Photophosphorylation in intact algae: Effects of inhibitors, intensity of light, electron acceptor and donors

The luciferin-luciferase method was used to determine ATP extractedfrom darkmaintained and light-exposed samples of the green algaChlorella pyrenoidosa and of the blue-green alga Anacystis nidulans.A few measurements on Synechococcus lividus (a bluegreen thermophile,clone 65?C) are also reported.

1. The light-minus-dark ATP levels (ATP) from aerobic cells ofChlorella and Anacystis were negative; however, ATP from Synechococcuswas positive. Large positive ATP was obtained in regularly grown(RG: moderate light) Chlorella treated with oligomycin; darklevels were reduced, light levels remained essentially unaffected.In high-light exposed (HLE) Chlorella, oligomycin reduced bothlight and dark ATP levels, but positive ATP was still obtained.However, in Anacystis, which has a different organization ofthylakoid membrane, oligomycin severely reduced both the lightand the dark ATP levels and the ATP remained negative.
2. Theoligomycin (12 µM) treated Chlorella and the untreatedAnacystis and Synechococcus show the presence of cyclic photophosphorylationunder conditions in which the non-cyclic electron flow fromphotosystem II to photosystem I is blocked by 10 µM 3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU), or not allowed to operate by the absence of CO₂. Cyclicphotophosphorylation ranged from 10–30% of the maximumATP in RG, to 40–50% in HLE Chlorella. In RG Chlorella,cyclic and non-cyclic (in the absence of DCMU) photophosphorylation(ATP) saturate at about 10³ ergs cm^–2 sec^–1 and10⁴ ergs cm^–2 sec^–1 and 10⁴ ergs cm^–2 sec^–1red (>640 nm) light, respectively; a lag was observed inthe light curve.
3. In Chlorella, the addition of the photosystemI electron acceptormethyl viologen (MV; 1 mM) increased ATPby twofold. Furtheraddition of DCMU (25 µm) reduced thisto the level observedwith DCMU alone. If 1 mM reduced dichlorophenolindophenol orphenazine methosulphate (DCPIPH₂ or PMSH₂, respectively)wasadded along with DCMU, the ATP level was 30–40% ofthecontrol. Further addition of MV increased the JATP to be70–80%of that of the control. These and other resultsconfirm thepresence of both non-cyclic and cyclic photophosphorylationin vivo, the former predominating in Chlorella, and the latterin Anacystis and Synechococcus.

(Received May 1, 1973; )     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Experimental Investigations on the Environmental Determination of {delta}13C at Different Altitudes

The influence of environmental variables which are known tochange with altitude on the ¹³C of leaves of Nardus stricta,have been investigated in controlled environment experiments.Low temperature and an increased incidence of freezing temperaturescaused ¹³C to increase. In contrast, lowered atmospheric pressurereduced ¹³C. Increased leaf surface wetness and soil water contentalso caused a decrease. Plants from different altitudes didnot differ consistently or significantly in their responses. Key words: Carbon isotope discrimination, altitude, temperature, atmospheric pressure, freezing     

EFFECT OF {alpha}-HYDROXYSULFONATES ON PHOTOCHEMICAL REACTIONS OF SPINACH CHLOROPLASTS AND PARTICIPATION OF GLYOXYLATE IN PHOTOPHOSPHORYLATION

1. The effect of -hydroxy sulfonates and sulfite, inhibitors ofglycolate oxidase, on the photochemical reactions of spinachchloroplasts was studied. The photo reduction of ferricyanideand NADP was not affected by the poisons, whereas the photophosphorylationand ¹⁴CO₂ fixation were inhibited.
2. Glyoxylate was photoreducedby the chloroplasts in the presenceof PPNR and glyoxylate reductase,and this reduction was acceleratedby the addition of NADP.ATP formation accompanied with thereduction of glyoxylate bythe illuminated chloroplasts wasobserved. It is supposed thatglyoxylate oxidizes the photoreducedNADPH₂ or PPNR and thusthe photophosphorylation is stimulated.

¹A part of this paper was presented at the annual meeting ofAgricultural Society of Japan, in August, 1964. ²Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka.     

