine nucleotides can be studied separately at nuclear and cytoplasmic sites of glass-grown ascites cells, by the use of a flow chamber in the microfluorimeter ofChance andLegallais.2. The addition of glucose to ascites cells leads to a reduction of pyridine nucleotides within the nucleus, thus providing evidence for the participation of nuclear pyridine nucleotides in cellular metabolism.3. Although generally nuclear and cytoplasmic pyridine nucleotides parallel each other in their response to different metabolic conditions, there are few instances (e.g., Amytal) where they do not show such parallelism. This is discussed with regard to the problem of reoxidation of nuclear reduced pyridine nucleotides.With 2 Figures in the Text     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

The cytoplasmic pH in photosynthesizing cells of the green alga Chlorella fusca,measured by P-31 NMR spectroscopy

P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar P_i indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a P_i-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this P_i-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal P_i and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG 2-Deoxyglucose - dG-6-P 2-deoxyglucose-6-phosphate - DCMU 3,4-dichlorophenyl-dimethylurea - MOPSO 3-(N-morpholino)-2-hydroxypropane sulfonic acid - P-31 NMR P-31 nuclear magnetic resonance     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Photoinhibition in seedlings of <Emphasis Type="Italic">Fraxinus</Emphasis> and <Emphasis Type="Italic">Fagus</Emphasis> under natural light conditions: implications for forest regeneration?

Ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.) seedlings were grown in the field under three levels of natural light: (1) open, (2) gap and (3) shade. Light acclimation of photosynthesis was characterized by means of modulated chlorophyll a fluorescence of intact leaves and growth parameters were measured at the end of the growing season. Measurements of maximum photochemical efficiency (F_v/F_m) of dark-adapted leaves at intervals through the day showed that ash had a higher F_v/F_m than beech in open and gap plots but not in shade plots. This indicated a larger build-up of photoinhibition in beech under gap and open conditions. Steady-state light response curves of the operating efficiency of PSII (F_q/F_m), the electron transport rate (ETR) and the photochemical efficiency factor (F_q/F_v) showed greater variability across light treatments in ash than in beech. Both species exhibited similar responses of non-photochemical quenching (NPQ) to light. When the data were normalized to the mean maximum irradiance in the growth environment, all photochemical parameters showed a reduction in variation across treatments, indicating that light acclimation in the two species occurred primarily through adjustments in rates of photochemistry. Adjustments in thermal heat dissipation were small in both species. This pattern was stronger in ash, suggesting a greater degree of phenotypic plasticity in photosynthetic capacity in this earlier successional species. Contrary to our expectations, the build-up of photoinhibition in beech did not appear to have a negative effect on total biomass accumulation relative to ash.Abbreviations ETR Electron transport rate - F_m Maximal fluorescence in the dark-adapted state - F_o Minimal fluorescence in the dark-adapted state - F_s Steady-state fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in the dark-adapted state - F_v/F_m Maximum photochemical efficiency of photosystem II in the dark-adapted state - F_m Maximal fluorescence in actinic light - F_o Minimal fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in actinic light - F_q=F_m–F_s; F_q/F_m Operating efficiency of photosystem II in actinic light - F_q/F_v Efficiency factor of PSII photochemistry (also referred to as qP—photochemical quenching) - F_v/F_m Maximum efficiency of PSII under actinic light if all reaction centres were open - NPQ Stern-Volmer non-photochemical quenching - PPFD Photosynthetic photon flux density (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a cosine-corrected quantum sensor - PPFFR Photosynthetic photon flux fluence rate (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a spherical quantum sensor. Fluorescence nomenclature follows Oxborough and Baker (2000).     

Dinuclear platinum complexes with<Emphasis Type="Italic"> N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>′-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis,cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells

A series of N,N-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl=2-aminoethyl, 3-aminoprop-1-yl and 4-aminobut-1-yl) was functionalized with trans-platinum DNA-binding moieties. Cytotoxicity testing in A2780 human ovarian carcinoma cells revealed high anticancer activity of the formed cationic dinuclear platinum complexes. The cationic dinuclear platinum complexes with the shortest aminoalkyl chain were shown to be the most active, which agrees with the structure–activity relationship found for the corresponding free ligands without platinum. The N,N-bis(aminoalkyl)-1,4-diaminoanthraquinones partly circumvent cisplatin resistance, whereas their dinuclear platinum complexes were found susceptible to the resistance mechanisms in A2780cisR. The platinum complexes have resistance factors comparable to the control dinuclear complex BBR3005 [{trans-PtCl(NH₃)₂}₂{-(NH₂(CH₂)₆NH₂)}](NO₃)₂. The 1,4-diaminoanthraquinone moiety is fluorescent, and thus the cellular processing of the compounds could be monitored by time-lapse digital fluorescence microscopy. The intercalators without platinum were shown to enter the cells within minutes. The platinum complexes enter the cells more slowly. Most likely, the positive charges of the platinum complexes hamper the diffusion through the membrane. Interestingly, the platinum complexes are processed differently than the platinum-free compounds by the cells. After 24 hours the fluorescent platinum complexes are encapsulated in large vesicles in the cytosol. Co-localization of the anthraquinone fluorescence with Lysotracker Green DND-26 shows that these vesicles are acidic compartments, probably lysosomes.Abbreviations AQ2 N,N-bis(2-aminoethyl)-1,4-diaminoanthracene-9,10-dione - AQ3 N,N-bis(3-aminoprop-1-yl)-1,4-diaminoanthracene-9,10-dione - AQ4 N,N-bis(4-aminobut-1-yl)-1,4-diaminoanthracene-9,10-dione - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide - PAQ2 [{trans-PtCl(NH₃)₂}₂(-AQ2)](NO₃)₂ - PAQ3 [{trans-PtCl(NH₃)₂}₂(-AQ3)](NO₃)₂ - PAQ4 [{trans-PtCl(NH₃)₂}₂(-AQ4)](NO₃)₂ - PBS phosphate buffered saline     

