Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K+ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H+ extrusion no longer requires the presence of K+ in the medium. The (ferricyanide reduced)/(H+ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H+ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K+ release to the medium. The ratio (ferricyanide reduced)/(H+ extruded + K+ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump...
Photosynthetic energy conversion was investigated in five species of marine unicellular algae, (Dunaliella tertiolecta, Thalassiosira pseudonana, T. weisflogii, Skeletorema costatum, Isochrysis galbana) representing three phylogenetic classes, which were grown under steady state conditions with either light or inorganic nitrogen as a limiting factor. Using a pump and probe fluorescence technique we measured the maximum change in variable fluorescence yields, the flash intensity saturation curves for the change in fluorescence yields and the kinetics of the decay in fluorescence yields. Under all growth irradiance levels nutrient replete cells exhibited approximately the same changes in fluorescence yields and similar fluorescence decay kinetics. The apparent relative absorption cross-section of photosystem II, calculated from the slope of the flash intensity saturation curves, generally increased as cells shade adapted. The decay kinetics of the fluorescence yield following a saturating pump flash can be expressed as the sum of three exponential components, with half-times of 160 and 600 microseconds and 30 to 300 milliseconds. The relative contribution of each component did not change significantly with growth irradiance. As cells became more nitrogen limited...
Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3* × Carlsberg II R and S, were used to test for differences in fluxes of Ca2+ across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca2+ postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca2+. Washout curves of 45Ca2+ from R and S protoplasts revealed three compartments—presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca2+). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca2+ and our initial rates of 45Ca-labeled Ca2+ uptake, we show that measurement of the unidirectional influx of Ca2+ across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly...
A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem II unit sizes than do bundle sheath chloroplasts. The larger photosystem II units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCl levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to DCMU, salinity altered photosystem II in bundle sheath cells but not in mesophyll cells. This result may indicate different ionic distributions in the two cell types or, alternatively, different responses of the two chloroplast types to environmental change.
We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H+ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (Ci). Light and Ci stimulated the uptake of sodium, but the stimulation by Ci was observed only when Ci was present at the time sodium was provided. Amiloride, a potent inhibitor of Na+/H+ antiport and Na+ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na+/H+ antiport and Na+ uniport.
The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent Km-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; Vmax app was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 106 vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H+-translocating enzyme activities revealed Km app values of 0.45 millimolar for ATPase (EC 22.214.171.124) and 23 micromolar for pyrophosphatase (PPase) (EC 126.96.36.199). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH+ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp...
Addition of 5 micromolar Cu2+, Cd2+, and Zn2+ was inhibitory to 10 micromolar H2O2-supported Hill activity (dichlorophenolindophenol reduction) and O2 evolution in membrane preparation from Anacystis nidulans. The reversal of Cd2+ and Zn2+ inhibition, in contrast to Cu2+, by exogenously added catalase (EC 188.8.131.52) suggested that the former cations were inhibitory to H2O2 degradation. Ascorbic acid (20 micromolar) supported 27% of the Hill activity which was insensitive to DCMU (10 micromolar) and the remaining activity, attributable to the DCMU sensitive process, was sensitive to inhibition by Cu2+ only. It is suggestive that the action site of Cd2+ and Zn2+ is located between the electron donation sites of H2O2 and ascorbic acid, while that of Cu2+ is located beyond it. Electron donation by reduced glutathione was insensitive to DCMU and Cu2+, indicating that the action site of Cu2+ is prior to its electron donation site. Further, the phenanthroline (10 micromolar) reversal of Cu2+ inhibition of Hill activity suggested a tentative action site of Cu2+ at the level of cytochrome.
The effect of anoxia on subsequent uptake and transport of K, Rb, and Na was examined with seedlings of barley (Hordeum vulgare L.), corn (Zea mays L.), and tall fescue (Lolium × Festuca hybrid derivative) to further our understanding of xylem loading. Roots were incubated in solutions depleted of O2 by flushing with N2 gas. After 1 hour exposure, plants were returned to aerated solutions for 16 hours prior to measuring uptake and transport. For each species, anoxia pretreatment significantly enhanced Na transport to the shoot. The rate of Na accumulation into roots, however, was not affected. There was no enhancement of either K or Rb accumulation in shoots, indicating specificity for Na transport. A minimum exposure to anoxia of 30 minutes and a minimum of 12 hours elapsed time was necessary to achieve the maximum rate of Na transport to the shoot in barley seedlings. Accumulation of Na in the shoot of both the control and anoxia pretreated barley plants was inhibited by anoxia and by addition of the proline analog, l-azetidine-2-carboxylic acid, during the uptake period. Enhancement of Na transport was associated with a proportional increase in the rate of synthesis of a membrane bound protein with a molecular weight of 78,000 daltons.
