Energetics of Active Phosphate Influx in Hydrodictyon africanum

The energy source for active phosphate influx in Hydrodictyonafricanum has been investigated using gas mixtures with andwithout O₂ and CO₂, light of various wavelengths, and metabolicinhibitors selective for respiratory or photosynthetic electrontransport and phosphorylation. It is concluded that, as in theother green algae studied, active phosphate transport requiresATP. In the dark this is supplied by oxidative phosphorylation;in the light the influx is much less sensitive to inhibitionof oxidative phosphorylation, and photophosphorylation (includingcyclic photophosphorylation) can act as energy source. Thissituation is more like that for active K influx (coupled toactive Na efflux) than to active Cl influx in H. africanum,except that the active dark influx is relatively greater forphosphate influx. The significance of these results for themechanism of regulation of light-stimulated ATP-requiring processes,and for the role of photosynthetic and oxidative phosphorylationin the energy metabolism of green cells, is discussed.     

The Role of Cyclic and Pseudocyclic Photophosphorylation in Photosynthetic 14CO2 Fixation in Hydrodictyon africanum

Experiments are reported in which the effects on photosynthesisof various inhibitors of cyclic photophosphorylation were investigated.These inhibitors, generally had only a small inhibitory effecton photosynthesis, and the inhibition was not increased by conditionswhich inhibit pseudocyclic photophosphorylation. These inhibitorsdo not inhibit the Emerson enhancement effect. From these resultsit was concluded that photosynthesis does not need any ATP otherthan that produced in non-cyclic photophosphorylation. The effectsof these inhibitors on active K influx in light-anaerobic conditionsin the presence or absence of CO₂ suggest that some of the ATPproduced by non-cyclic photophosphorylation can be used to supportactive K influx. The results are discussed in relation to themechanism of the Emerson effect, the stoichiometry of non-cyclicphotophosphorylation, and the ATP requirements for autotrophicgrowth.     

The Linkage of Light-stimulated Cl Influx to K and Na Influxes in Hydrodictyon africanum

There are reciprocal stimulations of Cl influx by K and Na,and of K and Na influx by Cl, in the light in Hydrodictyon africanum.The component of the K influx which stimulates, and is stimulatedby, Cl, is independent of the ouabainsensitive mechanism forK influx also found in H. africanum. The concentration dependenceof the cation effects on Cl influx and on the Cl-stimulatedportion of their own influxes are similar. The stimulation withK saturates at about 0.3 mM K; that with Na saturates at about2 mM Na. The Cl-dependent portions of the K and Na influxeshave similar responses to changes in photo-synthetic metabolism(far-red illumination, CDMU, and CCCP) as does the light-stimulatedCl influx. This suggests that Cl influx, and the Cl-stimulatedportions of K and Na influxes are both dependent on photosystem2 of photosynthesis, and are less sensitive to the uncouplerCCCP than is ¹⁴CO₂ fixation or the K-Na pump. It would thusappear that the Cl-dependent portions of the K and Na influxesin the light are linked to the cation-stimulated portion ofthe Cl influx. There is no very great change in the electricalcomponent of the inwardly directed passive driving force oncations under conditions in which Cl is being pumped comparedwith those under which it is not. It is not clear whether suchincrease in this driving force as do occur could account quantitativelyfor the increase in the cation influxes associated with Cl transport,or whether chemical coupling must be invoked. In addition tothe Cl-stimulated portions of the cation influxes, there arealso light-stimulated portions of K and Na influx which areindependent of Cl, not associated with the cation regulatingmechanism, and which seem to have a similar linkage to photosynthesisas does the Cl-K-Na pump. Since the light-stimulated portionof the K efflux appears to be similar to this portion of theK influx, these Cl-independent light-stimulated portions ofK and Na influxes are tentatively related to light-induced changesin cation permeability.     

Ouabain-insensitive K influx in Hydrodictyon africanum

Summary The occurrence is reported of cells of Hydrodictyon africanum which have, contrary to previous reports by the author, a K influx which is almost insensitive to ouabain. The conditions which govern the ouabain-sensitivity of the K influx have not yet been defined. The low ouabain sensitivity of the total K influx seems to be related to a smaller than usual activity of the component of K influx which is linked to Na efflux, and also to a smaller sensitivity of this component to inhibition by ouabain. The major components of K influx in ouabain-insensitive cells correspond to those components previously described as the passive and Cl-linked components.The occurrence is also reported of an increased sensitivity of active Cl influx (and the coupled cation influxes) in the light to uncouplers when the medium is aerated or otherwise stirred.     