Effects of Carbon Dioxide on Metabolism by the Glycollate Pathway in Leaves

When solutions of [¹⁴C]glycollate, glycine, serine, glycerate,or glucose were supplied to segments of wheat leaves throughtheir cut bases in the light, most of the ¹⁴C was incorporatedinto sucrose in air but in CO₂-free air less sucrose was made.The synthesis of sucrose was decreased because metabolism ofserine was partly blocked. Sucrose synthesis from glucose andglycerate in CO₂-free air was decreased but to a smaller extent;relatively more CO₂ was evolved and serine accumulated. Theeffects of DCMU and light of different wavelengths on metabolismby leaves of L-[U-¹⁴C]serine confirmed that simultaneous photosyntheticassimilation of carbon was necessary for the conversion of serineto sucrose. Of various products of photosynthesis fed exogenouslyto the leaves -keto acids were the most effective in promotingphotosynthesis of sucrose and release of ¹⁴CO₂ from ¹⁴C-labelledserine. This suggests that in CO₂-free air the metabolism ofserine may be limited by a shortage of -keto acid acceptorsfor the amino group. In CO₂-free air added glucose stimulatedproduction of CO₂ and sucrose from D-[U-¹⁴C]- glycerate andno competitive effects were evident even though glucose is convertedrapidly to sucrose under these conditions. In addition to asupply of keto acid, photosynthesis may also provide substratesthat can be degraded and provide energy in the cytoplasm forthe conversion of glycerate to sugar and phosphates and sucrose.     

In situ evidence that chilling in the light does not cause uncoupling of photophosphorylation or detachment of coupling factor in chilling-sensitive plants

The potential involvement of impaired photophosphorylation in the chilling sensitivity of photosynthesis in warm climate plant species has been a topic of investigation for more than two decades. With recent advances in the analysis of photosynthetic energy transduction in intact leaves, experiments are now possible that either address or avoid important uncertainties in the significance and interpretation of earlier in vitro work. Nevertheless, different laboratories using different techniques to analyze the effects of chilling in the light on photophosphorylation in intact cucumber (Cucumis sativus) leaves have come to very different conclusions regarding the role of impaired ATP formation capacity in the inhibition of net photosynthesis. In order to evaluate these discrepancies and bring this issue to a final resolution, in this investigation, we have made a detailed analysis of the decay of the flash-induced electrochromic shift and changes in chlorophyll fluorescence yield in cucumber leaves before, during and after a 5 h light-chill at chill temperatures of between 4 and 10°C. We feel that our findings address the major discrepancies in both data and interpretation as well as provide convincing evidence that photophosphorylation is not disrupted in cucumber leaves during or after light and chilling exposure. It follows that impaired photophosphorylation is not a contributing element to the inhibition of net photosynthesis that is widely observed in warm climate plants as a result of chilling in the light.Abbreviations CF chloroplast coupling factor or CF₁CF₀-ATP synthase - A₅₁₈ flash-induced electrochromic absorbance change measured at 518 nm - DCCD N,N'-dicyclohexylcarbodiimide - _H ⁺ transmembrane electrochemical potential of hydrogen ions - the electrical charge component of _H ⁺ - pH the hydrogen ion concentration component of _H ⁺ - F₀ and F_m the yields of chlorophyll fluorescence from dark-adapted material when all Photosystem II centers are open (F₀) or closed (F_m) - F₀' and F_m' F₀ and F_m measured in light-adapted material - F_s steady-state chlorophyll fluorescence yield in light-adapted material - Q_A primary quinone electron acceptor of Photosystem II - PPFD photosynthetic photon flux density     