We investigated H+ fluxes during circadian rhythmic movements of Samanea saman leaflets by monitoring the pH of a weakly buffered medium bathing extensor or flexor motor tissue excised at different times during 51 hours of darkness. Experiments were made in media of two different osmotic potentials: −0.3 megapascal (control medium) and −1.2 megapascals (control medium supplemented with 0.4 molar mannitol or sorbitol). Both extensor and flexor tissue took up H+ from the control medium at all times when the initial pH was 5.5. Rates of uptake by the extensor varied rhythmically in phase with the leaflet movement rhythm, whereas rates for the flexor were similar at all times. Addition of 0.4`molar mannitol (or sorbitol) to the medium magnified the amplitude of the rhythm in H+ uptake and release by extensor tissue and revealed a rhythm with flexor tissue. In the flexor, mannitol promoted H+ release (or reduced H+ uptake) at all times. We propose that mannitol reduces flexor cell turgor, and that low turgor activates the H+ pump. The magnitude and/or direction of pH changes varied with the initial pH of the medium. The pH values after 60 minutes converged to a narrow range, suggesting that cell wall pH might be regulated.
Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl− and H2PO4− uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K+ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K+ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl− FC induced intracellular acidification. Since H+ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K+/A− symport or HCO3−/A− antiport for anion uptake. Though not suitable to reveal the actual H+/A− stoichiometry...
The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization...
White light (25 watts per square meter) induced an increase in plasma membrane K+-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K+ on the cytoplasmic side of the patch and 50 millimolar K+ in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K+ over Na+ and Cl−. One (named PKC1) had a maximum conductance (Gmax) of 44 picosiemens at a membrane voltage (Vm) of −65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had Gmax = 66 picosiemens at Vm = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K+ and Na+ about equally well but not Cl−. It had Gmax = 109 picosiemens at Vm = 55 millivolts in (250/50 K) with 10 millimolar Ca2+ on the cytoplasmic side. Reducing Ca2+ to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar β-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.
Ion-sensitive microelectrodes were used to measure Cl− and H+ activities in the cytoplasm of the unicellular green alga Eremosphaera viridis de Bary. In the light, cytoplasmic Cl− activity was 2.2 millimolar at most and cytoplasmic H+ activity was about 5.4·10−8 molar (pH 7.3). Darkening resulted in a permanent increase of the Cl− activity to 3.2 millimolar and in a transient acidification, which was compensated within 3 to 5 minutes. Switching light on again decreased the Cl− activity to the light level (2.2 millimolar). Simultaneously, a transient alkalization of the cytoplasm was observed. The transient character of the light-dependent pH changes was probably caused by pH-stat mechanisms, whereas the light-dependent Cl− activity changes were compensated to a much smaller degree. Studies with different inhibitors (3-(3,4-dichlorophenyl)-1, 1-dimethylurea, piretanide, venturicidin) indicated a direct relation between the light-driven H+ flow across the thylakoid membrane and the observed light-dependent Cl− and H+ activity changes in the cytoplasm. It is suggested that light-driven H+ flux across the thylakoid membrane was in part electrically compensated by a parallel Cl− flux. The resulting Cl− and H+ activity changes in the stroma were compensated by Cl− and H+ fluxes across the chloroplast envelope giving rise to the observed Cl− and H+ activity changes in the cytoplasm.
The quantum efficiencies of photosystems I and II (PSI and PSII), [NADP]/[NADPH] ratios, and the activities of chloroplastic fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were measured in intact pea (Pisum sativum L.) leaves in air following the transition from darkness to 750 microeinsteins per square meter per second irradiance. PSII efficiency declined from a low value to a minimum within the first 10 to 15 seconds of irradiance, after which it increased progressively to the steady-state value. The resistance of electron flow between the photosystems was high at this time, but it was not the principal factor limiting electron flow. Oxidation of P700 was restricted by acceptor side processes for approximately the first 60 seconds of illumination. Once the acceptor side limitation was relieved, the oxidation state of P700 was used to estimate the quantum efficiency of electron transport by PSI. This was observed to increase progressively with time. The quantum efficiencies of both photosystems increased in parallel, consistent with a predominant role for noncyclic electron transport. Fructose-1,6-bisphosphatase activity increased in an approximately sigmoidal fashion with time of irradiance, paralleling the changes in the quantum efficiencies of the photosystems. In contrast...
A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (Fs) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, Fs is a very sensitive indirect measure of the balance of adenosine 5′-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that Fs under constant light is sensitive to manipulation of ambient CO2 concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO2 fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O2 concentration had a strong effect on fluorescence yield, and that O2 sensitivity was only evident when the concentration of CO2 was low. This finding provides evidence that both O2 and CO2 can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C3 plants. Indeed...
The bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1)
consists of two domains, a peripheral arm and a membrane arm. NuoH is a
counterpart of ND1, which is one of seven mitochondrially encoded hydrophobic
subunits, and is considered to be involved in quinone/inhibitor binding.
Sequence comparison in a wide range of species showed that NuoH is
comprehensively conserved, particularly with charged residues in the
cytoplasmic side loops. We have constructed 40 mutants of 27 conserved
residues predicted to be in the cytoplasmic side loops of Escherichia
coli NuoH by utilizing the chromosomal DNA manipulation technique and
investigated roles of these residues. Mutants of Arg37,
Arg46, Asp63, Gly134, Gly145,
Arg148, Glu220, and Glu228 showed low
deamino-NADH-K3Fe(CN)6 reductase activity, undetectable
NDH-1 in Blue Native gels, low contents of peripheral subunits (especially
NuoB and NuoCD) bound to the membranes, and a significant loss of the membrane
potential and proton-pumping function coupled to deamino-NADH oxidation. The
results indicated that these conserved residues located in the cytoplasmic
side loops are essential for the assembly of the peripheral subunits with the
membrane arm. Implications for the involvement of NuoH (ND1) in maintaining
the structure and function of NDH-1 are discussed.
Blue native gel electrophoresis purification and immunoprecipitation of F0F1-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F1 sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.
The mitochondrial permeability transition pore (MPTP) plays a key role in
cell death, yet its molecular identity remains uncertain. Although knock-out
studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the
adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP
opening can occur in the absence of either. Here we provide evidence that the
mitochondrial phosphate carrier (PiC) may also be a critical component of the
MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the
presence of carboxyatractyloside (CAT) that prevents ANT binding to
immobilized PAO. Only four proteins from solubilized CAT-treated beef heart
inner mitochondrial membranes bound to immobilized PAO, one of which was the
PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed
CsA-sensitive binding of PiC to CyP-D; this increased following diamide
treatment. Co-immunoprecipitation of the ANT with the PiC was also observed
but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone
analogues (UQ0 and Ro 68-3400) inhibited phosphate transport into
rat liver mitochondria with the same concentration dependence as their
inhibition of MPTP opening. UQ0 and Ro 68-3400 also induced the
“m” conformation of the ANT...
Microcystis aeruginosa, a unicellular cyanobacterium, contains small phycobilisomes consisting of C-phycocyanin, allophycocyanin, and linker polypeptides. SDS-polyacrylamide gels of the phycobilisomes were examined for fluorescent bands before and after spraying with a solution of ZnSO4, followed by Coomassie brilliant blue staining for protein. This procedure provides a rapid and sensitive method for detecting small amounts of phycobilin-containing polypeptides and distinguishing them from other tetrapyrrole-containing polypeptides and from `colorless' ones. Three polypeptide bands, in addition to the α and β phycobiliprotein subunits, have been detected under these conditions. An 85 kilodalton polypeptide was identified as a phycobiliprotein due to its enhanced fluorescence in the presence of ZnSO4. The other polypeptides do not contain chromophores and are colorless. They are approximately 34.5 and 30 kilodaltons in size.
Electrophysiological measurements were made on the mesophyll cells of wheat (Triticum aestivum L. cv Waldron) and oat (Avena sativa L. cv Garry) coleoptiles treated either with the herbicide diclofop-methyl (methyl 2-(4-(2′,4′-dichlorophenoxy)phenoxy)propanoate), or it's primary metabolite diclofop, (2-(4-(2′,4′-dichlorophenoxy)phenoxy)-propanoic acid). Application of a 100 micromolar solution of diclofop-methyl to wheat coleoptiles had little or no effect on the membrane potential (EM), however in oat, EM slowly depolarized to the diffusion potential (ED). At pH 5.7, 100 micromolar diclofop rapidly abolished the electrogenic component of the membrane potential in both oat and wheat coleoptiles with half-times of 5 to 10 minutes and 15 to 20 minutes, respectively. The concentrations giving half-maximal depolarizations in wheat were 20 to 30 micromolar compared to 10 to 20 micromolar in oat. The depolarizing response was not due to a general increase in membrane permeability as judged from the EM's response to changes in K+, Na+, Cl−, and SO42−, before and after treatment with diclofop and from its response to KCN treatment. In both plants, diclofop increased the membrane permeability to protons, making the EM strongly dependent upon the external pH in the range of pH 5.5 to pH 8.5. The effects of diclofop can best be explained by its action as a specific proton ionophore that shuttles protons across the plasmalemma. The rapidity of the cell's response to both diclofop-methyl (15-20 minutes) and diclofop (2-5 minutes) makes the ionophoric activity a likely candidate for the earliest herbicidal event exhibited by these compounds.