Light Stimulation of Active Transport in Hydrodictyon africanum

The mechanism of light stimulation of active K and Cl influx and active Na efflux, in Hydrodictyon africanum has been investigated using different wavelengths of red light and different gas mixtures, and the inhibitors DCMU and CCCP. The active Cl influx requires photosystem 2, since its relative quantal efficiency falls with increasing wavelength of red light, and it is as sensitive to the inhibitor DCMU as is photosynthesis; it is relatively insensitive to the uncoupler CCCP. The active K influx and active Na efflux are inhibited by CCCP, but the relative quantal efficiency of these processes increases with increasing wavelength of red light, and they are relatively insensitive to DCMU. These cation fluxes can be supported by cyclic photophosphorylation, whereas Cl influx needs photosystem 2 but probably not ATP.     

Measurement of Simultaneous Oxygen Evolution and Uptake in Hydrodictyon africanum

Oxygen uptake and evolution in illuminated and darkened cellsof Hydrodictyon africanum have been measured using ¹⁸O₂ massspeetrometry. Under conditions of light and CO₂ saturation forphotosynthesis, light stimulates oxygen uptake more than two-fold.This stimulation is prevented by DCMU but is not affected bycyanide or the uncoupler CCCP. The data are consistent withthe occurrence of a pseudocyclic electron flow and photophosphorylationin vivo in H. africanum; this agrees with data on light-dependentactive phosphate influx in this alga. Part of the light-stimulatedoxygen uptake might be involved in glycolate synthesis by thepathway proposed by Coombs and Whittingham.     

Measurement of Cytoplasmic pH by the DMO Technique in Hydrodictyon africanum

The 5, 5-dimethyl-[2-¹⁴C]oxazolidine-2, 4-dione (DMO) distributiontechnique for the measurement of intracellular pH has been appliedto giant cells of Hydrodictyon africanum. Significant metabolism of DMO was found in this alga; the free[DMO + DMO–] in subcellular samples is thus derived fromthe total label in cells equilibrated in [¹⁴C]DMO solutionsby measuring and subtracting the label in metabolic productsof DMO. A further problem arises from the observation that theDMO concentration in the vacuolar sap is always lower than thatpredicted by the transmembrane equilibration of undissociatedDMO from the bathing medium. This is interpreted in terms ofa finite permeability to the anion DMO–. Since the effectof P_DMO– on the DMO distribution is much smaller at thetonoplast (where the transmembrane electrical potential differenceis small) than at the plasmalemma, the values of cytoplasmicpH are computed assuming equilibration of undissociated DMOacross the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decreaseor increase of external pH by 1 unit causes a decrease and anincrease in cytoplasmic p11 respectively of about 0.2 pH units.Determinations of _vo at pH 6, 7, and 8, together with an assumedconstant value of _cv, permit calculations of µ_H+ at theplasmalemma and tonoplast. The values are relatively independentof external pH in the range pH 6–8 at 21–25 and12–14 kJ mol^–1 respectively. The significance ofthese results for the regulation of intracellular pH, and forthe regulation and energising of the fluxes of ions, is discussed.     

The Action of Phlorizin on Photosynthesis and Light-stimulated Ion Transport in Hydrodictyon africanum

Phlorizin at 1 mM inhibits the coupled active influx of K andefflux of Na in Hydrodictyon africanum in the light. It doesnot inhibit the coupled influx of Cl and monovalent cations,nor the passive ion fluxes. Photosynthesis can be stimulatedup to 30 per cent under light-saturated conditions. It is concludedthat the effects of phlorizin cannot be solely due to an inhibitionof the membrane ATPase, and that photophosphorylation must insome way be affected. The nature of this effect is discussedin the light of the effects of phlorizin on photophosphorylationin isolated chloroplasts.     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Regulation of K+ Influx in Barley: Evidence for a Direct Control of Influx by K+ Concentration of Root Cells

Siddiqi, M. Y. and Glass, A. D. M. 1987. Regulation of K⁺ influxin barley: Evidence for a direct control of influx by K⁺ concentrationof root cells.—J. exp. Bot. 38: 935–947. The kinetics of K⁺ (⁸⁶Rb⁺) influx into intact roots of barley(Hordeum vulgare L. cv. Fergus) seedlings having different combinationsof root and shoot [K⁺], different growth rates and differentroot:shoot weight ratios were studied. K⁺ influx was stronglycorrelated with root [K⁺]; shoot [K⁺], growth rates, and root:shoot ratios appeared to have little effect on K⁺ influx. Adetailed study showed that both V_max and K_m for K⁺ influx wereaffected by root [K⁺] but not by shoot [K⁺]. We have suggestedthat factors such as growth rates and root: shoot ratio mayaffect K⁺ influx indirectly primarily via their influence onroot factors such as root [K⁺]. We have reiterated that othertypes of kinetic control, e.g. increased or decreased synthesisof ‘carrier systems’, may operate in addition todirect (allosteric?) control of K⁺ influx by root [K⁺]. Thenegative feedback signal from root [K⁺] appeared to be the primeeffector in the regulation of K⁺ influx. Key words: Barley, K⁺ influx     