Respiration Rate and Adenosine Triphosphate Formation in Tissues4

When leaves of Nicotiana tabacum L. var. Xanthi were inoculatedwith TMV, local lesion formation gave enhanced ATP concentrationaccompanied by increased respiration. Phosphorus metabolismin healthy and infected leaves was investigated either by floatingleaf tissue on ³²P₁-phosphate buffer or by allowing intact leavesto take up ³²P₁-phosphate buffer through the petioles. Bothmethods gave increased ³²P₁ incorporation into inorganic andorganic acid-soluble phosphorus fractions of the infected leaf.Comparison of the specific activity of ³²P₁-phosphate buffersupplied with the specific activity of the -phosphate groupof ATP demonstrated that 50 per cent of the ATP in the healthyleaf compared with over 78 per cent of the ATP in the infectedleaf was metabolically active in respiration. As the ATP concentrationin the infected leaf is much greater than that in the healthyleaf, this means that the amount of ATP being utilized and resynthesizedas a result of respiratory metabolism in the infected leaf ismore than twice that in the healthy leaf. Pulse-labelling experimentsdemonstrated that the rate of ATP turnover was very similarin healthy and infected leaves; therefore the increased respirationin the infected leaf results from the larger ATP pool with aturnover rate of ATP similar to that in the healthy leaf. Withtobacco callus tissue cultures infected by TMV, where infectiondoes not result in local-lesion formation, phosphorus metabolismwas unaltered in the infected tissue. It is concluded that necroticlocal-lesion formation results from increased availability ofATP at sites of ATP utilization, and this is aggravated by over-productionof ATP in the infected leaf.     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Variations of Natural 13C Abundances in Leguminous Plants

The natural ¹³C abundance (¹³C value) of the field-grown leguminousplants (soybean, kidney bean, pea, azuki bean, mung bean, peanutand cowpea) was investigated by mass spectrometry with a precisionbetter than %0.2 for ¹³C. Among organs of premature plants,the leaves had the most negative values, and the nodules generallyhad the least negative values, and other organs, fruits, stemsand roots, showed intermediate values. In the soybeans so farinvestigated, the grains of nodulating plants exhibited higher¹³C values than nonnodulating lines. The ¹³C values of the grainsvaried depending on the species: peanuts showed the most negativevalues. Possible causes underlying these variations are discussed. ³Institute of Biological Sciences, University of Tsukuba, Sakura-mura,Ibaraki 305, Japan. (Received December 3, 1982; Accepted May 25, 1983)     

Ammonia Regulation of Intermediary Metabolism in Photosynthesizing and Respiring Chlorella pyrenoidosa: Comparative Effects of Methylamine

The natural ¹³C abundance (¹³C value) of the field-grown leguminousplants (soybean, kidney bean, pea, azuki bean, mung bean, peanutand cowpea) was investigated by mass spectrometry with a precisionbetter than %0.2 for ¹³C. Among organs of premature plants,the leaves had the most negative values, and the nodules generallyhad the least negative values, and other organs, fruits, stemsand roots, showed intermediate values. In the soybeans so farinvestigated, the grains of nodulating plants exhibited higher¹³C values than nonnodulating lines. The ¹³C values of the grainsvaried depending on the species: peanuts showed the most negativevalues. Possible causes underlying these variations are discussed. (Received March 2, 1983; Accepted May 27, 1983)     

Investigations of the {delta}13C Pattern in Leaves of Fagus sylvatica L.

The natural abundance of ¹³C in different parts of beech (Fagussylvatica L.) leaves was analysed. Values for leaf ribs wereconsistently higher than those for intercostal tissue. Similardifferences occur between petiole and stem, with petiole beingless negative. The pattern of results is the same, independentof the position in the tree. However, the absolute values differby up to 6%. from the bottom to the top of the tree. Valuesof ¹³C are in the range of – 29 to – 32%. for thelower leaf strata; while values between – 24 and –26%. have been measured for the top of the tree. Absolute ¹³Cvalues of the whole tissue and cellulose differ by about 2%.,but relative ¹³C trends are almost identical. However, ¹³C trendsare not identical for different leaf parts. A comparison ofcellulose and whole tissue ¹³C data makes it unlikely that the¹³C variations are primarily due to different compositions ofchemical compounds. No fractionation seems to exist betweenleaf and wood cellulose. Tissue from different areas of a leafrevealed identical carbon isotope compositions. Key words: Carbon isotope ratio, Fagus sylvatica L., beech leaves     

Direct Evaluation of Effects of Fatty-Acid Unsaturation on the Thermal Properties of Photosynthetic Activities, as Studied by Mutation and Transformation of Synechocystis PCC6803