Gibberellic acid induces cytoplasmic acidification in maize coleoptiles

Indole-3-acetic acid (IAA), fusicoccin and weak acids all lower the cytoplasmic pH (pH_i) and induce elongation growth of maize (Zea mays L.) coleoptiles. Gibberellic acid (GA₃) also induces elongation growth and we have used confocal laser scanning microscopy to study the effects of GA₃ on pH_i employing the pH-indicator dyes, 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein and carboxy-semi-naphthorhodafluor-1. We confirm that GA₃ induces growth significantly in light-grown but only slightly or not at all in dark-grown coleoptiles. The growth induced by IAA treatment was similar in light- and dark-grown coleoptiles. The pH_i decreased by up to 0.6 units during the first 7 min of GA₃ or IAA treatment of both light- and dark-grown coleoptiles. Gibberellic acid inhibited IAA-induced growth of dark-grown coleoptiles. Hence, in dark-grown coleoptiles GA₃ may activate either directly or indirectly reactions that interfere with the signalling pathway leading to elongation growth. The possible role of pH_i in growth is discussed.Abbreviations ABA abscisic acid - AM acetoxymethyl ester - BCECF 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein - [Ca²⁺]_i cytoplasmic free calcium - GA_(n) gibberellin A_(n) - GA₃ gibberellic acid - IAA indole-3-acetic acid - PGR plant growth regulator - pH_i cytoplasmic pH - Pipes piperazine-N,N-bis[2-ethanesulfonic acid] - Snarf-1 carboxy-semi-naphthorhodafluor-1 We thank Dr R. King (CSIRO, Canberra) for providing the GA₁ and T. Phillips for processing the photographic material. H.R. Irving was supported by an Australian Research Council Research Fellowship and the work was supported by an Australian Research Council grant.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Photosynthetic electron transfer and membrane energization in spheroplasts and membrane vesicles of the thermophilic cyanobacterium Synechoccus 6716

The higher the incubation temperature, the higher the light intensity that membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 require for the saturation of O₂-production. If membrane vesicles are incubated at temperatures at which intact cells are growing optimally, photosynthetic O₂-production and membrane energization decrease rapidly, suggesting that the thermophilic properties are rapidly lost. If membrane integrity is maintained (spheroplasts) the harmful effect of higher temperatures is much less. The effects of 2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone (DBMIB), 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide (S-13), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and N,N-dicyclohexylcarbodiimide (DCCD) are the same as in chloroplasts, be it that DCCD acts as an electron transfer inhibitor at higher concentrations. The supposed alternative site of DCMU inhibition in cyanobacteria is rejected.Spheroplasts show a reversible energy-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) caused by illumination. ATP hydrolysis only give rise to fluorescence quenching in membrane vesicles. Long incubation at higher temperatures reduces the fluorescence quenching of membrane vesicles and spheroplasts, the latter being more stable than the former.Abbreviations 9AA 9-aminoacridine - ACMA 9-amino-6-chloro-2-methoxyacridine - Chl chlorophyll - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DCP 1,5-diphenylcarbazide - PMS methyl-phenazoniummethosulfate - PS-I photosystem I - PS-II photosystem II - S-13 5-chloro-3-t-butyl-2 chloro-4-nitrosalicylanilide     

Reduced levels of cytochrome b 6/f in transgenic tobacco increases the excitation pressure on Photosystem II without increasing sensitivity to photoinhibition in vivo

We have examined tobacco transformed with an antisense construct against the Rieske-FeS subunit of the cytochromeb ₆ f complex, containing only 15 to 20% of the wild-type level of cytochrome f. The anti-Rieske-FeS leaves had a comparable chlorophyll and Photosystem II reaction center stoichiometry and a comparable carotenoid profile to the wild-type, with differences of less than 10% on a leaf area basis. When exposed to high irradiance, the anti-Rieske-FeS leaves showed a greatly increased closure of Photosystem II and a much reduced capacity to develop non-photochemical quenching compared with wild-type. However, contrary to our expectations, the anti-Rieske-FeS leaves were not more susceptible to photoinhibition than were wild-type leaves. Further, when we regulated the irradiance so that the excitation pressure on photosystem II was equivalent in both the anti-Rieske-FeS and wild-type leaves, the anti-Rieske-FeS leaves experienced much less photoinhibition than wild-type. The evidence from the anti-Rieske-FeS tobacco suggests that rapid photoinactivation of Photosystem II in vivo only occurs when closure of Photosystem II coincides with lumen acidification. These results suggest that the model of photoinhibition in vivo occurring principally because of limitations to electron withdrawal from photosystem II does not explain photoinhibition in these transgenic tobacco leaves, and we need to re-evaluate the twinned concepts of photoinhibition and photoprotection.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlophenyl)-1,-dimethylurea - Fo and Fo minimal fluorescence when all PS II reaction centers are open in dark- and light-acclimated leaves, respectively - Fm and Fm maximal fluorescence when all PS II reaction centers are closed in dark- and light-acclimated leaves, respectively - Fv variable fluorescence (Fm-Fo) in dark acclimated leaves - Fv variable fluorescence (Fm-Fo) in lightacclimated leaves - NPQ non-photochemical quenching of fluorescence - PS I and PS II Photosystem I and II - P680 primary electron donor of the reaction center of PS II - PFD photosynthetic flux density - Q_A primary acceptor quinone of PS II - q_p photochemical quenching of fluorescence - V+A+Z violaxanthin+antheraxanthin+zeaxanthin