Cyclic Photophosphorylation in Vivo and its Relation to Photosynthetic CO(2)-Fixation

Salicylaldoxime (2 × 10⁻³m and less) inhibits cyclic photophosphorylation in intact Chlorella cells severely whereas photosynthetic O₂-evolution and ¹⁴CO₂-fixation is hardly affected. Cyclic photophosphorylation in vivo was measured by following anaerobic light dependent glucose uptake. A similar difference in susceptibility has been observed with carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Various controls exclude the possibility that the difference in inhibition was caused by differing experimental conditions or, in the case of glucose assimilation, by an inhibition of a reaction other than photophosphorylation.     

Evidence for Endogenous Cyclic Photophosphorylation in Intact Chloroplasts: CO(2) Fixation with Dihydroxyacetone Phosphate

This study examines the capacity of intact spinach (Spinacia oleracea L.) chloroplasts to fix ¹⁴CO₂ when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial ¹⁴CO₂ fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of ¹⁴CO₂ fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no ¹⁴CO₂ fixation occurred when 3-phosphoglycerate was used. ¹⁴CO₂ fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O₂ (25-50 micromolar) stimulated ¹⁴CO₂ fixation, but the activity decreased with increasing O₂ concentrations. The fixation of ¹⁴CO₂ in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts.     

A Model for the Regulation of K Influx, and Tissue Potassium Concentrations by Negative Feedback Effects upon Plasmalemma Influx

By means of a modified Michaelis-Menten equation for K⁺ influx, which includes terms for root and external K⁺ concentrations (root [K⁺] and [K⁺]₀, respectively) it is possible to predict the manner in which short-term (perturbation) fluxes of K⁺ into roots of barley plants (Hordeum vulgare cv Fergus) vary with root [K⁺] and [K⁺]₀. Influx values derived from this equation were used to predict changes of root and shoot [K⁺] and K⁺ absorption rates (as functions of time and [K⁺]₀) from a knowledge of K⁺ efflux, relative growth rates of roots and shoots, and the partitioning of absorbed K⁺ between these organs. A microcomputer program was employed to model these changes in low-salt plants following transfer to solutions in which [K⁺]₀ was maintained at values ranging from 5 to 1000 millimoles per cubic meter. The model was operated on the basis of 10 minute absorption periods which provided data for continuous `updating' of tissue [K⁺]. The simulations were undertaken for periods corresponding to 30 days. During this time the model accurately predicted the manner in which K⁺ influx and root and shoot [K⁺] gradually approach values which are essentially independent of [K⁺]₀. The computer program was also used to predict the outcome of changing various external and internal parameters of the proposed regulatory system. The results of these simulations are discussed in the context of current models for negative feedback control of ion fluxes.     

Titration of Photophosphorylation, Oxidative Phosphorylation and Glycolysis in Scenedesmus with Desaspidin: Regulation between Pathways and Sites for Photophosphorylation

Narrow concentration intervals were used, covering 10^?6– 10^?4M desaspidin. The interaction with glycolysis involves three steps, the inhibitor constants (K_i:s) being in turn 2.7 × 10^?5M, 1.3 × 10^?4M, and high. About 18% of total glycolysis is inhibited in each of the two first steps, and 65% left for the third reaction. After compensation for glycolysis, oxidative phosphorylation may show a sudden jump to about 10% inhibition at 1.5 × 10^?5M desaspidin, the possible K_i of the reaction starting here being very high. Correcting for glycolysis, desaspidin affects total Photophosphorylation in two steps, with the K_i values of 7.8 × 10^?5M and 4.6 × 10^?4M respectively. Inhibition in the first step is about 27% of the total photophosphorylation. By applying 10^?6M DCMU[/3-(3, 4-dichlorophenyl)-l, l-dimethy lurea], one can abolish non-cyclic photophosphorylation. Desaspidin then reacts in a single step with a K_i of 1.4 × 10^?4M. At 5 × 10^?5M DCMU, also the pseudocyclic photophosphorylation is abolished. The remaining, true cyclic photophosphorylation has a single K_i of 2.3 × 10^?5M for desaspidin. Under non-cyclic conditions, the true cyclic process contributes about 25% to total Photophosphorylation. Under pseudocyclic conditions, no cyclic photophosphorylation occurs. Under true cyclic conditions, the non-cyclic and pseudocyclic processes are inoperative. This indicates a regulative system, so that either (1) the (non-cyclic + true cyclic), (2) only the pseudocyclic, or (3) only the true cyclic systems can be traced, dependent on the level of DCMU applied. There are two sites for non-cyclic Photophosphorylation, one of them common to the pseudocyclic pathway. Cyclic photophosphorylation has a third site, different from the other two.     