Glycerolipids of thylakoid membranes isolated from the cyanobacteriumSynechocystis PCC6803 contained high levels of dienoic and trienoicC₁₈ fatty acids, in addition to saturated C₁₆ and monoenoicC₁₈ fatty acids. A mutant (Fadl2) of this cyanobacterium wasdefective in the desaturation of C₁₈ fatty acids at the ¹² position,and its thylakoid membranes lacked trienoic acids and containeda very reduced level of dienoic acids. A derivative strain ofFadl2 (Fadl2/desA), which had been transformed with a gene fordesaturation at the ¹² position, fully recovered the abilityto desaturate the fatty acids in the glycerolipids of thylakoidmembranes. The thermal properties of the photosynthetic activitiesof the mutant and the transformant were compared with thoseof the wild-type strain. Despite great diversity in the extentof unsaturation of fatty acids between the wild-type, Fadl2,and Fad12/desA strains, no significant differences were foundeither in the temperature dependence of photosynthesis or inthe heat stability of photosynthetic, photosystem II and photosystemI activities. These results demonstrate that the trienoic fattyacids and, probably, the dienoic acids of the lipids in thethylakoid membrane do not affect the thermal properties of theabove-mentioned activities of photosynthesis. ³Permanent address: Institute of Plant Physiology, BiologicalResearch Center of Hungarian Academy of Sciences, H-6701 Szeged,P.O. Box 521, Hungary (Received August 9, 1990; Accepted December 7, 1990)     

STUDIES ON PHOTOPHOSPHORYLATION: I. TWO-STEP EXCITATION KINETICS OF PHOTOPHOSPHORYLATION

Based on the observations on the time lag and the low efficiencyof photophosphorylation at low light intensities, a ‘two-stepexcitation kinetics’ of photophosphorylation of chloroplastswas developed, which could explain some of the hitherto unexplainedfacts concerning photophosphorylation. By analyses of the experimental results along the line of thetheory, it was inferred that the assumed substance (X) responsiblefor ATP formation occurs in a relatively large quantity of about1 mole/5–20 moles of chlorophyll. It was concluded thatthe high energy intermediate (X^**) of ATP formation which wasassumed to accumulate on pre-illuminating the chloroplasts (inthe absence of ADP and Pi) does not represent a side pool ofextra energy, but a substantial intermediate in the normal courseof ATP formation. Alternative possibilities for the explanation of the experimentalfacts were also discussed. ¹This paper is submitted to the University of Tokyo to fulfilla part of the requirements for the doctorate of H. SAKURAI.     

Temporal variations in carbon isotope ratio of phytoplankton in a eutrophic lake

Temporal variations in carbon isotope ratio of phytoplanktonand dissolved inorganic carbon (DIC) in Lake Suwa were reported.In summer, blooming of Microcystis spp. resulted in low concentrationsof DIC and high pH, and HCO₃^– was the prominent speciesof DIC. Chlorophyll-specific rates of photosynthesis were relativelyconstant irrespective of the algal biomass during summer. Carboxylationin photosynthesis of Microcystis spp. was mainly catalyzed byribulose bisphosphate carboxylase (RuBPCase). Carbon isotopediscrimination between ¹³C of phytoplankton and DIC was considerablysmall in early summer and appeared to be negatively correlatedto DIC concentration. We concluded that carbon fixation by phytoplanktonin Lake Suwa is controlled not by the switch of photosyntheticpathways, but by low DIC concentration and high pH, suggestingthat photosynthesis of Microcystis spp. in Lake Suwa is governedby uptake kinetics other than the carboxylation step.     

ENZYMIC FORMATION OF GLUTAMINE IN RICE-PLANT SEEDLINGS

1. Purified preparation from rice-plant seedling catalyses a stoichiometricreaction between ATP, glutamate, and NH₂OH in the presence ofMg⁺⁺ to form glutamyl hydroxamate, ADP and inorganic phosphate.
2. The method of purification and some of the properties of theenzyme are described. Co⁺⁺ can be substituted for Mg⁺⁺. Mn⁺⁺,NaF, and PCMB inhibit the enzyme strongly.
3. Inorganic orthophosphateis liberated from ATP by the additionof or cysteine in the presence of glutamate, Mg⁺⁺ andthis preparation. Glutamine was detectedin the reaction productsby paperchromatography.
4. The same preparation catalyses a reactionbetween gluta mineand NH₂OH in the presence of Mg⁺⁺ or Mn⁺⁺,ADP and inorganicphosphate, to form glutamyl hydroxamate.

¹ Present address: The Department of Chemistry, Faculty of Science,Kanazawa University, Kanazawa. (Received October 31, 1960; )     

PKC-delta and CaMKII-delta 2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-₂, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-Ras^N17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-₂ coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- ₂; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus     

Stomatal Control of Photosynthesis of Eucalyptus globulus Labill. Trees under Field Conditions in Portugal

Pereira, J. S., Tenhunen, J. D. and Lange, O. L. 1987. Stomatalcontrol of photosynthesis of Eucalyptus globulus Labill. treesunder field conditions in Portugal.—J. exp. Bot. 38: 1678–1688. Stomatal behaviour of adult leaves of Eucalyptus globulus treeswas studied under field conditions in Portugal. In the absenceof severe plant water stress stomata were open when the summedtotal of photosynthetically active photon flux density incidenton both leaf surfaces was above 100 µmol m²s¹ and leafconductance to water vapour reached 245 mmol m ² s¹ on a total(both epidermes) leaf area basis. The stomata of both leaf epidermesresponded similarly to changes in solar radiation and waterstress. Water stress resulted in decreasing daily maxima inleaf conductance as predawn leaf water potential decreased.Maximal leaf conductance decreased to less than 50 mmol m ²s ¹ when predawn leaf water potential decreased below —1·0MPa. At similar values of predawn leaf water potential stomatawere more closed as the leaf to air water vapour partial pressuredifference increased. The effect of increasing air dryness onstomata was greatest at high predawn leaf water potential. Dailymaxima in photosynthetic rates and in leaf conductance werelinearly related to one another in spring and summer. Both decreasedwith increase in leaf water stress. In autumn and winter, increasesin leaf conductance occurring under natural conditions duringthe course of the day were not necessarily accompanied by increasesin net photosynthesis. Stomata were more closed in the afternoonthan in the morning at the same rates of net photosynthesis,temperature or leaf to air water vapour partial pressure difference. Key words: Eucalyptus globulus,, photosynthesis, stomata, water stress.     

Interspecific variability and environmental influence on particulate organic carbon {delta}13C in cultured marine phytoplankton

The stable carbon isotope composition of particulate organicmatter expressed as ¹³C was measured in cultures of 13 speciesof marine microalgae in different phylogenetic groups. The effectsof salinity variations and changes in photoperiod were alsoassayed for three of them (i.e. Skeletonema costatum, Amphidiniumopercularum and Isochrysis galbana); the effect of nature ofnitrogen supply (nitrate. ammonium) was studied for one (S.costatum).These environmental parameters were chosen because of theirvariability in the ocean and their possible effects on ¹³C valuesof phytoplankton organic carbon. Batch culture conditions andsampling time after inoculum were strongly controlled in orderto provide cells in good physiological state which were comparablefrom one culture to the other. In the same way, sampling waslimited to the first 2 days of exponential growth, in orderto avoid a possible dissolved inorganic carbon (DIC) limitation.Carboxylase activities [of the enzyme ribulose 1,5-bisphosphatecarboxylase oxygenase (Rubisco), and the three ß carboxylases:phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvatecarboxykinase (PEPCK) and pyruvate carboxylase (PC)] and totalchlorophyll a concentrations were assayed simultaneously. The¹³C values observed were between –30.2 and –12.7i.e. comparable to those observed in the world's oceans. Theisotopic composition of phytoplankton organic carbon was shownto be under the influence of the parameters tested but ¹³C variationsare specific to the species considered. The nature of ßcarboxylase found in each species, or systematic position, couldnot be linked to the isotopic composition of organic carbon.No linear or single correlation between ¹³C variations and environmentalmodifications were observed and there is no evidence for a simpleand universal relation between ¹³C of phytoplankters and theirenvironment. In monospecific cultures as in the field, ¹³C fractionationby Rubisco (and eventually by PEPCK) may be counterbalancedby other mechanisms.     

The development of photophosphorylation and photosynthesis in greening bean leaves

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10⁻⁵m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10⁻⁵m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.