Influx and efflux of K(+) in sunflower roots after transfer between solutions with different K(+) concentrations

It was investigated whether K(+) efflux, like K(+) influx, is affected when roots are transferred between solutions with different K(+) concentrations. Sunflower plants (Hehanthus annuus L. cv. Uniflorus) were grown on complete nutrient solutions with 0.1, 1.0, 10 or 25 mM K(+) . This produced plants with K(+) concentrations in the roots varying between 9 and 110 μmol (g fresh weight)(-1) . At the beginning of the experiments the plants were transferred to an (86) Rb-labelled experimental solution initially containing 0.1 mM K(+) . At intervals during 6.5 h samples were removed from the solution and analyzed for K(+) and radioactivity. Based on the analyses K(+) ((86) Rb) influx, K(+) net uptake and K(+) efflux could be computed. In'low K(+) 'roots, K(+) ((86) Rb) influx and K(+) net uptake agreed, suggesting a very low K(+) efflux. This was contrary to'high K(+) 'roots, where K(+) efflux was initially higher than K(+) ((86) Rb) influx. After about 4 h, K(+) efflux declined to a low value also in these roots. When 2-4-dinitrophenol was included in the experimental solution, K(+) ((86) Rb) influx was generally depressed, whereas K(+) efflux was high throughout the experiment and directly proportional to the K(+) status of the roots. Our hypothesis is that after transfer of'high K(+) 'roots to a solution with low K(+) concentration, the K(+) efflux from the vacuoles of root cells transiently increases, until a new electrochemical equilibrium is attained.     

Relative Importance of H2 and H2S as Energy Sources for Primary Production in Geothermal Springs

Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophy-based primary production. Thermodynamic predictions of energy yields for specific electron donor and acceptor pairs in such systems are available, although direct assessments of these predictions are rare. This study assessed the relative importance of dissolved H₂ and H₂S as energy sources for the support of chemolithotrophic metabolism in an acidic geothermal spring in Yellowstone National Park. H₂S and H₂ concentration gradients were observed in the outflow channel, and vertical H₂S and O₂ gradients were evident within the microbial mat. H₂S levels and microbial consumption rates were approximately three orders of magnitude greater than those of H₂. Hydrogenobaculum-like organisms dominated the bacterial component of the microbial community, and isolates representing three distinct 16S rRNA gene phylotypes (phylotype = 100% identity) were isolated and characterized. Within a phylotype, O₂ requirements varied, as did energy source utilization: some isolates could grow only with H₂S, some only with H₂, while others could utilize either as an energy source. These metabolic phenotypes were consistent with in situ geochemical conditions measured using aqueous chemical analysis and in-field measurements made by using gas chromatography and microelectrodes. Pure-culture experiments with an isolate that could utilize H₂S and H₂ and that represented the dominant phylotype (70% of the PCR clones) showed that H₂S and H₂ were used simultaneously, without evidence of induction or catabolite repression, and at relative rate differences comparable to those measured in ex situ field assays. Under in situ-relevant concentrations, growth of this isolate with H₂S was better than that with H₂. The major conclusions drawn from this study are that phylogeny may not necessarily be reliable for predicting physiology and that H₂S can dominate over H₂ as an energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy.     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.     

Evaluation of Hydrodictyon reticulatum as protein source in feeds for Oreochromis (Tilapia) niloticus and Tilapia zillii

The green filamentous alga Hydrodictyon reticulatum was evaluated as a protein source in fish-meal substituted diets for Oreochromis niloticus and Tilapia zillii fingerlings. The fingerlings were fed in duplicate groups each of six different diets for 50 days. Five of the diets contained 30% crude protein supplied by varying proportions of fish meal and H. reticulatum meal. The five diets were formulated to supply fishmeal protein: H. reticulatum meal protein ratios of 30:0 (diet 1), 15:5 (diet 2), 20:10 (diet 3); 15:15 (diet 4), 10:20 (diet 5) respectively. A sixth diet containing only 25% crude protein supplied entirely by H. reticulatum meal was also fed. The best growth and protein utilization was obtained at lower levels of H. reticulatum substitution for both species of fish. Carcass analysis revealed a decrease in lipid contents of the fishes with increasing levels of the alga in the diet.